JPH0326967A - Analysis of nitrite ion and analytical element - Google Patents

Abstract

PURPOSE:To measure the nitrite ion in body fluids with high sensitivity by allowing electrolyte halide to be present in an analytical system. CONSTITUTION:Electrolyte halide is allowed to be present in an analytical system. The electrolyte halide is contained in the analytical element 2 constituting the analytical system due to a nitrite ion developing reagent. A diazo component is a compound reacting with nitrous acid to form a diazonium salt, that is, an amine compound having a primary amino group. The electrolyte halide is allowed to be present in the nitrite ion analytical system in an amount of 0.1 - 20wt. per 1wt. of a diazo color forming reagent as sodium chloride. As the form of the nitrite ion analytical element 2, a monolithic multilayer analytical element wherein a reagent-containing reaction layer 12 and a porous developing layer 13 are provided on a liquid impervious transparent support 11 is pref. designated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a body fluid analysis method and an analysis element used in clinical chemical tests of various body fluids, and particularly relates to the analysis of nitrite ions.

[Background of the invention]

Various body fluids are overwhelmingly used as samples for clinical chemical analysis. Among them, blood, especially serum, is commonly used as a sample because of its many advantages, such as normally maintaining homeostasis, making it easy to obtain information on whole-body metabolic dynamics and various organs, and being relatively less invasive to collect. It is being Urine is a relatively commonly used specimen, and gastric juice, pancreatic juice, intestinal fluid, bile, and cerebrospinal fluid may also be used as samples.

Saliva is an analysis sample that has been completely ignored until recently in the field of clinical medicine, although its secretory physiology is relatively well understood.

However, it is a sample that can be collected at any time with the least burden on the patient, can be obtained in sufficient quantity except in special cases, does not require special pretreatment, and has many advantages as a sample for clinical chemistry. It is thought that there will be more opportunities to use it in the future as research develops. Detection of saliva components is not only for clinical chemistry purposes, but also extends to the legal maintenance of social order, quick and simple judgments in emergency situations, and simple daily hygienic checks at home. There are many different types of analytical elements (for example, Japanese Patent Application Laid-open Nos. 60-178828 and 63-5).
No. 8257, etc.) have been proposed and disclosed, and even check for bad breath.

Volatile sulfur compounds, alcohol compounds, aldehyde compounds, amine compounds, ammonia, etc. have been proven to be the main odors in bad breath. In particular, some odors emitted from the oral cavity are caused by hydrogen sulfide, methylmerkab. Tan,
It is known that mercabuto compounds such as dimethyl sulfide are the main component. A method for detecting mercabuto compounds in exhaled breath or saliva for the purpose of measuring bad breath is disclosed in JP-A-57-135360 and JP-A-57-148252.
No., JP-A-58-191957 and JP-A-60-17
No. 8828.

However, these methods have drawbacks such as the measurement sensitivity being too low to adequately detect bad breath, and the stability of the reagents being poor even if the measurement sensitivity is sufficient, making it difficult to accurately measure bad breath. I couldn't do that.

Furthermore, JP-A-63-1-464 states that it is impossible to detect halitosis using mercabuto compounds as an indicator due to its sensitivity, and that a nitrite ion calibration curve in saliva, which has a correlation with the above-mentioned halitosis, is used as an indicator to detect nitrite ions. A method for detecting bad breath using a reagent that develops color through a diazo reaction, a test paper impregnated with the reagent, and an analytical element has been proposed.

This method has been developed based on the rapidity and simplicity of the reaction and its stability between a compound that reacts with nitrite ions in diazo and a reagent containing a polymeric acid that develops color by diazo coloring (hereinafter simply referred to as color reagent). There is.

However, the methods proposed so far are still insufficient in sensitivity.

[Purpose of the invention]

An object of the present invention is to provide an analytical method for determining nitrite ions present in body fluid samples with high sensitivity, quickly, simply, and with high precision, and an analytical element used in the analysis.

[Structure and effects of the invention]

The object of the present invention is to provide a nitrite ion analysis system based on a nitrite ion coloring reagent containing a compound that develops diazo color with nitrite ions and an acid that does not liberate halogen ions. This is achieved by a nitrite ion analysis method characterized by the presence of an electrolyte halide and an analytical element in which the nitrite ion coloring reagent further contains an electrolyte halide.

The compound that develops diazo color with nitrite ion according to the present invention is composed of a diazo component and a coupling component.

