JPS62296897A - Integrated type of analysis element with multilayer structure - Google Patents

Abstract

PURPOSE:A liquid-impermeable and light-transmissible support is laminated with a reagent layer containing oxidase and a color-reaction composition for detecting hydrogen peroxide and with a porous development layer to provide a layer containing a catalase inhibitor, freeing the measurement from the influence of hemolysis. CONSTITUTION:A liquid-impermeable, light-transmissible support is laminated with at least one of reagent layer and porous development layer. The reagent layer contains oxidase and a color reaction composition for detecting hydrogen peroxide which is formed by participation of oxidase. In addition, a catalase inhibitor such as azide, fluorine compound, aminotriazole is included, substantially in an immobilized form, in the layer closer to the development layer than the reagent composition layer and/or in the development layer.

Description

[Detailed Description of the Invention] 3. Detailed Description of the Invention [Field of Application in the Fish Industry] The present invention uses a liquid as a sample, and the first analyte (analysis target component)? This invention relates to a film-like multilayer analysis element with a fixed lid.

[Conventional technology]

Is the sheet-like or film-like analytical element part of the fractional distillation reagent or necessary h-product? By incorporating it in a dry form into an element and depositing liquid U#+ thereon, it is possible to easily and quickly analyze all the analytes in a sample.

Among these analytical elements, the materials for the single-layer reagent-supporting layer include IP paper and membrane filters.
What to use (Special Publication No. 36-4198, U.S. Percent Liver No. 56
No. 07093 ¥? ).

As a primary, non-integral multilayer analysis element that assumes simple lamination or peeling, a glass filter or membrane filter may be used (Japanese Patent Application Laid-Open No. 11395/1983), or two filters may be used.
', shaded over i (% 1977-15109
No. 6, U.S. Pat. No. 3,526,480), etc. are known.

These single-layer or simply laminated multi-layer analysis elements are used for limb fish, but it is difficult to say that they have achieved sufficient reliability for quantitative use.

Arranging multiple layers on an adult-impermeable, light-transparent support;
A multilayer analytical element in which each layer is laminated in a state where each layer is in contact with each other without any gaps, as indicated by the foliage of fluid contact, is disclosed in Japanese Patent Publication No. 53-21677, Japanese Patent Application Laid-open No. 55-164559,
No. 55-90859, No. 57-197466, No. 5
7-1 [No. 11761, No. 57-ICM760] Publications C are disclosed. These include one side of a transparent support, a porous development and a multilayered functional layer.
'iIt is integrated in a state that it cannot be peeled off. The outermost layer of the porous spreading layer, that is, the top layer, is the layer to which the liquid sample is applied. . In other words, does the unit surface spread out the entire liquid sample that has been supplied point by point, so that the sample is distributed in just one direction? This layer is also called the metering layer.
The CQL surface 81 to 9 is supplied with a sample of approximately one foot -bi uniformly distributed in the functional layer located at the top of the surface.

The functional layer is typically a reagent layer;
There are barrier layers, traps, water absorption layers, pretreatment layers, migration prevention layers, and many other wires, which are responsible for the test samples and machine stops necessary for analysis. There are also cases where the development layer has a single function and cases where it has multiple functions. In some cases, it is a developing layer that is contained in the entire analysis wipe and serves as a reagent layer, a reaction layer, and a purple layer.

As materials for forming each layer'(f''), the spreading layer includes one layer of Planche polymer described in Japanese Patent Publication No. 53-21677 bα, woven fabrics and knitted fabrics described in Japanese Patent Application Publication No. 55-164559, and Japanese Patent Application Publication No. 57-1989 - 4 super-fiber development layers made from disjointed microfibers and reactive bacterial molecular materials described in Publication No. 197466, and JP-A No. 55-90859, 1b-J 57-1017
Particular structures such as those shown in Publications No. 60 and No. 57-101761 are common.

Further, hydrophilic polymer materials such as Iso Y stop layer nigeratin, polyacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, acarose, cellulose conductors, etc. can be mentioned.

In the multilayer analytical element described in iJ, oxidase is used to generate all hydrogen peroxide, and the action of this hydrogen peroxide is used to create a composition (coloring reagent) that can generate all of the detectable colored substances. ) may be used for quantitative determination. These include, for example, Trinder-containing reagent compositions used for glucose detection and the like. For example, Ann, Chin, Bioch
em.

6, pp. 24-27 (1969), US Pat. No. 5,992,158 E! Reference is made to the descriptions of A Gad Cao and Japanese Patent Publication No. 56-45599.

