JPS6252461A - Multilayered analytical element - Google Patents

Abstract

PURPOSE:To obtain the titled analytical element wherein a hydrophilic polymer layer does not change with the elapse of time or heat and by a swelling characteristic and the penetration of a component to be analyzed are uniform and the reproducibility of an analytical result is excellent, by bringing the hydrophilic polymer layer of a multilayered analytical element to a phase separable structure. CONSTITUTION:The kind of the support used in a multilayered analytical element is not asked if said support is impermeable to a liquid and pervious to light but, for example, various polymer materials such as cellulose acetate, polyethylene terephthalate, polycarbonate, polystyrene or the like are suitable. A hydrophilic polymer layer having a phase separable structure is one utilizing such a phenomenon that phase separation is generated if two or more kinds of polymers mutually different in compatibility are mixed in the good solvents of the polymers and it is necessary to select a pref. combination corresponding to a purpose. The embodiments of combinations are shown in a table.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a multilayer analytical element for detecting specific components in a fluid sample.

BACKGROUND OF THE INVENTION In recent years, a number of methods have been developed for analyzing specific components in biological fluid samples. Particularly in the field of clinical chemistry, various analytical vessels have been developed and have been introduced into clinical test seedlings in many hospitals. Among these, the multilayer analysis element disclosed in the specification of Japanese Patent Publication No. 53-21677@ has attracted attention because of its ease of operation and high leg circumference.

The basic structure of this type of multilayer analytical element includes a liquid-impermeable and light-transparent support, a hydrophilic polymer layer, and a porous spreading layer for spreading the sample.

The hydrophilic polymer layer of the multilayer analytical element is usually used as a reagent layer containing various reagents depending on the component to be analyzed.
Various polymers are selectively used as the hydrophilic polymer depending on the component to be analyzed.

An important property of the hydrophilic polymer layer is that it maintains constant permeability to biological fluid samples.

However, in conventionally used hydrophilic polymer layers, the physical properties governing permeability tend to deteriorate due to heat and aging, resulting in a decrease in water absorption and degree of swelling. ``This inhibits the prompt absorption of the target component to be analyzed in the biological fluid sample and the release of reaction reagents, which poses a major problem in the simultaneous reproducibility of quantitative analysis results as an analytical element.

In particular, alkaline phosphatase, γ-glutamyl transpebutidase, leucine aminopeptidase, etc., which react in minute amounts, are strongly affected by this.

Therefore, even if the reaction amount of the target component to be analyzed is small, such as alkaline phosphatase, leucine bebutidase, γ-glutamyl transpebutidase, etc., the hydrophilic polymer layer will not release the reaction reagents or cause biological It has been desired to develop a multilayer analytical element that can rapidly absorb the components to be analyzed in a fluid sample within a certain period of time, and has stable permeability and swelling properties even with heat and aging.

[Objective of the Invention] The first object of the present invention is to provide good analytical results even when the reaction amount of the target component is minute, such as alkaline phosphatase, leucine bebutidase, γ-glutamyl transbebutidase, etc. The object of the present invention is to provide a multilayer analytical element.

The second object of the present invention is that the hydrophilic polymer layer does not change due to heat or aging, has stable swelling properties, and has permeability of components to be analyzed in the layer and rapid release of reaction reagents.
A multilayer analytical element having At properties is provided.

[Structure of the Invention] The above-mentioned object of the present invention is to provide a multilayer analytical element having a hydrophilic polymer layer and a porous spreading layer on a liquid-impermeable and light-transmitting support, in which the hydrophilic polymer layer is This is achieved using a multilayer analytical element with a phase-separated structure.

[Specific Structure of the Invention] The support used in the multilayer analytical element of the present invention is a liquid-impermeable and light-transparent support (hereinafter referred to as the support of the present invention), and its type may be A variety of polymeric materials are suitable for this purpose, such as, but not limited to, cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene. 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 of the present invention on the observation side can be arbitrarily processed depending on the purpose.

When the hydrophilic polymer layer used in the present invention is provided on the above support, it can be coated directly, but in some cases, a light-transparent undercoat layer may be used to bond the hydrophilic polymer layer and the support. It is effective to increase the adhesion between the two.

Next, the hydrophilic polymer layer having a phase-separated structure used in the multilayer analytical element of the present invention will be explained.

The hydrophilic polymer layer having a phase-separated structure used in the present invention utilizes the phenomenon that phase separation occurs when two or more kinds of polymers having different compatibility with each other are mixed in a good solvent for the polymers. Since this phase separation varies depending on the type of polymer used, its mixing ratio, solvent, etc., it is necessary to select a preferable combination depending on the purpose of the present invention.

Examples of combinations of polymers that form a hydrophilic polymer layer having a phase-separated structure will be described below.

Below is a blank space. Each of the above polymers 1 and 2 may be a homopolymer or a copolymer.

When the above-mentioned polymers form a copolymer, the comonomers for the copolymer include, for example, vinyl acetates, acrylic esters, and methacrylic esters (however,
Preferred are hydrophobic comonomers such as lower alkyl groups whose alkyl group has 4 or less carbon atoms), styrenes, acrylonitriles, methacrylonitriles, vinyl chlorides, and nylidene chlorides.

Further, the proportion of the copolymer is preferably about 25% by weight or less, more preferably about 20% by weight or less of the comonomer.

The solvent to be used can be selected from solvents that have a sufficient solubility to dissolve each of the polymers 1 and 2 used in the present invention. Water is generally used as a solvent that satisfies this condition. Furthermore, water-miscible organic solvents, such as lower alcohols (methanol, ethanol, propatool, butanol, etc.), tetrahydrofuran, etc., may be used as a mixed solvent with water, unless the solubility of the polymer is significantly impaired. things are possible.

Further, these concentrations can vary depending on the coating method, desired film thickness, etc., but are usually 20 to 1% by weight of polymers (combined concentration, preferably 12 to 5% by weight). .

Adjusting solutions of two or more polymers to desired concentrations,
This should be added to the final concentration at the end of the night. Further, stirring may be carried out to the extent that the desired polymer solution is uniformly mixed.

If these are left to stand still for an extremely long time before application, they should be stirred continuously (for example, for more than one day), or
Alternatively, stirring may be performed for a certain period of time before application.

The hydrophilic polymer layer of the present invention can be applied by various coating methods used in the photographic industry, such as slide hopper method, dip coating method,
A desired film thickness can be easily obtained by curtain coating, doctor blade coating, or the like.

Further, the drying temperature is not particularly specified, but when the hydrophilic polymer layer of the present invention contains an enzyme, a complement, or a substrate, it is preferable to dry at 55° C. or lower.

The dry thickness of the hydrophilic polymer layer of the present invention is generally preferably from about 5 microns to about 50 microns, more preferably from about 10 microns to about 30 microns.

According to the present invention, the hydrophilic polymer layer of the present invention and the porous spreading layer are sequentially laminated on the support of the present invention to obtain a multilayer analytical element of the present invention.

As the porous spreading layer applicable to the multilayer analytical element of the present invention, one or more layers are provided directly or indirectly on the hydrophilic polymer layer provided on the support of the present invention.

This porous spreading layer may have the function of (1) uniformly distributing a certain volume of a biological fluid sample per unit area within the hydrophilic polymer layer; (2) has the function of removing substances or factors that inhibit analytical reactions in biological fluid samples; It may also have a function of performing a background effect of reflecting the light.

The porous spreading layer used in the present invention may have all of the above three functions in one, or it is also possible to separate the three functions as appropriate and use a separate layer for each function. .

Furthermore, it is also possible to use a combination of a layer having two of the three functions and a layer having the remaining functions.

The thickness of the porous spreading layer used in the present invention can be arbitrarily selected depending on the purpose, but is preferably about 30 microns to about 600 microns, more preferably about 50 microns to about 400 microns. be.

The porous spreading layer used in the present invention is
Plush polymer layer described in No. 77, JP-A-55-1
Hydrophilized fabric described in No. 64356, JP-A-57
Porous layer comprising a mixture of discrete fibers and a polymer containing a reactive group as described in No. 197466, JP-A-55
- Granular structure consisting of polymer beads and adhesive described in JP-A-90859, JP-A-57-101760 and JP-A-57-1017-
Examples include particle conjugates in which polymer beads having reactive groups as shown in No. 101761 are bonded directly or via a low-molecular compound.

In particular, the porous spreading layer made of discrete fibers and a high molecular weight polymer having a reactive group, described in JP-A-57-197466, is easy to manufacture and has excellent performance.

When the porous spread layer according to the present invention is a 11-fiber structure spread layer, examples of the material forming the spread layer include natural cellulose or its derivatives, and synthetic fibers such as polyethylene, polypropylene, and polyamide; Examples include those constructed by three-dimensional entanglement of random fibers of various types, whether natural or synthetic.

Furthermore, one or more materials of any size can be selected as the above materials, but JIS standard sieves have a size of 50 to 325 meshes, preferably 100 to 3.
20 meshes, more preferably 200 to 300 meshes.

In the multilayer analytical element of the present invention, the hydrophilic polymer layer of the present invention is usually used in such a manner that the hydrophilic polymer layer of the present invention contains reaction reagents that undergo a predetermined reaction with a specific component in a biological fluid sample. These reaction reagents are arbitrarily selected depending on the characteristic component to be measured.

When the component to be analyzed is alkaline phosphatase, these reaction reagents have a pH of about 9.5 to 1.
0.5 is contained in the hydrophilic polymer layer, and the substrate p-nitrophenyl phosphate is contained in the porous spreading layer or other separate layer. Ru.

In the case of leucine aminobebutitase, it is also a substrate, and it is preferable to contain leucil-p-nitroanilide and the four rules in different layers.

That is, the hydrophilic polymer layer of the present invention can contain the compound, and the substrate can be contained in the porous spreading layer or other layers. When the substrate is contained in a layer other than the spreading layer,
It is preferable to contain it in a further layer between the spreading layer and the hydrophilic polymer layer. Further, magnesium ions (usually magnesium chloride) as an activator may be contained in any layer, but should preferably be contained in the same layer as the substrate.

In the case of γ-glutamyl transbebutidase, it is possible to contain the substrate γ-glutamyl-p-nitroanilide, glycylglycine, and a buffer in the hydrophilic polymer layer. Preferably γ-.

It should contain glutamyl-p-nitroanilide and glycylglycine separately from the M four rules.

At this time, it is preferable to contain a buffering agent in the hydrophilic polymer layer and the other layers in the porous development layer or other layers, as in the case of the leucine aminobebutidase.

Further, magnesium ions (magnesium chloride) as an activator may similarly be contained in any layer, but should preferably be contained in the same layer as the substrate.

In order to incorporate the above-mentioned reaction reagents into the hydrophilic polymer layer having a phase separation structure of the present invention, after dissolving the two types of polymers causing the phase separation in the good solvent and before coating, By adding and dispersing it, it can be uniformly contained in the hydrophilic polymer of the present invention.

It will be appreciated that the reagents contained in the hydrophilic polymer layer of the present invention will depend on the component to be analyzed in the biological fluid sample and the analytical reaction selected to analyze this component. Additionally, if the selected analytical reaction is composed of two or more reagents, it is possible to contain these reagents mixed together in one hydrophilic polymer layer; It may be contained as the above hydrophilic polymer layer.

These may be determined by the mechanism of action of the analytical reaction itself, and their configuration is arbitrary as long as it does not have an undesirable effect.

On the other hand, it is possible to perform analytical reactions on two or more components to be analyzed in a sample within the same hydrophilic polymer.

In this case, it is necessary to select the analytical reactions so that two or more analytical reactions do not interfere with each other, and similarly, when measuring the generated reaction products, so that they do not affect each other.

Moreover, the above-mentioned reaction reagents can also be contained in the porous development layer as necessary.

In the multilayer analytical element of the present invention, acids, alkalis, salts, buffers, dissociating agents, surfactants, etc. can be appropriately contained in the porous spreading layer and the wood-philic polymer layer depending on the purpose of the present invention. .

The pH, amount, and type of the buffer used in the present invention should be selected as necessary. For example, generally phosphorus
Saw agent (Na2HPO4-Na0)(), carbonate buffer (NaHCO2-Na2C○3), borate buffer (Na8+07-Na0l-1), Tris buffer [Tris(hydroxymethyl) [aminomethane-hydrochloric acid], solid buffers (eg, CAPS-KOH, etc.), and the like.

Examples of the surfactant that can be used in the present invention include alkylphenoxypolyethoxyethanol [e.g. Torito X-100 (manufactured by A-M&Haas)], polyethylene oxide alkyl ester [e.g. Emulgen-120 (manufactured by Kao Atlas Co., Ltd.]) Co, alkylphenoxypolyoxyglycidol E, e.g. surfactant 1
0G (manufactured by Olean Corporation)] is cited as a typical example.

The multilayer analytical element of the present invention can have various functional layers and layer configurations as desired. For example, the reagent layer, reflective layer, and subbing layer described in U.S. Pat. No. 3,992,158, U.S. Pat. oa2.33s @ radiation blocking layer as described in U.S. Patent No. 4,066.403; registration layer as described in U.S. Pat. No. 4,144,306; Migration inhibition layer, U.S. Patent No. 4°127.4
Scintillation layer described in No. 99, JP-A-55-908
Scavenger layer described in '59 and U.S. Pat.
It is possible to construct a multilayer analytical element suitable for the purpose of the present invention by arbitrarily combining the destructive bond-like members described in No. 10,079.

The above-mentioned various layers can be coated in layers of arbitrary thickness by using the slide hopper coating method, extrusion coating method, dip coating method, etc. used in the photographic industry.

In addition, as for the layer structure, for example,
No. 4,110,079, Japanese Unexamined Patent Publication No. 1983-1
It is possible to arbitrarily select the layer structure described in No. 31565 and the like.

The multilayer analysis element manufactured in this way can be made into various shapes depending on the analysis method. It can take a variety of shapes including, for example, a stretchable tape of the desired width, a sheet, or a slide mounted on a plastic mount.

Examples of fluid samples applicable to the multilayer analysis element of the present invention include biological fluid samples such as serum (including plasma and serum), lymph fluid, and urine. Also, ■ of the fluid sample used
is arbitrary, but is preferably about 5μ2 to 50μ2, more preferably about 5μ2 to about 20μ2.

It is preferred to apply a typical 10 μ2 fluid sample.

Also, if necessary, 30℃~50'C1 preferably 35℃
After incubation at ~40°C for 1 to 20 minutes, preferably ~10 minutes, light in the range of 500 to 650 nm is applied from the liquid-impermeable and light-transparent support side as appropriate depending on the conditions. The reflected optical density can be measured through a filter that allows light of a specific wavelength to pass through, and the analyte concentration can be determined from a calibration curve. Furthermore, by creating color samples in advance and visually comparing them, it is also possible to perform measurements on a limb-like basis.

[Specific Effects of the Invention] As explained above, in the multilayer analytical element of the present invention,
Even when the target component to be analyzed has a small reaction port, such as alkaline phosphatase, leucine aminobebutitase, γ-glutamyl transbebutitase, etc., 1. It was possible to provide a multilayer analytical element in which the hydrophilic polymer layer does not change with time or heat, the swelling characteristics of the layer and the permeation of the component to be analyzed are uniform, and the reproducibility of analysis results is excellent.

[Specific Examples of the Invention] Hereinafter, the present invention will be specifically explained using Examples, but the embodiments of the present invention are not limited to these.

Example 1 Multilayer analytical element sample 1 was prepared by sequentially coating layers having the following composition on a transparent undercoated polyethylene terephthalate support having a thickness of 180 microns.

(Phase-separated hydrophilic polymer layer of the present invention) Polyacrylamide (10% aqueous solution) (Polymer 1) 336 g Poly-N-vinylpyrrolidone (10% aqueous solution) (Polymer 2) 84a Sodium carbonate 8.45 g Sodium hydrogen carbonate 3. 36g Triton ■X-100 (nonionic surfactant.

After adjusting to PL-110.5 with 3 g of 4% aqueous solution of sodium hydroxide (produced by Rohm and Haas), the amount was adjusted to 600 g with distilled water, and a doctor blade coater with a diameter of 250 microns was used. It was coated on the support using the above-mentioned material and dried.

(Porous development layer) p-nitrophenyl phosphate disodium salt 0.73g Magnesium sulfate 7820 3.20G Triton X-10060 Styrene-glycidyl methacrylate copolymer weight ratio 9:1) g, og xylene 120g was placed in a stainless steel pot and dispersed using a sand stirrer with glass beads added for 13 hours.

Thereafter, the glass beads were filtered and a dispersion liquid was collected.

Furthermore, the above dispersion 137.93CI1. : Add 38 g of il1 paper powder D (manufactured by Toyo Roshi ■) and stir and disperse.
Coating was performed on the hydrophilic polymer layer using a doctor blade coater having a gap of 500 microns, followed by drying.

Samples 2 to 7 were prepared in the same manner as Multilayer Analytical Element Sample 1 obtained above, except that the combination of Polymer 1 and Polymer 2 constituting the hydrophilic polymer layer was replaced with the combination shown in Table 1 below. did.

Below is a normal human serum as a margin sample and enzyme 〇a in this serum.
(Product name 1 manufactured by Nippon Pharmaceutical ■) and the alkaline phosphatase activity units are 5 King-Armstrong units (hereinafter abbreviated as KA-tJ), 15KA-U, 30K.
Three types of standard specimens A-U were prepared.

Furthermore, the above serum was applied to the porous spreading layer of each analytical element sample.
After dropping 2 μ2 and incubating at 37°C, the reflection density was measured 2 and 4 minutes after dropping with a reflection densitometer using a 405 nm filter, and the difference in the reflection density was calculated, and the alkaline phosphatase activity value of each serum was calculated. A calibration curve was created by −order regression.

Furthermore, each n-10 of this serum was subjected to the above procedure,
The measurement was performed and applied to the above-mentioned calibration curve, and the alkaline phosphatase activity value was calculated to determine simultaneous reproducibility. The results are shown below in Cloth-2.

However, in the calibration curve (y = ax + b) in Table 2,
x-KA-U and y-ΔD are respectively represented. Here Δ
D means the difference in reflection density at 405 nl between 2 minutes and 4 minutes after dropping the sample solution.

In addition, in the analysis results, the upper row or average value is shown, the middle row SD is the standard deviation, and the lower row CV is the simultaneous reproducibility expressed by the following formula.

As is clear from the results in Table 2 above, the multilayer analytical element samples (Nos. 1 to 4) using the hydrophilic polymer layer according to the present invention
) is a comparative multilayer analytical element sample (No.
, 5 to 7).

Example-2 Multilayer analytical element sample of the present invention used in Example-1 (N
o, 1 to 4), and comparative multilayer analytical element samples (N 0
．． Serum samples with alkaline phastase activity values of 5 KA-U and 30 KA-tJ were stored at 40°C and 55% relative humidity for 0, 2, 5, and 7 days. Evaluation was carried out using the same operations as in Example 1.

However, the calibration curves for each analytical element shown in Table 2 of Example-1 were used. The results are shown in Table-3.

However, in Table 3, ΔD in the upper row represents the difference in reflection density at 405 nm between 2 minutes and 4 minutes after dropping the sample, and % in the lower row represents the rate of change from KA-U on day 0. Also, F
og indicates the reflection density at 405nffi of an unused multilayer analytical element sample.

As is clear from the results in Table 3 above, the multilayer analytical element samples of the present invention (N 0.1 to 4) showed an extremely small increase in FogC fog, and storage at 40°C with a relative humidity of 55% for 10 days.
Also shows the activity value on day 0, which is almost the same value as ΔDF<. However, it is clear that the comparative multilayer analytical element samples (No. 5 to 7) have large fluctuations in ΔD and large fluctuations in FOA upon storage, and cannot be put to practical use.

Example 3 Layers having the following composition were sequentially coated onto a transparent subbed polyethylene terephthalate support having a thickness of 180 microns.

(Hydrophilic polymer layer) A layer having the composition shown in Table 4 below.

Below margin (Porous development layer) A layer having the composition shown in Table 5 below.

The following margins were prepared using normal human serum (γ-GTP activity 20 IU, To this, purified human γ-GTP enzyme was added, and 581
Human serum with activities of U/ffi and 801U# was prepared.

Using this serum, drop 10 μ2 of the above-mentioned serum onto each multilayer analytical element sample, and while incubating at 37°C, measure the reflection density at 405 nm for 2 minutes and 4 minutes after dropping the serum using a reflection densitometer. Find the above γ-G
Corresponding to the TP activity value, a test line was created by linear regression in the same manner as in the example. Further, each serum was similarly measured at n-10 for each analytical element, applied to the calibration curve of each analytical element, γ-GTP activity was calculated, and then simultaneous reproducibility was calculated. The results are shown in Table 6 below.

However, in Table 6, X of the test m-line (y = ax + b) represents the concentration of the specimen (IU/l, V = △Dk, and x
, 5o1cv have the same meaning as in Example 1.

As is clear from Table 6 above, the multilayer analytical element samples (No. 8 to 10) for measuring γ-G-P of the present invention have 20 to 80 I
All exhibit good simultaneous reproducibility at U/ffi. On the other hand, the comparison multilayer analytical element samples (NO, 10, 11) have larger variations than the multilayer analytical element samples of the present invention.

Example-4 Multilayer analytical element sample of the present invention prepared in Example-3 above (
No. 8 to 10), Comparative multilayer analysis element sample (No.
The following study was conducted using 10.11).

The multilayer analytical element sample was left at 40°C and 55% relative humidity for 5 and 10 days, and normal human serum (
Purified human γ-G'TP enzyme was added to γ-GTP activity 20 IU/II, 381 U/II! and γ of 761Uzl
-Add 10μ2 drops of serum adjusted to GTP activity37
2 minutes and 4 minutes after dropping while incubating at °C.
The reflection density at 405 nm was measured, and the difference was found and applied to the calibration curve shown in Example-3.
Activity was determined. The results are shown in Table-7.

As is clear from Cloth-7 above, the multilayer analytical element samples of the present invention (Nos. 8 to 10) show no change in their discrimination ability even under the conditions of 40°C and 55% relative humidity for 10 days. Also, no significant increase was observed in Fog.

On the other hand, the comparative multilayer analysis element sample (No. 11.12) was F
It was found that og increased significantly and the performance deteriorated significantly.

Claims (1)

[Claims] A multilayer analysis element having a hydrophilic polymer layer and a porous spreading layer on a liquid-impermeable and light-transparent support, wherein the hydrophilic polymer layer has a phase-separated structure. element.