Diazo bokun is a compound that reacts with nitrous acid to form a diazonium salt, and is an amine compound with a primary amino group. Preferred amine compounds include heterocyclic amines and aromatic amine derivatives.

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 the diazonium salt, and is an aromatic compound having an amino group and/or a hydroxyl group, and is a derivative thereof.

Furthermore, when the sosoling component is an aromatic amine, organic acid salts or inorganic acid salts may be used.

These two components are dissolved or dispersed in water or a water-soluble solvent to form a diazo reaction coloring reagent. In this case, the ratio of the two components and the concentration of the coloring reagent are appropriately adjusted to cover the concentration range of nitrite ions to be detected. An acid is required for the reaction of the diazo reaction coloring reagent.

Acids that do not liberate halogen ions according to the present invention include acids that do not contain halogen or do not dissociate halogen ions even if they contain, such as inorganic acids such as sulfuric acid, nitric acid, sulfamic acid, and phosphoric acid. , for example, acetic acid,
Carboxylic acids such as chloroacid, trichloroacetic acid, probionic acid, succinic acid, benzoic acid, salicylic acid, organic sulfonic acids such as p-toluenesulfonic acid, benzenesulfonic acid, and polymeric acids are used.

Polymer acids include polyacrylic acid, polymethacrylic acid, acrylic acid-butyl acrylate copolymer' (50:
50), ethylene-monobutyl maleate copolymer, polystyrene sulfonic acid, acrylamide-2-methylethanesulfonic acid-butyl-styrene acrylate copolymer (15:80:5), poly-2-methacryloxyethyl Phos 7A 1, 2 Acrylamide 2-
Methylpropanesulfonic acid monobutyl acrylate copolymer. (50:50) etc.

Note that the numbers in parentheses represent the polymerization ratio.

A predetermined amount of the former two is mixed to form the above-mentioned coloring reagent. When saliva is supplied to the analysis system, the pH of the analysis system is 0.
It is sufficient that the content is 1 to 2.

Examples of the electrolyte halides according to the present invention include sodium fluoride, sodium chloride, sodium bromide, potassium fluoride, potassium chloride, potassium bromide, magnesium chloride, magnesium bromide, calcium chloride, calcium bromide, Examples include lithium chloride and lithium bromide.

The electrolyte halide is present in an amount of 0.1 to 20 wL in terms of sodium chloride per 1 vL of the diazo coloring reagent in the analysis system.

The nitrite ion analysis element of the present invention is preferably in the form of an integrated multilayer analysis element (specially Publication No. 53-21677, JP-A-55-
No. 164359, No. 55-90859, No. 57-19
No. 7466, No. 57-101760, No. 57-101
No. 761, No. 58-90167, etc.).

The reaction layer can be provided as a layer by applying a water-soluble polymer or a hydrophilic and organic solvent-soluble polymer as a binder onto the support. Examples of water-soluble polymer binders include gelatin, gelatin derivatives such as pentatalated gelatin, water-soluble cellulose derivatives such as hydroquine ethyl cellulose and carboxymethyl cellulose sodium salt, polyvinyl alcohol, poly(N-vinylpyrrolidone), polyacrylamide, and polymethacrylamide. , a copolymer of acrylamide and acrylic ester,
Examples include poly (mono- or dialkyl-substituted) acrylamide, poly (mono- or dialkyl-substituted) methacrylamide, and water-soluble copolymers thereof, and preferably gelatin, polyacrylamide, and acrylamide and acrylic acid ester ρ copolymers are used. . Hydrophilic and organic solvent-soluble polymer binders include poly(N-vinylpyrrolidone) and poly(N-vinylimidazole).
, poly(N-vinyltriazole), derivatives thereof or copolymers thereof, cellulose derivatives such as ethyl cellulose and methyl cellulose, and the like.

In addition, when two or more types of reagents are contained in a reaction layer, even if these reagents are mixed together in the same reaction layer, two or more types of reagents may be mixed together in the same reaction layer. They may be contained separately or in combination in separate reaction layers.

The thickness of the reaction layer can be arbitrarily selected as desired, but is preferably 1 to 200 μm, and more preferably 5 to 100 μm.

The liquid-impermeable transparent support (hereinafter abbreviated as support) may be of any type as long as it is liquid-impermeable and transparent, but examples include cellulose acetate and polyethylene terephthalate.
- It is possible to use various polymeric materials such as polycarbonate or polystyrene, but also inorganic materials such as glasses. The thickness of the support is arbitrary, but
Preferably it is 5 to 250 μm.

Furthermore, a transparent undercoat layer may be used on the inner surface of the support on which the reaction layer is laminated, as the case may be, to improve the adhesion between the reaction layer and the support.

The porous spreading layer according to the present invention has a porous structure having interconnecting pores (preferably a short diameter of 5 μm to 300 μm) that can freely contact the body fluid sample in order to uniformly and quickly spread the body fluid sample. It is necessary to be present.

Preferable examples include granules having a size of 10 to 350 μm;
Another example is a structure made of one or more materials selected from 40 to 400 mesh fibers.

Materials for the granules include diatomaceous earth, titanium dioxide, barium sulfate, zinc oxide, lead oxide, microcrystalline cellulose, silica sand, glass, silica gel, cross-linked dextran, cross-linked polyacrylamide, agarose, cross-linked agarose, and various synthetic resins (such as polystyrene). ), etc.

Further, the fibers used in the porous spreading layer according to the present invention include bulb, powder filter paper, cotton, hemp, silk, wool, chitin, chitosan, cellulose ester, viscose rayon, ammonia rayon, polyamide (6-nylon, 66
-nylon, 610-nylon, etc.), polyester (
polyethylene terephthalate, etc.), polyolefins (
polypropylene, vinylon, etc.), glass fiber, asbestos, and other vegetable, animal, and mineral fibers;
．． A single recycled fiber may be used, or a mixture of these may be used.

By coating and/or forming a film with such granules, fibers, or a mixture of granules and fibers, a porous structure in which a porous structure with interconnecting pores that can freely contact the body fluid sample is created. Form a fake. These granules, fibers, etc. are formed into a film using the above-mentioned binder in such a way that the particles adhere to each other at points.
The methods of No. 90859 and No. 57-67860 can be applied.

The porous spreading layer has the function of (1) uniformly distributing a certain volume of body fluid sample to the reaction layer per unit area; Furthermore, the performance described in Japanese Patent Publication No. 53-21677, i.e. (2) the ability to remove substances or factors that inhibit reactions in body fluid samples, and/or (3) support when performing spectrophotometric analysis. It is preferable that it has the function of performing a background effect of reflecting measurement light transmitted through the body.

For example, a spread layer of a non-fibrous porous medium called brush volima made of titanium dioxide and cellulose diacetate is described in Japanese Patent Publication No. 53-21677;
No. 94658, No. 57-12847, No. 57-197
Examples include the fiber structure spreading layer described in Japanese Patent Application Laid-open No. 466 and No. 58-70161, and the particle combination structure spreading layer described in JP-A-58-90167.

The thickness of the spreading layer in the analytical element of the present invention should be determined by its porosity, but is preferably about lOO
~600μ, more preferably about 150-400/11
It is 11. In addition, the porosity is preferably about 20 to 85%.
It is.

Further, various other additives such as preservatives and the like may be added to the developing layer as desired.

The integrated type multi-counterfeit analysis element described in lnJ may be prepared by using, for example, the adhesive layer described in Japanese Patent Application No. 63-207282, or the adhesive layer described in U.S. Pat.
,992. 158, a subbing layer, a radiation blocking layer as described in U.S. Pat. No. 4,042,335,
The barrier layer described in U.S. Pat. No. 4,066,403, the migration prevention layer described in U.S. Pat. No. 4,166,093, the scavenger layer described in JP-A-55-90859, etc. can be arbitrarily combined to meet the objectives of the present invention. Any structure can be used.

The various layers of these analytical elements are arranged on the support according to the invention.
In accordance with the desired W4fR, it is possible to apply a layer of any thickness by sequentially laminating layers using the slide hopper coating method, extrusion coating method, dip coating method, etc. that have been conventionally used in the photographic industry. can.

Next, the analytical element of the present invention and its use will be explained using an example of its embodiment.

In FIG. 1, 1 is the main body of the clinical chemistry analyzer, and in FIG. 2, 2 is the analytical element. Analysis element 2 is observation window 21
A film 23 in which a developing layer is laminated on a reaction layer impregnated with a certain reagent is interposed between a mount base 2l having a body fluid spotting hole 22a and a mount force bar 22 having a body fluid spotting hole 22a. A reference coloring code 24 for identifying reagent data is installed on the surface of the mounting force bar 22 so that it can be identified in 5 bits. The analytical element 2 is inserted through the element insertion opening IT provided on the front surface of the main body I, is held between a pair of rollers for carrying the element installed in the main body I, and is carried into the incubation means. The incubation means maintains the analytical element 2 at a set temperature and transfers the analytical element 2 on which the body fluid sample has been deposited to the photometry section after a set time.