It has been reported that analysis using this reagent composition is affected by the bath surface. It is believed that these are mainly caused by the consumption of hydrogen perbide by catalase in red blood cells in whole blood and heme jt7 protein. As a method to completely eliminate such effects, US Patent No. 555506
No. 9, No. 3349006, No. 3862885, etc. are based on a reagent containing catalase inhibitor gold and oxidase to remove the influence of substances that have catalase activity when the liquid sample and oxidase react. Furthermore, it is disclosed that a color-forming reagent 1r which can be inspected is reacted with a lightning reagent.

Furthermore, ′p! Japanese Unexamined Patent Publication No. 59-109192 discloses that a catalase inhibitor is contained in a layer close to the developing layer of a multilayer analytical element. According to the publication, this means that the developing layer material is completely impregnated with a catalase inhibitor bath solution.
It is disclosed that it can be incorporated into the spreading layer by drying or by spraying. These can eliminate the influence of bath blood like the US patent and 1 mJ.

[Problems that the invention attempts to resolve]

However, catalase inhibitors are also known to inhibit peroxidase, which is the catalyst for the conversion of hydrogen peroxide into a color-forming substance that can be detected by a color-forming reagent, resulting in a total decrease in the concentration of color-forming dyes, As a result, sensitivity is significantly reduced.

This results in improved analytical precision, ie, reproducibility.

The object of the present invention is to provide a multilayer analytical element that eliminates the influence of the bath surface in a multilayer analytical element that contains all of the reaction m acid reagent systems that can be detected by the action of hydrogen peroxide produced by oxidase. It is about providing.

Another object of the present invention is to provide an integrated multilayer analytical element containing a catalase inhibitor that avoids a decrease in reflection density, a decrease in sensitivity, and a deterioration in simultaneous reproducibility.

[Means for solving problems]

SUMMARY OF THE INVENTION 1 The present invention relates to an integrated multilayer device comprising at least one reagent layer and a porous layer on a liquid-impermeable and light-transparent support. An integrated multilayer analysis in which layers are laminated, and a part of the reagent layer contains oxidase and a hydrogen peroxide detection coloring reagent composition for detecting hydrogen peroxide generated under the involvement of oxidase. In the element, the catalase activity inhibitor is contained in a substantially immobilized form in a layer closer to the spreading layer than the layer containing the hydrogen peroxide detection coloring reagent composition and/or As a result of all the experiments, the present inventor discovered that the catalase inhibitor is contained in the spreading layer.
The present invention has been achieved by discovering that the above-mentioned drawbacks can be overcome by including the porous spreading layer and/or a layer close to the spreading layer in a substantially immobilized form.

The catalase activity inhibitor used in the multilayer analytical element of the present invention may be any known inhibitor of catalase activity, such as cyanide compounds, azide compounds, fluorine compounds, formic acid and its salts, acetic acid and its salts, aminotriazole, etc. Examples include ascorbic acid, divalent copper ion, semicarbazide, pyrazole, diethyldithiocarbamate, nitrite, N-ethylmaleimide, and the like. In particular, azide compounds, fluorine compounds, aminotriazoles, acetates, and the like are preferred because of their strong inhibitory effects.

The multilayer analytical element of the present invention comprises a reagent layer containing a reactive composition that produces a detectable substance under the action of hydrogen peroxide, and a porous support on a water-impermeable and light-transparent support. It has a spreading layer as an essential constituent layer, but if necessary, a functional layer having other functions (for example, a light reflecting layer, a filtration layer, a barrier layer, a migration prevention layer, etc.) or a structural auxiliary layer (for example, a contact layer) may be added. etc.) The catalase activity inhibitor may be present in a layer closer to the surface than the reagent layer,
Alternatively, it can be contained in two or more of these layers, if necessary.

Furthermore, it is also effective to newly provide a layer containing a catalase activity inhibitor. Particularly preferably, the porous spreading layer or the layer adjacent thereto (
(for example, an adhesive layer), it is preferably incorporated into a porous spreading layer. It is not preferable to include the lfc inhibitor in a layer containing peroxidase because it inhibits the action of peroxidase and causes a decrease in sensitivity.

In the present invention, the catalase activity inhibitor must be contained in a desired layer in a substantially immobilized form. In the present invention, "substantially immobilized" means time 1 at the end of the reaction.
This means that there is enough of it in the specified layer. This can be confirmed from the difference in reflection quality when all of the same liquid samples are deposited on an analytical element that has the entire layer containing the catalase inhibitor and an analytical element that does not contain the catalase inhibitor.

