JPH0255954A - Multilayered analysis element - Google Patents

Abstract

PURPOSE:To measure the specific component in a biological sample with good accuracy by greatly improving the adhesiveness between a reagent layer and porous development layer of the analysis element. CONSTITUTION:At least one kind of the copolymers selected from the group consisting of the copolymers expressed by the formula [I] [where R1 denotes a lower alkyl group; x denotes 20-95mol%; y denotes 5-80mol%] and the formula [II] [where R2 is a hydrogen atom or methyl group; R3 is a phenyl group, cyano group or the group expressed by the formula -COOR4 (where R4 denotes a lower alkyl group); x1 denotes 50-95mol%, y1 denotes 5-50% number] is incorporated into the layers between the porous development layer and the reagent layer and the porous development layer in the multilayered analysis element which has the reagent layer on a base and the porous development layer thereon and is used to analyze the specific component in a fluid sample, by which the adhesiveness is greatly improved. The multilayered analysis element greatly improved in the measurement accuracy is obtd. in this way.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an analytical element for analyzing a specific component in a fluid sample, and more particularly to a multilayer element for analyzing a specific component in a biological sample. Regarding analytical elements.

[Conventional technology]

Various configurations of multilayer dry analytical elements for analyzing specific components in biological fluid samples are known. Among them, an intermediate layer made of a polymer such as poly(N-vinylpyrrolidone) or poly(N-inglobil acrylamide) is provided between the reagent layer and the porous development layer, and the reagent layer and the porous development layer are Methods for improving the adhesiveness with
No. 52-131786, No. 53-24893,
It is described in Examples of Japanese Patent Application No. 54-26795, No. 60-256068, Japanese Patent Application No. 197569/1980, etc.

[Problem to be solved by the invention]

Coating the intermediate layer made of a polymer as described above certainly has a certain effect on improving the adhesion between the reagent layer and the porous spreading layer, but still, in order to create a multilayer analytical element (slide), When cutting into small pieces, peeling was observed between the reagent layer and the porous spreading layer, and the adhesion was still far from satisfactory, and further improvement is desired.

An object of the present invention is to provide a multilayer analytical element in which the adhesion between the reagent layer and the porous spreading layer is significantly improved, and the measurement volume (simultaneous reproducibility) is also significantly improved accordingly. be.

[Means to solve the problem]

To summarize the present invention, the present invention relates to a multilayer analytical element for analyzing a specific component in a fluid sample, which has a reagent layer on a support and a porous spreading layer above the reagent layer. In, the following general formula! and lI: [in the formula, R1 is a lower alkyl group, X is 20 to 95 mol%
, 7 represents a number of 5 to 80 mol%" [wherein R1 is a hydrogen atom or a methyl group, R, rj phenyl group, cyan group, or -00014, -M5<wherein R4 is lower alkyl group), xI rl, 50-95 moA/
%,y,d! At least one copolymer selected from the group consisting of copolymers represented by: It is characterized by containing between no.

Hereinafter, the present invention will be fully explained in detail.

Specific components in a fluid sample that can be measured in the present invention include, for example, glucose ((flu), total cholesterol (T-choL-moglobin (Hb), albumin (Alt+), total protein (TP), and uric acid (Um)). , total bilirubin (T-Eil), creatinine (OR),
Glutamate oxaloacetate transaminase (GO
T), glutamine! 1! The components include pyrubimbo transaminase (GPT), lactate dehydrogenase (LDi), γ-glutamyl transpeptidase (y-GTP), alkaline phosphatase (ALP), and amylase (MY).

The reagent layer according to the present invention contains reagents that directly or indirectly interact with specific components in the fluid sample, and these reagents include pigment-forming precursors, enzymes, coenzymes, buffers, and necessary components. Substrates and the like are included depending on the situation.

As a reagent contained in the reagent layer according to the present invention, for example, the 4h group for measuring C11u is 4-aminoantiviral 7
．． 1.7-hydroxynaphthalene, glucose oxidase (GOD), peroxidase (POD), phosphate buffer, etc., with T-Ch.

In the case of 1, nb, add sodium azide, sodium nitrite, Tris buffer, etc. to Alb. In case, query/
For TPO, use steel sulfate, potassium sodium tartrate, sodium hydroxide, etc. For Um, use cablauricase, POD, borate buffer, etc. For T-Bil, use diazonium salt. , polymeric acid substances, etc., and in the case of OR, creatinine amide hydrolase, creatine amidinohydrolase, sarcosine oxidase, Kabra-POD, Gud buffer, etc.
In the case of GO'l', glutamate dehydrogenase (GID
H) Diaphorase, a tetrazolium compound, Tris buffer, etc. are used for GPT O, GIDH, diaphorase, a tetrazolium compound, Tris buffer, etc. are used for LDH, and a tetrazolium compound, Tris buffer, etc. are used for γ-GTPO. Silglycine,
Magnesium chloride, Tris buffer, etc., in the case of ALP, tetrazolium compound, Tris buffer, etc.
In the case of OPK, diaphorase, tetrazolium compound, gut buffer, etc., in the case of AMY, p-nitrophenylmaltohebutaoside, α-glucosidase,
Examples include good buffer. Various other additives, such as preservatives and surfactants, can also be added to the reagent layer as desired.

The specific components in the fluid sample that can be measured in the present invention are not limited to those mentioned above, and various components other than those mentioned above may also be used. The reagents are not limited to those described above, and when using different types of components or different analytical reactions even for the same components, reagents other than those similar to those described above can be used.

The above-mentioned reagent layer can be provided as a layer by coating a water-bathable polymer or a hydrophilic and organic solvent-soluble polymer as an inder onto the support. Water-soluble polymers (inders include gelatin, gelatin fJj conductors such as phthalated gelatin, water-soluble cellulose d conductors such as M droxyethyl cellulose and carboxymethyl cellulose sodium salt, polyvinyl alcohol, poly(
N-vinylpyrrolidone), polyacrylamide, polymethacrylamide, coelectrolyte of acrylamide and acrylic acid ester, poly(mono- or dialkyl-substituted) acrylamide, poly(mono- or dialkyl-substituted) methacrylamide, and water-soluble copolymers thereof, etc. Preferably, gelatin, polyacrylamide, and a copolymer of acrylamide and acrylic ester are used. Examples of the hydrophilic and organic solvent-soluble polymer binder include poly(N-vinylpyrrolidone), poly(N-vinylimidazole), poly(N-vinylimidazole) and their derivatives or coelectrolytes thereof, ethyl cellulose, Examples include cellulose d-conductors such as methylcellulose. These polymers (inders are mainly alcohols,
For example, it is a hydrophilic polymeric substance that dissolves in ethanol, gropanol, butanol, etc.

The above polymer binder can be arbitrarily selected depending on the specific component selected and its analytical reaction. Furthermore, if the analytical reaction chosen is composed of two or more reagents, it is also possible to contain two or more reagents mixed together in the same reagent layer. may be contained as separate reagent layers. These components may be determined depending on the working structure of the analytical reaction itself, but their configuration is arbitrary as long as they do not have undesirable effects.

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

The porous spreading layer according to the present invention has the function of (1) uniformly distributing a fixed volume of fluid sample to the reagent layer per unit area; In addition, it further performs the performance described in Japanese Patent Publication No. 55-21677, that is, (2) the function of removing substances or factors that inhibit analytical reactions in fluid samples and/or performing RIT33 spectrophotometric analysis. It is preferable that it has the function of performing a pack-ground effect of reflecting the measurement light that sometimes passes through the support. Therefore, the porous spreading layer according to the present invention may be either a layer having only the dA function described in (1) above, or a layer having the functions of (1) and/or (3) in addition to (1). or (
It is possible to appropriately separate a plurality of functions including 1) and use a separate layer for each function. Furthermore, (11, (2
) and (3), a layer having two functions;
It is also possible to use a combination of layers having one of the remaining functions. For example, the spread layer of a non-fibrous porous medium called a plush polymer consisting of titanium dioxide and cellulose diacetate, which is described in the above-mentioned Japanese Patent Publication No. 55-21677; Developed layer of hydrophilized fabric, JP-A-57-9465
No. 8, No. 57-125847, No. 57-197466
JP-A No. 513-9016, a spreading layer formed by applying a fiber dispersion as described in the specifications of No. 58-70161, etc.
Examples include the particle combination structure development layer described in No. 7 specification. In particular, the spreading layer and particle aggregate structure spreading layer formed by coating the fiber dispersion are particularly useful as materials that can quickly transport blood cell portions. The thickness of the developing layer in the multilayer analytical element of the present invention should be determined by its porosity, but is preferably about 100 to 600 μm.
1 More preferably about 150 to 400 μm. Further, the porosity is preferably about 20 to 85%.

Depending on the specific component selected and its analytical reaction, the porous spreading layer can contain a reagent that directly or indirectly interacts with the specific component in the fluid sample, as in the case of the reagent layer described above. .

For example, when measuring T-Oho, cholesterol esterase (coI!i), cholesterol oxidase (COD), etc. are used, for Alb, bromcresol green, etc., for Um, hydrogen donor, etc. T-
In the case of Bil, dyphyllin, etc. are administered to QOT (in the case of D, - sodium aspartate, α-ketoglutarate, nicotinamide adenine dinucleotide (!aAD
”) etc. (in the case of eSGPT, L-alanine, α-
Ketoglutaric acid, 1IAD+, etc., LDH (D, lithium lactate, MAD+ diaphorase, etc.,
For LP, use 5-bromo-4-indolyl phosphate, magnesium sulfate, etc. For OPK, use creatine phosphate, ADP, glucose-6-phosphate, dehydrogenase, hexokinase, glucose, 1jAD+, etc. , r-G'
In the case of rP, examples include r-glutamyl-p-nitroanilide.

These are in the - column. If the type of specific component or the same specific component as above but a different analytical reaction is used,
Reagents other than those mentioned above may also be contained.

Furthermore, various other additives such as preservatives and surfactants may be added as desired.

In particular, surfactants can be effectively used to adjust the permeation rate when a fluid sample is applied to the element of the present invention.

As usable surfactants, both ionic (anionic or cationic) and nonionic surfactants can be used, but nonionic surfactants are effective. Examples of nonionic surfactants include alkyl surfactants such as 2,5-di-t-buterphenoxybolyetelene glycol, p-octylphenoxybolyeterene glycol, and p-isononylphenoxypolyethylene glycol.
Examples include polyalkylene glycol derivatives of converted phenols and polyalkylene/glycol esters of higher fatty acids. These surfactants have the effect of regulating the rate of penetration of the fluid sample into the reagent layer and at the same time suppressing the occurrence of undesirable "chromatographic phenomena."

The above-mentioned surfactant can be used in a widely selected amount, but may range from 25IILk% to 0% by weight based on the weight of the coating solution.
．． 005 cod 0, preferably 15% by weight to α05% by weight can be used.

As the copolymer according to the present invention, a copolymer represented by the general formula (1) and/or d(II) can be used.

Typical specific examples of the copolymer according to the present invention are shown below, but the present invention is not limited thereto.

Internal copolymer (1111-vinylpyrrolidone-vinyl acetate copolymer (
Molar ratio 95:5) (2) N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 80:20) (3) N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 70:50) (4) N -Vinylpyrrolidone-vinyl acetate copolymer (molar ratio 60:40) (51kl-vinylpyrrolidone-vinyl acetate copolymer (
Molar ratio 50:50') (6) N-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 40:60) (N-vinylpyrrolidone/vinyl acetate copolymer (molar ratio 50:0) Pyrrolidone-vinyl acetate copolymer (molar ratio 20:80) (9) N-vinylpyrrolidone-vinyl propionate copolymer (mole ratio 60:40J (till la-vinylpyrrolidone-vinyl acetate copolymer (molar ratio 70) :50) un n-vinylpyrrolidone-methyl acrylate copolymer (molar ratio 95:5) 03) l-vinylpyrrolidone-methyl acrylate copolymer (molar ratio 90:10) (13N-vinylpyrrolidone-acrylic acid Methyl copolymer (molar ratio 75:25) αXun N-vinylpyrrolidone-ethyl acrylate copolymer (molar ratio 85:15) tlj M-vinylpyrrolidone-acrylic acid-n -
Butyl copolymer (molar ratio 90:10') (lb) N
-Vinylpyrrolidone-methyl methacrylate copolymer (molar ratio 90:10) (17) N-vinylpyrrolidone-ethyl methacrylate copolymer (molar ratio 80:20) (18) N-vinylpyrrolidone-methacrylic acid-n
-Butyl copolymer (molar ratio 95:5) (11N-vinylpyrrolidone-acrylonitrile copolymer (molar ratio 85:15)) (4) N-vinylpyrrolid/-styrene copolymer (molar ratio 90:10) ?ρ N-vinylpyrrolidone-styrene copolymer (molar ratio 75:25) N-vinylpyrrolidone-styrene copolymer (molar ratio 50:50) The copolymer according to the present invention can be produced by a known polymerization method. can be easily obtained using

In addition, when the copolymer according to the present invention is used in the intermediate layer, in the general formula 1, rl and x are preferably 50 to 95 moles or more, and y is preferably 5 to 50 moles.

In the general formula (2), xl is preferably 70 to 95 moles, and yl is preferably 5 to 30 moles. Also, if it is contained in the developing layer, the general formula! In formula (2), X is preferably 20 to 50 mol, and y is 50 to 80 mol.
, is 50 to 70 mol%%7. is preferably 30 to 50 moles.

The layer made of the above copolymer can be arbitrarily selected as desired, but preferably [11 to 50 μm]
More preferably α5 to 30 μm. Further, the layer made of the above-mentioned copolymer may optionally contain the above-mentioned reagents.

The above-mentioned copolymer can be coated by dissolving it in alcohols such as ethanol, pronophenol and butanol, ketones such as acetone and methyl ethyl ketone, and organic solvents such as tetrahydrofuran, toluene, xylene, chloroform and dichloroethane.

The support related to the analytical element of the present invention (hereinafter abbreviated as the support related to the present invention) may be of any type as long as it is liquid-impermeable and light-transmissive, but for example, cellulose acetate, polyethylene terephthalate, Various polymeric materials are suitable for this purpose, such as polycarbonate or polystyrene. Furthermore, in addition to the above polymer materials, inorganic materials such as glass can be used as well. The thickness of the support according to the present invention is arbitrary, but preferably 50 to 25
It is 0 μm. Further, one side surface of the observation side of the support according to the present invention can be arbitrarily processed depending on the purpose.

Furthermore, it is possible to improve the adhesion between the reagent and the support by using a light-transmitting undercoat layer on the side of the support on which the reagent layer is laminated, depending on the case.

The analytical element of the present invention may be used as required, for example, in US Pat.
Reflective layers as described in US Pat. No. 992.158, subbing layers, radiation blocking layers as described in US Pat. No. 4,042,535, barrier layers as described in US Pat. No. 4,06 & 405, US Pat. , 166.093, JP-A-55-9085
Nucavenger skin described in No. 9 and U.S. Patent No. 4
．． The destructible bond-like members described in No. 11α079 can be arbitrarily combined to form an arbitrary structure that meets the purpose of the present invention.

The various layers of these analytical elements are sequentially laminated on the support according to the present invention using a slide hopper coating method, an extrusion coating method, a dip coating method, etc., which are conventionally known in the photographic industry, and are appropriately selected according to the desired configuration. By doing so, a layer of arbitrary thickness can be applied.

Using the analytical element of the present invention, t of a specific component in a fluid sample can be measured.
t1 It can be measured from the support side according to the present invention by reflection spectrophotometry according to a modified initial velocity method or a reaction end point method. The amount of the specific component can be determined by applying the measured value thus obtained to a calibration curve prepared in advance.

The amount of fluid sample applied to the analytical element of the present invention can be determined arbitrarily, but is preferably about 5 μt to about 50 μt.
It is preferably 5 μt to 20 μL.

Usually 10 μt of fluid test #1 rt- is preferably applied.

The analytical element of the present invention can be used for the analysis of whole blood, serum, and plasma without any disadvantage. Furthermore, other body fluids such as urine, lymph, spinal fluid, etc. may be used without any disadvantage. In cases where whole blood is used, a radiation blocking layer as described above or other reflective layer may be provided, if necessary, to prevent radiation for detection from being interfered with by blood cells.

The analytical reaction used in the analytical element of the present invention can be arbitrarily determined depending on the purpose, but it is useful, for example, in the field of clinical chemistry, and is particularly useful for analyzing components of biological fluid samples, such as blood or urine. used for.

In addition, in the analytical element of the present invention, the copolymers used for the spreading layer and the intermediate layer may be the same or different, but it is preferable that the copolymers be of the same type because they can make the adhesiveness stronger.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Implementation ff1J-1 (Multilayer analytical element for HDL-cholesterol) A reagent layer having the following composition was provided on a transparent subbed polyethylene terephthalate support having a film thickness of 180 μm.

Reagent layer (R-1-1) Gelatiny IAOf7wI”7-
Chloro-3-(2-hexyldecylsulfonylethyl)-6-methyl-virazolo[42-c-8 monotriazole peroxidase ascorbic acid oxidase triingrovir naphthalene sodium sulfonate? , 2-vinylsulfonylethane) Sodium dibutyl azide phthalate M-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid intermediate layer (Z) 1.89t/m"1500007m" 9500 [7/m"&56 f 7m"α201/m"t029/m"α161/m"4.0097m" Spreading layer (8) FJfE raw material fiber styrene-glycidyl methacrylate cojt@body (overlapping ratio 9:1) Polyoxyethylene lauryl ether Cholesterol oxitasecholene terol esterase hydrochloride 4-kl, M'-diethylamino-2(2'-methanesulfonamide ether)aniline 01897m"
Bovine serum albumin 50,697m”91.0
W/m" 22.8 f/m" 15Of/m2 18000/m" 180007 m" Developing layer table-1 In addition to the above composition, a developing layer containing the following exemplified copolymer was prepared, and a spreading layer was placed on the reagent layer. The intermediate layer shown in the upper table and the development layer shown in the lower table were sequentially provided to the analytical elements-1 to 1 of the present invention shown in Table-1.
6 and comparative analysis elements-1 and 2 were prepared.

The above analytical elements-1 to 6 of the present invention and comparative analytical element-1
10 μL of human serum with a HDL-cholesterol concentration was applied to each column of HDL and 2, and after incubation at 57°C for 7 minutes, the reflection density was measured using a 546 nm filter. A calibration curve was prepared in advance from the EDL-cholesterol concentration values of human serum assayed with -Cholesterol-N (manufactured by Chugai Pharmaceutical Co., Ltd.).

Furthermore, human serum with normal value (50q/dt) and abnormal value (25xq/dt) was spotted 10 times each.
The same operation as above was performed to measure the reflection density, and the HD and L-cholesterol concentration values were calculated by applying it to a calibration curve prepared in advance, and then the coefficient of variation CVS was determined. The results are shown in Table-2.

Shishi-2 As is clear from the results in Table-2, the analytical element of the present invention-
It can be seen that Samples Nos. 1 to 6 exhibit better simultaneous reproducibility than Comparative Analysis Elements-1 and 2.

In addition, in Comparative Analytical Elements-1 and 2, rt peeling was observed between the reagent layer and the developing layer when cutting into small pieces to prepare analytical elements (slides), but in Analytical Element-1 of the present invention, rt peeling was observed between the reagent layer and the developing layer. In samples 6 to 6, no peeling was observed and good adhesion was exhibited. In particular, the analytical element-5ri of the present invention exhibited good adhesion and good simultaneous reproducibility.

〔Effect of the invention〕

As explained above, the analytical element of the present invention has the remarkable effect that the adhesion between the reagent layer and the porous spreading layer is significantly improved, and the measurement accuracy (simultaneous reproducibility) is also significantly improved accordingly. It can be played.

Claims (1)

[Claims] 1. A multilayer analytical element for analyzing a specific component in a fluid sample having a reagent layer on a support and a porous spreading layer above the reagent layer, which has the following general formulas I and II: ▲Math. There are chemical formulas, tables, etc. ▼... [I] [In the formula, R_1 is a lower alkyl group, x is a number from 20 to 95 mol%, and y is a number from 5 to 80 mol%] ▲ Numerical formulas, chemical formulas, tables, etc. ▼... [II] [In the formula, R_2 is a hydrogen atom or a methyl group, R_3 is a phenyl group, a cyano group, or a group represented by the formula -COOR_4 (in the formula, R_4 represents a lower alkyl group), x_1 is a 50
~95 mol%, y_1 represents a number of 5 to 50 mol%] At least one copolymer selected from the group consisting of copolymers represented by: A multilayer analytical element characterized in that the content is contained in a layer between the porous spreading layer.