Next, FIG. 3 shows an embodiment of an analytical element that easily collects body fluid (for example, saliva) and analyzes it without requiring special analytical equipment.

In the above example, a reaction layer is provided on a transparent support, and a porous spreading layer is provided on top of the reaction layer, which is inert to each of the various body fluids and can uniformly spread the body fluids.

A body fluid (e.g., saliva) collection member (abbreviated as collection member) is capable of collecting a predetermined amount of body fluid, contains the collected body fluid, and can easily supply the required amount of body fluid to a detection member consisting of at least a reaction layer and a development layer. A flexible porous material is chosen that can provide release.

The detection member and the collection member are connected, and the holders are connected by a flexible or bendable connection member so that the collection member is in contact with the developed layer of the detection member. The surfaces can be freely moved in and out.

Furthermore, various auxiliary members, auxiliary structures, or layers can be added to supplement or maintain the performance of the analytical element and to facilitate measurement operations. For example, it is preferable to include a hook for detecting the cover when the analytical element is not in use and for maintaining pressure on the sampling member, a mount for holding both members, and the like.

In FIG. 3, reference numeral 1 denotes a detection member, which has a structure in which a reaction layer 12 containing a reagent is laminated on a support 11, and a development layer 13 is further laminated thereon. , and is clamped to a mount 4 having an observation window 42.

Reference numeral 2 denotes a collection member, which is bonded to a connecting member 3 that connects the detection member 1 and the collection member 2 with an adhesive 123;
It covers the body fluid spot 4l and faces the development layer l3.

The connecting member 3 is fixed to the mount 4 at one end so as to be openable and closable with respect to the developing layer l3, and is fitted onto the mount 4 by a hook 3l provided at the other end. The connecting member closes when the analysis element is not in use and serves as a cover for the detection member 1 and collection member 2, opens to serve as a "hunting point" when collecting body fluids, and closes again to fit the hose 3l to collect the body fluid. Member 2 and development layer 1
A pressure contact between 3 and 3 can be maintained.

The amount of the body fluid sample to be used is arbitrary as long as it is sufficient to be impregnated with the body fluid sample, but it is preferably 5 to 50 μQ per lcm2, and more preferably about 5 to 20 μQ.
It is. It is usually preferred to apply a sample of body fluid of about 10μQ.

〔Example〕

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

Example 1 (1) Preparation of analytical element The following coating solution was coated on a transparent subbed polyethylene terephthalate support having a thickness of about 180 μm and dried to form a reaction layer. The film thickness after drying is approximately 2 0 11 rrr
Met.

(Reaction layer) N-1-naphthyl-N'-ethylethylenediamine oxalate 3.0 g sulfanilic acid 1.3 g 2-acrylamide 2-methylpropanesulfonic acid-butyl acrylate copolymer (copolymerization ratio 50:50) 7 .7g bird
Tons X - 100
2.9 g (manufactured by Rohm & Haas) Polyacrylamide 8.5 g Distilled water was added to make 200.0 g.

A coating solution having the following composition was applied and dried to laminate a porous development layer. The film thickness after drying is approximately 280 μm.

(Development layer) Filter paper powder D (manufactured by Advanten Toyo Co., Ltd.) 24.0g Triton X-100
2.4 κ (manufactured by Rohm & Haas) Styrene-glycidyl methacrylate copolymer (t ratio 9:l) 7.4 g HoIJ (N-vininolepyrrolidone) 1.3 g xylene
40.0g pork
20. 1. The multilayer film produced in this way. 5cmXl. It was cut into 5 cm pieces and sealed in a plastic mount to prepare analytical element 1 (1) for comparison.

Furthermore, an analytical element of the present invention was prepared in the same manner as above except that 1.00 g of sodium chloride was added to the reaction layer coating solution.
I created l.

In addition, 2.40% of sodium chloride was added to the developing layer coating solution.
Analytical element-2 of the present invention was prepared in the same manner as above except that g was added.