As a method for substantially immobilizing the catalase activity inhibitor contained in the porous spreading layer, there are various methods depending on the material of the porous spreading layer.

For example, in a fiber expanding horn made of a mixture of loose fibers and a reactive polymeric material disclosed in Japanese Patent Application Laid-Open No. 57-197466, the fibers used are i! i After soaking and impregnating in a catalase activity inhibitor bath solution of a suitable concentration and drying to create a catalase activity inhibitor-impregnated fiber,
It can be formed by preparing a coating solution consisting of a mixture of the above-mentioned impregnated fibers and a reactive polymeric material according to a conventional method, and applying and drying the coating solution.

In addition, in the case of a non-fibrous porous media layer, that is, a single layer of plush polymer, as disclosed in Japanese Patent Publication No. 53-21677, a catalase activity inhibitor is dispersed in a uniform binder during the manufacturing process. After adding and dispersing a white pigment (as required), a poor bath is added to cause so-called phase separation, and the coating can be applied as a slurry.

In the case of using the fabric disclosed in JP-A No. 55-164359, for example, after impregnating and drying the catalase activity inhibitor, the above-mentioned catalase activity inhibitor? Il-
A polymer dissolved in a non-dissolving solvent is impregnated, dried, and then used to generate 7'CH2O, which is produced under the involvement of O oxidase, and has a peroxidase-like activity in order to convert it into a detectable color-forming substance. Alternatively, a substance exhibiting peroxide activity is required.

Generally, peroxidase is used, but the present invention also uses synthetic peroxidase, hemin, methemoglobin, oxyhemoglobin, hemoglobin, hemochromogen, alkaline hematin, hemin derivatives, iron sulfocyanate, iron tannate, iron ferrocyanide, chromate, etc. useful for implementation. Color-forming compositions that exhibit a detectable change in the presence of hydrogen peroxide and peroxides may optionally employ a suitable m-acid. Typical examples that can be used include those containing the following substances along with a coloring agent if necessary.

Monoamines such as aniline and its derivatives, diamines such as 0-phenylenediamine, benzidine, etc.
Phenols, such as phenol, thymol, cresol, naphtholpebolyphenols, such as catechol, guaiacol, virocalol, etc., aromatic carboxylic acids,
For example, salicylic acid, gallic acid, etc., leuco dye, flJ
t Halocomalachite Green, U.S. Patent No. 4089
Triarylimidazoles and triarylmetas 7 described in Specification No. 747 [B, E, Babb (B, g, Babb), S, Dani x
198 in the name of A/ (D, S, Daniel) et al.
U.S. Patent Application No. 61250, filed May 21, 2013
9], colored dyes, such as 2,6-cyclophenol indophenol, various biological substances, such as epinephrine, flavonic acid, tyrosine, etc., and others, clinical Those known to those skilled in the art of chemistry.

Particularly useful color-forming compositions are the leuco dyes mentioned above and the triarylimidazole compounds described in particular in the US Pat.

Other preferred color-forming compounds include all color-forming color formers that can react with oxidizable complexion compounds to produce a color change. Useful color-forming color formers include U.S. Pat. No. 4,251,629.
No. 4260679 and No. 4396714, Publication No. 58-22200, European Patent Application No. 68556, British Patent No. 210786
Substituted anilines (i.e., toluidines) including those described in the specification of No. 3 and Japanese Patent Publication No. 58-898. Representative oxidizable color developer compounds include benzidine and its congeners, p-phenylene diamine, p-amine phenol, aminoantipyrine, such as 4-aminoantipyrine, and the like. A preferred color developing compound is 4-aminoantipyrine.

Oxidase enzymes that can be incorporated into the present invention include glucose oxidase, uricase, cholesterol oxidase, sarcosine oxidase, pyruvate oxidase, glycerin oxidase, glycerophosphate oxidase, choline oxidase, lactate oxidase, and the like.

These oxidase enzymes are selected according to the analyte and used in combination with other enzymes. Historically, when used in combination with a buffer at a preferred pH range and concentration,
Of course this is desirable.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these examples.

Example 1 A reagent layer having the following composition was coated on a transparent undercoated polyethylene terephthalate support having a thickness of 180 μm and dried.

Sera f 7 14.0 f
7m”1,7-hydroxynaphthalene
Q, 835f/m"4-aminoantipyrine hydrochloride
1.273t/m2 bellogysidase
61500 U/m” Glucose Oxidase 119000 U/m” Dimedone
0.185 f/,,2
Potassium phosphate buffer (pH 6,1~&2) 2
．． 8241! /m'Alkanol XC (product name)
(Dry film thickness 16μm consisting of 121897m)
Reagent layer A coating solution prepared as below was applied onto the above reagent layer to form a developing layer.

Triton X-100 (part name) in xylene 3002
9.75 r'i bath, styrene-glycidyl methacrylate copolymer (polymerization ratio 9:1) was dissolved at 24.5
After thoroughly mixing and stirring, add P paper powder (more than 300 mesh) for 975ft and stir further.

Furthermore, a desired amount of an aqueous sodium azide solution is added to the above dispersion. If necessary, a known dispersion method such as ultrasonic dispersion was used to perform all dispersion to obtain a coating solution.

The above coating liquid was applied and dried on the reagent layer, and the development N1
All created.

Sawagami powder (more than 300 meshes) 101.7
? / m2 Toridor X-1001 [Ll 797
m2 Sodium azide Quantity as shown in Table 1 of Table 1. Development term for a dry film thickness of 300 am and a dry film thickness of 300 am. It was dried and used as comparative analysis elements (2) to (5).

The analytical elements (1) to (5) of the present invention and the comparative analytical elements +11 to (51 have a glucose concentration of 50.100°50
0.500 q/az) Serum 'ff: 1oμ trough n=1
After incubation at 37° C. for 7 minutes, the total reflection density was measured using a 546 nm filter.

Furthermore, catalase (Sigma) kO, 1ooo, 2500.5000.
7500. Separated to 10000S, U/tnl, 10μ was spotted in the same way, and after fc incubation at 37°C for 7 minutes, reflection gold was measured using a 546mm filter.

All results are shown in Table 2 and No. 6.

Table 2 Glucose concentration (my/az) so　　　iooo　　5oo　　so.

Table 6 Catalase quality (S, U/m/) 0 1000 2500 5000 75 [1010
,0DDt31 [LaB5 CL88! l α
884 CL8B2 Q, 884 0.883 (4
) α8[10,889α885 G, 883 α8
84 α884 (5) α886 α879 α8
82 α883 Q, 882 [L878(3)[
L824 α81D Q13j3 l1809
α800 CL782 (41rl, 803 α7
98 (L789 Q, 7i 11774 0.
776 (51Q, 798 α794 α783 0.
771 α764 Q, 766 From the above results, it is possible to eliminate the influence of catalase while maintaining good simultaneous reproducibility with the analytical element of the present invention, whereas with the comparative analytical element It can be seen that there is a major problem with either or both of the effects of catalase.

Example-2 A reagent layer having the following composition was formed on a transparent polyethylene terephthalate support with a thickness of 18,077 m that had been subbed. ! “Apply and dry.

Gelatin 15.09 t
/,,2 peroxidase 1
220007m”4-aminoantipyrine hydrochloride
182597m"1,y-dihydroxynaphthalene α612j'/m"Dimedy
(113797m" Potassium phosphate buffer (pH6,7~&9) 3.227 97m
“Alkanol XC (product name) 250
Wit/m"1.2-bis(vinylsulfonyl)ethane 100 Iv/FM" dry film thickness 16 μm
reagent layer.

A coating solution prepared as described below was applied onto the reagent layer to form a developing layer.

1xoor2 of Triton X-100 in xylene 3002
Then, 24.5 f of styrene-glycidyl methacrylate copolymer (polymerization ratio 9:1) was dissolved. To this solution, add sodium azide of fcIi as shown in Table 4 below, and add f paper powder (500 mesh or more) to the solution in which sodium azide is finely dispersed using a known method. Or do everything. If necessary, the material may be further dispersed using a known method such as an ultrasonic dispersion machine. During stirring, cholesterol oxidase, cholesterol esterase, ox serum albumin 2.42
(bovine serum albumin 1), cholesterol oxidase, cholesterol esterase both 1000
Add ItJ containing) and stir? went.

flI flour bed (600 mesh or more) 101
．． 717m” Triton X-10013,56ri/rn2
Cholesterol oxyp'-se 300007m"Cholesterol esterase 300007m"
Bovine blood 7 and albumin 50
〇? 7m2 Sodium azide
A spread layer with a dry film thickness of 300 μm consisting of the material listed in Table 4.

4th Clothing Furthermore, as in Example-1, the comparative analytical element (6) was sprayed with sodium azide to a spreading layer of 1.28017 m'', and this was used as the comparative analytical element (7). Next.

The analytical element of the present invention (61, (7L comparative analytical element (6)
), (7) has a total cholesterol layer of 180, '500.
Used on serum of 450 wq/az, applied 10μti,
After incubation at 57°C for 7 minutes, 546n
Reflection density using m filter! It was determined to be 141J.

The measurement results of simultaneous reproducibility of reflection density are all shown in Table 5. Furthermore, red blood cells were separated from human whole blood, hemolyzed by ultrasonication, and the red blood cell membrane was completely removed to obtain a hemoglobin bath solution, and the amount of hemoglobin was 0.50.100.200.300.50.
The addition was made so that the concentration was 0 cape/dt. However, the total cholesterol amount at this time was set to be 180 μ/dt. The results are shown in Table 6.

Table 5 (n=10 each) No. 6 Split layer r index of the blowing invention 6) α748 α739 α74
7 α748 Q, 751 α756(7) C
L756 (1754α756 [1749Q,
76D Q, 762 ratio analysis system +61 Q, 765
0.73 α517 [L488α4760.462
(7n α733 α692 Q, 6B7
Q, 703 0.710 11709 or above Results (The analytical element of the present invention is azide) No decrease in reflexivity was observed due to cooling of IJ, and the simultaneous reproducibility was good.

Furthermore, with regard to hemolysis, it is clear that the analytical element of the present invention exhibits sufficiently stable performance and is not affected by hemolysis. On the other hand, the comparative analytical element (6) without sodium azide shows sufficiently stable simultaneous reproducibility for each cholesterol once, but for hemolysis, the reflection concentration decreases as the amount of hemolysis increases. It will be done. The comparative analysis element (6), which is not immobilized due to primary sodium azide, not only shows a significant decrease in reflectance and deterioration in simultaneous reproducibility, but also eliminates the effects of hemolysis. It's hard to say.

Example 3 Reagent fIII having the following composition was applied onto a transparent polyethylene terephthalate support having a thickness of 180 Am and which had been prime coated.

Gelatin (hardened) 10f/m2
Uri 77-ze 250
U/m” peroxidase 7
000U/m"pHast't phosphate buffer
7.5 y7. Film thickness FJ1 consisting of z
2 μm reagent layer.

Further, on the above-mentioned trial layer, a developing layer was prepared using sodium azide polyethylene resin prepared as described below.

Powdered paper (50%
0 mesh or more) and stir well? It was lightly filtered, the powdered F paper was separated, and only the water content was completely removed using a freezing VL dryer to prepare powdered Kido paper impregnated with sodium azide. Containing sodium azide 11i100f
&J powder fQ per paper, 120f.

TRITON

Further, as comparative analytical elements (8) and (9), the former had sodium azide removed from the developing layer, and the latter had sodium azide contained in the reagent layer at α12617 m2.

For the analytical element (8) of the present invention and comparative analytical elements (8) and (9), human serum with known uric acid concentration t-10μ
was added and incubated at 37°C for 7 minutes, then 6
Reflection density was measured at 30 nm. The results are shown in Table 7 below.

Table 7 The upper row is the average value, the middle row is the standard deviation, and the lower row is C and V.

Historically, catalase total OS, U/d, 1000 S, U/d, 2500
S, U/7+7! , 5 0 0 0
S, U/ad, 1 0 0 lo
Serum added to 03-U/m/? 6111 was determined in the same manner as before. The results are shown in Table 8 below.

Looking at the results in Table 8 and above, it can be seen that the analytical element of the present invention is better than the comparative analytical element.

〔Effect of the invention〕

As explained above, according to the analytical element of the present invention, mm
This has the remarkable effect of eliminating the effects of the above, and also completely avoiding a decrease in sensitivity and, ultimately, a deterioration in simultaneous reproducibility.

Claims (1)

[Claims] 1. At least one reagent layer and a porous development layer are laminated on a liquid-impermeable and light-transparent support,
and a part of the reagent layer contains an oxidase and a hydrogen peroxide detection coloring reagent composition for detecting hydrogen peroxide generated under the involvement of the oxidase, wherein a catalase activity inhibitor is used. is contained in a substantially immobilized form and in a layer closer to the developing layer and/or in the developing layer than the layer containing the hydrogen peroxide detection coloring reagent composition. An integrated multilayer analysis element characterized by: