JPS62265995A - Production of dry-type liquid analysis element - Google Patents

Abstract

PURPOSE:To obtain a dry-type liquid analysis element for the quantitative determination of alkaline phosphatase, having high storage stability and analytical accuracy, by precipitating a specific salt of p-nitrophenyl phosphate from its solution by the addition of a poor solvent and coating or impregnating the precipitate. CONSTITUTION:An amine salt or an amino alcohol salt of p-nitrophenyl phosphate (preferably 2-amino-2-methylpropane-1,3-diol salt or 2-amino-2- ethylpropane-1,3-diol salt) is dissolved in a good solvent (preferably an alcohol) and the obtained solution is added with a poor solvent (e.g. acetone) to separate the solution into a liquid phase and a solid phase. The solid phase is dissolved or dispersed in a liquid and coated to or impregnated in a liquid-permeable sheet (woven, knit or nonwoven cloth) to obtain the objective dry-type liquid analysis element.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a dry analytical element useful for quantifying the activity of alkaline phosphatase in biological fluids such as serum.

Salts of paranitrophenyl phosphate (p-NPP) are known as substrates for alkaline phosphatase. It was already well known around 1965 that disodium salts have poor stability in dry conditions.

Furthermore, it has been shown that certain amine salts, such as tris(hydroxymethyl)aminomethane salt and dicyclohexylammonium salt, are relatively stable reagents.
It is known as No. 872.

In addition to the above, amines listed as sample examples in the same publication include methylamine, ethylamine, dipropylamine,
1 to butylamine, dichlorohexylamine, dicyclohexylamine, tricyclohexylamine, monoethanolamine, benzylamine, dimethylamine, tritylamine, furfurylamine, aniline, jetanolamine, and morpholine. It has been shown that these amine salts can be easily dissolved in water.

However, these water-soluble salts are
When applied as an aqueous solution to the dry multilayer liquid analysis element described in No. 1983 and JP-A-55-164356, the stability is poor, which is also pointed out in JP-A-60-237999. It is.

It has been desired to realize an analytical element for determining alkaline phosphatase with good storage stability, and for this purpose, in 1982-237999, the substrate was not dissolved in the solution but made into granules and dispersed in the coating solution. We are proposing a method to do so. However, with this method of mechanically dispersing the substrate in a dry state or in a poor solvent, it is difficult to obtain results with good reproducibility, that is, it is difficult to obtain high analytical precision.

[Object of the Invention] An object of the present invention is to provide a dry analytical element for quantifying alkaline phosphatase that has good storage stability and high analytical accuracy, and a method for producing it.

[Summary of the Invention] The above object is to produce a dry analytical element for quantifying alkaline phosphatase having at least an 8I body permeable layer such as a liquid spreading layer.
+)-NPP) amine or amino alcohol salt,
A solution of a good solvent (e.g. water, methanol) and a poor solvent (e.g. water, methanol).
For example, by precipitating a liquid phase and a solid phase, and dissolving or dispersing the separated solid phase, a liquid permeable layer such as a liquid spreading layer is coated or immersed. This was achieved by a method for producing a dry analytical element for quantifying alkaline phosphatase, which is characterized in that it is used as

The dry analytical element manufactured by this method has extremely good storage stability and high analytical accuracy.

As the amine or amino alcohol salt of p-NPP, for example, tris(hydroxymethyl)aminomethane described in the above-mentioned Japanese Patent Publication No. 45-34872
(2-amino-2-hydroxymethyl-1,3-propanediol), dicyclohexylamine, methylamine, ethylamine, dipropylamine, tributylamine, cyclohexylamine, synchlohexylamine, tricyclohexylamine, monoethanolamine, benzylamine , dimethylamine, tritylamine, furfurylamine, aniline, jetanolamine, morpholine and the like. These are water soluble. Salts soluble in alcohol, especially methanol, include 2-amino-2-methylpropane-1,3'-diol salt and 2-amino-2-methylpropane-1,3'-diol salt.
-amino-2-ethylpropane-1,3-diol salt.

Either of these salts is dissolved in an appropriately concentrated solution in a good solvent.

Since the above salts have few good solvents, a wide range of poor solvents can be used. For example, acetone, methyl ethyl lactone, cyclohexane, benzene',!・Ruen et al. Add the solvent to the salt solution in an amount sufficient to precipitate the salt.

Well-known methods can be used to separate the precipitated solid phase from the liquid phase. That is, any method such as gravity sedimentation, centrifugal sedimentation, inertial collision, or filtration can be used, but gravity sedimentation, centrifugal sedimentation, or filtration is convenient. Pressure filtration or vacuum filtration can also be used for filtration.

It is better to disperse the precipitated salt in a poor solvent than to redissolve it in a good solvent.Dispersing the precipitated salt does not require a special dispersion machine (such as a colloid mill), and can be easily stirred using a stirring rod. It is enough.

The liquid spreading layer is preferably a spreading layer made of a woven, knitted or cellulosic non-woven fabric, but is also applicable to dry analytical elements with spreading layers made of plush polymers or particulate structures.

The present invention can be applied to various known dry analysis elements. In particular, it can be applied to an element containing a solid carrier through which both an autochromic substrate and a test liquid can pass. The elements are formed on a light-transparent/liquid-impermeable support, in addition to a porous liquid spreading layer, an undercoat layer, a water absorption layer, an adhesive layer, a reactive reagent layer, a light guide layer, a filtration layer, and other known components. Such analysis elements, which may be multi-layered luck, are disclosed in U.S. Patent No. 3,992,158, U.S. Pat. There is something.

When a support is used, the practical configurations of the dry analytical element produced according to the present invention are (1) one having a liquid spreading layer on the support.

(2) A support having a reagent layer and a liquid spreading layer in this order. In this case, the NPP substrate may be contained in either the reagent layer or the Wt body I71 layer, but it is preferably contained in the liquid spreading layer. , the reagent layer may consist of two or more reagent layers.

(3) A support having a reagent layer, a light guide layer, and a liquid developing layer in this order, in which case the NPP substrate is reagent XNl,
Although it may be included in either the light guide layer or the liquid spreading layer, it is preferably included in the liquid layer 17FIJCil.

(4) A support having a detection layer, a light guide layer, and a liquid developing layer in this order, the NPP substrate may be included in either the light guide layer or the liquid developing layer, but it is included in the liquid developing layer. is preferable.

(5) A support having a detection layer, a light guide layer, a reagent layer, and a liquid developing layer in this order, the NPP base material may be contained in any of the reagent layer, light guide layer, and liquid developing layer. , is preferably included in the liquid spreading layer.

(6) A support having a detection layer, a second reagent layer, a light guide layer, a first reagent layer, and a liquid developing layer in this order. Although the NPP substrate may be contained in any of the reagent layers, the light guide layer, and the liquid spreading layer, it is preferably contained in the liquid spreading layer.

In (1) to 6) above, between the detection layer and the reagent layer or the liquid development layer, between the reagent layer and the liquid development layer, between the light guide layer and the detection layer, the reagent layer or the liquid development layer, or between the two A filtration layer may be provided between the reagent layers. A water absorption layer may be provided between the support and the detection layer, the reagent layer or the liquid development layer; between the support and the detection layer, the reagent layer or the liquid development layer; between the reagent layer and the liquid development layer; A pH)l'fFr layer or a dura layer, etc. may be provided between the light guide layer and the detection layer, the reagent layer or the liquid spreading layer, or between two reagent layers. In the present invention, the above ( 1)(2) or (
3) layer structure.

In the dry analytical element manufactured according to the present invention, the NPP substrate contains two or more N (for example, a reagent layer and a liquid developing layer, or a second reagent layer and a first reagent layer, or a first reagent layer and a liquid developing layer).
In this case, it is also possible for each one of the substrates to be distributed more heavily either in a layer farther from the support or in a layer closer to the support.

Examples of light-transparent/water-impermeable supports that can be used in the dry analytical element produced in the present invention include polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose esters (e.g., cellulose triacetate, cellulose acedate propionate, etc.) with a thickness of about 50 μl to about 11
'M, preferably in the range of about 80 .mu.m to about 300 .mu.l, may be mentioned as a transparent support in the form of a film or sheet. If necessary, an undercoat layer may be provided on the surface of the support to strengthen the adhesion between the support and the water absorption layer and reagent layer provided on the support.

Further, instead of the undercoat layer, the surface of the support may be subjected to physical (eg, glow discharge, corona discharge) or chemical activation treatment to improve the adhesive strength.

In the integrated multilayer analytical element produced in the present invention, a water absorption layer may be provided on the light-transparent/water-impermeable support (in some cases, via another layer such as an undercoat layer). The water-absorbing layer provided in the dry analytical element of the present invention is preferably a layer made of a hydrophilic binder and containing as a layer-forming component a hydrophilic polymer that swells by absorbing water.

Hydrophilic polymers that can be used in the water absorbing layer are generally natural or synthetic hydrophilic polymers having a swelling ratio upon absorption of water ranging from about 1.5 to 20, preferably from about 2.5 to 15 at 30°C. . Examples of such hydrophilic polymers include gelatin (e.g., alkali-treated gelatin, acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (e.g., phthalated gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol,
Examples include polyvinylpyrrolidone.

The dry thickness of the water-absorbing layer is preferably in the range of about 1 μm to about 100 μβ, more preferably in the range of about 3 μ5 to about 30 μl. Furthermore, the water absorption layer can contain a surfactant (cationic, amphoteric, or nonionic) and an y1 buffering agent, if necessary.

The water absorption layer of the dry analytical element manufactured by the present invention contains light! !
Masking (reflective or absorbing) particulates can be included if desired.

Water absorption layer, reagent layer, depending on the location, light path layer, FAN,
An adhesive layer for adhering and laminating the spreading layer may be provided on the layer such as when the adhesive layer is wet with water or contains water! Examples of hydrophilic polymers that can be used in the adhesive layer, preferably consisting of a hydrophilic polymer that is capable of adhering the rx opening layer when moistened, include hydrophilic polymers similar to those used in the water absorption layer. Examples include polymers. Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred, and the dry smoke film thickness of the adhesive layer is generally in the range of about 0.5 .mu.l to about 20 .mu.m, preferably about 1 .mu.s to about 10 .mu.l. In addition, the adhesive layer improves the adhesion force between the eyebrows other than MN.
The adhesive layer, which may be provided on a desired layer, can be provided by applying an aqueous solution containing a hydrophilic polymer and a surfactant added as necessary onto the water-absorbing layer, etc. using a known method. .

Examples of buffering agents that can be contained in the reagent layer of the dry analytical element manufactured according to the present invention include carbonates, borates,
Mention may be made of known tll'I agents such as phosphate and Good's buffer.

These buffers can be selected and used with reference to known literature such as "Basic Experimental Methods for r-Proteins and Enzymes" (Takeshi Horio et al., Nankodo, 1981).

The light shielding layer is preferably water-permeable waste in which light-reflecting fine particles are dispersed using a hydrophilic polymer having film-forming ability as a binder. When measuring changes in color (color change, color development, etc.) from the light-transmitting support side, the color of the aqueous liquid dotted onto the developing layer, especially when the sample is whole blood, is measured by the color of the hemoglobin. It blocks red and other colors and also functions as a light reflective layer or background layer.

Examples of light-reflecting fine particles include pigment fine particles, such as titanium dioxide fine particles (rutile type, anatase type, or brutzite type) with a particle size of about 0.1 μl to about 1.0 μl.
(2 μm microcrystalline particles, etc.), barium sulfate, microparticles, and aluminum microparticles T1. Among these, titanium dioxide fine particles and barium sulfate fine particles are preferred. Particularly preferred are rutile titanium dioxide fine particles.

Examples of hydrophilic polymer binders with film-forming ability include weakly hydrophilic regenerated cellulose, cellulose acetate, etc. in addition to the hydrophilic polymers used in the production of the detection layer described above. Among these, gelatin , gelatin derivatives, polyacrylamide, etc. are preferred.
In addition, a known hardening agent may be added to gelatin or gelatin derivatives.

light! ! The shielding layer can be provided by applying an aqueous dispersion of light-reflecting fine particles and a hydrophilic polymer onto the detection layer, reagent layer, etc. by a known method and drying it.

In the present invention, the above-mentioned light-reflecting fine particles may also be included in the developing layer if necessary.

It is preferable that the spreading layer is a spreading layer having a liquid meter 1 action. Having a liquid meter 2 action means that the part containing the 11-body sample dotted onto its surface is substantially It has the effect of spreading in the structural (horizontal) direction at a rate of a substantially constant amount per unit area without causing uneven distribution.

The materials that make up the matrix of Exhibition #IN include paper, non-woven fabrics, laminated fabrics (e.g., plain weaves such as broadcloth and boblin), knitted fabrics (e.g., tricot knitting, double tricot knitting, Milanese knitting, etc.), and glass. A three-dimensional lattice structure made of fibrous paper, a membrane filter 1 made of plush polymer, polymer microbeads, etc. can be used. Among these, it is preferable to use fibrous layers typified by woven fabrics and knitted fabrics. For details of these, see Japanese Patent Application Laid-Open No. 55-1643.
5G No. 57-66359 and No. 60-2227
Please refer to No. 69.

The m fabric or knitted fabric that can be used for the dry analytical element produced in the present invention is processed by degreasing such as washing with water to remove threads.
It is preferable that oils and fats attached during the production of the woven or knitted fabric be substantially removed.

The integrated multilayer analytical element produced in accordance with the present invention is approximately -15
i+a+ is cut into small pieces such as squares of about 30ax or circular pieces of approximately the same size, and the pieces are cut into small pieces such as squares of about 30ax or circles of approximately the same size, and are cut into small pieces such as JP-A-57-634523. ? ? 3.58-5
It is preferable from all viewpoints of manufacturing, packaging, transportation, storage, measurement operations, etc. to use it as an analysis slide by storing it in a slide frame or the like as disclosed in each publication No. 01144.

The dry analytical element manufactured according to the present invention can be prepared from approximately 5 μl to approximately 3 μl.
0μ! An aqueous liquid sample, preferably from about 8 .mu.l to about 15 .mu.r, is spotted onto the spreading layer and optionally incubated at a substantially constant temperature ranging from about 20.degree. C. to about 45.degree. Thereafter, detectable changes such as color changes and color development within the dry analytical element are measured by reflectance photometry from one side (from the light-transmitting support side in the case of integrated multilayer analytical elements), and the colorimetric method is used to detect detectable changes in the liquid sample. Analyze the component to be measured.

[Example 1] Recrystallization of fil p-nitrophenylphosphate bis(tris) salt p-nitrophenylphosphate bis[) IJs(
hydroxymethyl)amine] salt 13. Completely dissolve in distilled water with rtf≠Odl. Add 300 ml of acetone to this solution.
115 (Add while stirring well. After about 1 minute, suction the precipitate and wash with ethanol several times. Wash with acetone and suction dry.

(2) Production of analytical elements (Part 1) Transparent polyethylene terephthalate support (thickness: /10
A coating solution having the following composition was applied onto the treated surface and dried to form a buffer layer with a dry thickness of is μm.

Coating liquid for buffer layer formation: Alkali-treated deionized gelatin -4tg water
YuhiroOg nonylphenoxy polyglycitol /, Gg poly(N-[
[Dimethylaminopropylcoacrylamide 11% aqueous solution (average molecular weight approximately 10,000 io) JOgN-(2-
hydroxyethyl) ethylenediamine-N, N'.

N'-triacetic acid 2 mmol Zn (CH3
COO12-2H20/mmol MgSO2・7H20
Add a NaOH aqueous solution of N to a solution of λ mmol or more to adjust the pH to /1.
．． Adjust to 0 and then.

On top of the above buffer layer, apply the following coating solution to a dry film thickness of 3.6 μm.
The coating was applied to a thickness of m and dried to form a dura layer.

Coating liquid for forming dura layer: Gelatin 12g water
26? g Nonylphenoxy polyglycitol l 3 g 1 l Corbis(hynylsulfonylacetamide) ethane 0.7 λg Water/acetone (mixing ratio =/:/) /Ig Apply the following coating solution on the above dura layer to a dry film thickness. It was coated so that the thickness was 3 μm to form an adhesive layer.

gelatin 12g water
, 2rJ, 7g nonylphenoxyφ polyglycitol l・3g On the above adhesive layer, apply an aqueous solution of O9≠tinonylphenoxy polyglycitol, and then apply a tricot knitted fabric made of 36 gauge jOD polyethylene terephthalate spun yarn. It was crimped to form a spread layer.

(3) Fabrication of analytical element (its λ) Using the recrystallized substrate of +11 for the development layer,
A coating solution f/204/rrL having the following composition was coated and dried.

p-nitrophenylphosphate bis(tris) salt prepared with self-developing substrate-containing coating solution (1) /J, 1g polyvinylpyrrolidone (average molecular weight 100,000) 100g ethanol ≠00g acetone
For the method of 100 g or more (Xt), an integrated multilayer analytical element for measuring ALP activity according to the present invention was produced (hereinafter abbreviated as alkaline phosphatase 1ALP).

[Comparative Example 1] In Example 1, except that the recrystallization method of p-nitrophenylphosphate bis(tris) salt, which is the substrate in the coating solution containing the self-developing substrate, was changed to the pulverization method shown below. ,
Analytical elements for ALP measurement according to the present invention were prepared in the same manner as in Example 1.

p-nitrophenylphosphate bis() IJs)
Salt/J, Itf first crushed in a mortar, then crushed nf200
Sift through a mesh sieve.

This material is used as a substrate.

[Comparative Example 2] In Example 1, the acetone root 1 in the coating solution containing the self-developing substrate was replaced with water joot, and without further recrystallization, the substrate was directly dissolved in the water !00? An analytical element for measuring ALP activity according to the present invention was prepared in the same manner as in Example 1, except that the coating liquid was prepared as a fake solution.

[Measurement Example 1] In order to compare and examine the storage stability of the analytical element for measuring ALP activity produced in Example 1 and Comparison Example 1.2, we tested the analytical element for ALP activity measurement immediately after production. After storage at 0C for 3, 5 and 7 days, the reflected optical density was measured when all liquid samples not containing NLP were deposited. The results are shown in Table 1.

The values of each △OD shown in Table 1 are calculated for each temperature (≠6CS
Ko! 0C1≠j0c) for each number of days (3, !, 7 days) after spotting the liquid sample on the analytical element and incubating it at 37°C for 6 minutes] and [the analytical element immediately after fabrication at 37°C] (increase in reflected optical density).

As is clear from the above results, the shelf life of the analytical element for ALP activity measurement of Example 1 manufactured using the method of the present invention was as follows.
It turned out to be very good.

[Example 2] In Example 1, p-nitrophenylphosphate bis(tris) salt kp-nitrophenylphosphate bis(copmin-2-ethyl/13- An analytical element for ALP measurement according to the present invention was prepared in the same manner as in Example 1, except that the prono-ξ diol) salt was used and the following recrystallization was performed.

Recrystallization of p-nitrophenylphosphate bis(2-aminocoethyl/,!-propanediol) salt in 200111 methanol Completely dissolve. Add 200ml of acetone to this solution while stirring well. After about 10 minutes, all the precipitate was filtered through the suction port and washed several times with ethanol. After washing with acetone, it was suction-dried on paper. .

[Measurement Example 2] A similar storage stability test was conducted for the analytical layer for measuring ALP activity of Example 2, and as shown in Table 2 below, good storage stability was found.

Table λ

Claims (5)

[Claims] (1) Prepare an amine salt or amino alcohol salt of paranitrophenyl phosphate as a solution in a good solvent, mix it with a poor solvent to precipitate it, separate the liquid phase and solid phase, and dissolve or disperse the separated solid phase, A method for producing a dry analytical element for quantifying alkaline phosphatase having a liquid permeable layer, which is used as a coating or dipping composition for the liquid permeable layer. (2) Make a solution of the amine salt or amino alcohol salt of paranitrophenyl phosphate in a good solvent, mix it with a poor solvent to precipitate it, separate the liquid phase and solid phase, disperse the separated solid phase, and develop it into a liquid. The manufacturing method according to claim 1, characterized in that it is used as a layer coating or dipping composition. (3) Make an alcohol-soluble salt of paranitrophenyl phosphate into an alcohol solution, mix it with a poor solvent, precipitate it, separate the liquid phase and solid phase, and dissolve or disperse the separated solid phase to achieve liquid permeability. The manufacturing method according to claim 1, characterized in that it is used as a layer coating or dipping composition. (4) 2-amino- of paranitrophenyl phosphate
2-Methylpropane-1,3-diol salt or 2-amino-2-ethylpropane-1,3-diol salt is made into a solution of alcohol, mixed with a poor solvent to precipitate, and the liquid phase and solid phase are separated. and dissolve or disperse the separated solid phase,
The manufacturing method according to claim (1), characterized in that it is used as a coating or dipping composition for a liquid-permeable layer. (5) Make an alcohol-soluble salt of paranitrophenyl phosphate into an alcohol solution, mix it with a poor solvent, precipitate it, separate the liquid phase and solid phase, disperse the separated solid phase, and apply a spreading layer or The manufacturing method according to claim (1), characterized in that it is used as a dipping composition.