Furthermore, an analytical element of the present invention was prepared in the same manner as above except that 1.00 g of sodium chloride was added to the reaction layer coating solution and 2.40 g of sodium chloride was added to the development layer coating solution.
3 was created.

(2) Measurement of nitrite ion using analytical element 10μg/m
f2, prepare a sodium/lium nitrite aqueous solution with a concentration of 40 ug/mQ and 80 ug/mQ, and add the above analytical element (1).
And on the developing layers 1 to 3, the above sodium nitrite aqueous solution 1011 (2 spots was applied. 30# fi, 546
Reflection density (Dr) was measured using a ni interference filter.

The results are shown in Table I.

Table 1 As is clear from the above table, the analytical element of the present invention exhibits a higher reflection density than the comparative analytical element, especially for sodium chloride.
- It is clear that Analytical Elements 2 and 3 of the present invention, in which lium is added to the developing layer, exhibit significantly high reflection concentrations, and it is clear that nitrite ions can be measured with high sensitivity using the analytical element of the present invention.

Example 2 (1) Preparation of analytical element A reaction layer coating solution having the composition described in Example 1 was coated on a transparent subbed polyethylene tere-7 tallate support having a thickness of 180 μm. The thickness of the reaction layer after drying was 21 μm.

Next, on this upper layer, a 5% butanol solution (containing 0.1mM bromophenol blue) of N.vinylpyrrolidone-vinyl acetate copolymer (copolymerization ratio 80:20) was applied and dried to form an adhesive layer ( The dry film thickness was 3 μm).

Furthermore, applying a certain spreading layer coating solution as described in Example-1,
Drying (dry film thickness 260μ+n) L, analytical element-(2) for comparison was prepared.

Also, sodium chloride (1.75g), potassium chloride (2.24g), magnesium chloride hexahydrate (3.05g)
g), chloride (1.66 g) and sodium sulfate (4.26 g) were respectively added to the above-mentioned developing layer coating solution, which was coated and dried in the same manner as above to form analytical elements 4 and 5 of the present invention. ,6. 7 and analytical element for comparison (3
).

(2) Example of measurement of nitrite ion using analytical element-1
In the same manner as (2), 10.40 and 80 μg/m
Reflection density was measured using an aqueous sodium nitrite solution with a concentration of I2. The results are shown in Table-2.

As is clear from Table 2, the analytical element of the present invention can detect nitrite ions with significantly higher sensitivity than the comparative analytical element.

Example 3 (1) Preparation of analytical element In the same manner as the analytical element 2 of the present invention in which sodium chloride was added to the developing layer prepared in Example 1, sodium chloride was added to the developing layer coating solution. The amount of 1.20g. 4.8
0g. 9.60 g of each was added to prepare analytical elements 8.9.10 of the present invention.

(2) Measurement of nitrite ions using an analytical element Sodium nitrite was added to saliva collected under natural conditions, and 10,4
0. Prepare a mixed saliva containing sodium nitrite with a concentration of 80 μg/mQ, and place 10 μg of it on the spreading layer of the analytical element.
I scored each Q.

After 1 minute of spotting, the reflection density (D') was measured using a 546 nm interference filter.

Table 3 As is clear from Table 3, the analytical element of the present invention allows
Nitrite ions can be measured with extremely high sensitivity.

〔Effect of the invention〕

The present invention provides a highly sensitive method for analyzing nitrite ions and a structure of an analytical element.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a clinical chemistry analyzer according to the present invention. FIG. 2 is an exploded explanatory view of one embodiment of the analytical element of the present invention, and FIG. 3 is a sectional view of the analytical element for simple measurement used for body fluid testing. In Fig. 2; 21...Mount base, 22...Mount force bar 23...Detector (film), In Fig. 3; 1...Detection member, 2... Body fluid collection member, 3... Connecting member, 4... Mount, 1l... Support, l2... Reaction layer, l3... Deployment layer.

Claims (2)

[Claims] (1) In a nitrite ion analysis system based on a nitrite ion coloring reagent containing a compound that develops diazo color with nitrite ions and an acid that does not liberate halogen ions, an electrolyte halide is present in the analysis system. A nitrite ion analysis method characterized by: (2) An electrolyte halogen is contained in the analytical element constituting a nitrite ion analysis system based on a nitrite ion coloring reagent containing a compound that develops diazo color with nitrite ions and an acid that does not release halogen ions. A nitrite ion analysis element characterized by: