JPS62175199A - Dry analytical element for measuring amylase activity - Google Patents

Abstract

PURPOSE:An analytical element, containing separately provided water-permeable layer containing glucosidase and water-penetrative layer containing a special self-developing oligosaccharide substrate and having little dissociation by the glucosidase and low background density. CONSTITUTION:A dry analytical element having at least two water-permeable layers on a light-transmitting support. A self-developing oligosaccharide substrate capable of dissociating trisaccharide, disaccharide or monosaccharide (as necessary, tetrasaccharide) linked to p-nitrophenol in the presence of amylase is contained in either one of the layers. A water-penetrative layer containing glucosidase is provided between the layer and the light-transmitting support.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a dry multilayer analytical element for measuring enzyme activity, particularly amylase activity.

[Background of the invention] The amylase analysis method using a self-developing oligosaccharide substrate is described in Nature, Vol. 182, 525-526.
Page (1,958) and JP-A-53-1.1092 Kasa are known. In particular, various oligos W (e.g. G 5 ,
For example, PNP-G5 and PNP-
G7 and the amylase analysis method using glucosidase enzyme use conventional pigmented starch substrates in which starch is dyed with reactive dyes, such as blue starch and amylopectin azure, to generate a precipitate and quantify using the difference in molecular weight distribution. do"
"Digital starch method" (e.g. H, U, Berbmeyert I
It is superior in terms of quantitative performance and accuracy compared to methods (Methods of Enzymatic Analysis, by U., Ier8+neyer, page 894), and is also suitable for continuous monitoring analysis of enzyme reactions (late assay in the narrow sense).
It has the advantage of being able to

This method uses dry multilayer analysis elements, such as JP-A-57-66
It can also be applied to the dry multilayer analysis element described in No. 359. However, if a self-developing substrate such as PNP-G5 or PNP-G7 and glucosidase enzyme are contained in the same layer, even if there is no amylase to be detected,
Dissociation of the self-developing substrate by the glucosidase enzyme occurs, and coloration due to PNP is observed, forming a background density.

[Object of the Invention] The object of the present invention is to provide a dry multilayer analysis for measuring amylase activity in which there is no background concentration due to PNP that is generated due to the dissociation of a self-developing substrate by the glucosidase enzyme even in the absence of amylase to be detected. It is in the provision of elements.

[Structure of the Invention] The method described in 1- above has at least two water-permeable layers on the north side of the light-transparent support, and at least one of the water-permeable layers has amylase in the presence of Parfait 1 - trisaccharide, trisaccharide or monosaccharide (sometimes tetrasaccharide) with lophenol attached
comprising a self-developing oligosaccharide substrate capable of dissociating , and containing curcosidase in a water-permeable layer provided between the water-permeable layer containing the self-developing oligosaccharide substrate and the support. By multi-layer dry liquid fE analysis element featuring
It was soiled.

In particular, the advantages of the present invention were preferably achieved by using the multilayer dry liquid (this analytical element, in which the layer containing the oligosaccharide substrate does not substantially contain curcosidase).

As glucosidase, α-talcositase or β
- Cucurcidase can be used.

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 the self-developing substrate and the test liquid permeate. Elements include support, reaction reagent layer, detection layer, reflection layer, porous liquid spreading layer, adhesive layer, filtration layer, water absorption layer, undercoat layer, +
') j: and other known layers. As such an analytical element, U.S. Patent No. 3.992
．． ], No. 58, No. 4,042,335-SJ] and Japanese Patent Application Laid-open No. 164356/1983.

When a support is used, the practical configuration of the dry analytical element of the present invention is (1) a detection layer on the support and a liquid development layer thereon. In this case, the detection layer contains curcosidase and the liquid development layer contains an oligosaccharide substrate.

(2) A support having a detection layer, a reagent layer, and a liquid development layer in this order. In this case, glucosidase,
The liquid development layer contains an oligosaccharide substrate, or the detection layer contains glucosidase and the reagent layer contains an oligosaccharide substrate.

(3) A device having a detection layer, a second reagent layer, a first reagent layer, and a liquid development layer in this order on a support. In this case, the second reagent layer contains curcosidase, and the first reagent layer contains the oligosaccharide substrate.

Preferred in the present invention is a two-layer structure (1). In 1) to 3) above, there is a light shielding layer and/or a filter between the detection layer and the reagent layer or the liquid developing layer, between the reagent layer and the liquid developing layer, or between the second reagent layer and the first reagent layer. A vortex layer may also be provided.

In the present invention, the self-developing substrate may be contained in two or more layers (for example, the first reagent layer and the liquid spreading layer 17i'1 layer). In this case, the self-developing substrate is far from the support. It is also possible to have a greater distribution either in the layer or in the layer closer to the support.

In the present invention, glucocytase may also be contained in two or more circular layers (for example, a detection layer and a reagent layer, or a second reagent layer and a first reagent layer), and in this case, the layer far from the payment unit or the support It can also be distributed more heavily in one of the layers closer to the body.

Light transmittance that can be used in the dry analytical element of the present invention
Examples of water-impermeable supports include polyethylene terephthalate, polycarbonate of bisphenol A, borisdyrene, cellulose nisdel (e.g., cellulose 1-acetate, cellulose 1-helacetate-I, cellulose acetate-1-propionate, etc.). Made of polymer with a thickness of approximately 50 μm, approximately 1. zz, preferably in the range of about 80 μM to about 300 μI, may be mentioned.

A subbing layer is provided on the surface of these supports, and the -)
- Placement of the detection layer and other layers on the support (4) can be strengthened.Also, instead of an undercoat layer, the surface of the support can be coated with physical The adhesive force may be improved by performing a chemical activation treatment (discharge, corona discharge) or a chemical activation treatment.

The integrated multi-purpose polyurethane I of the present invention (the analysis element includes a light-transmissive hydraulic
A detection layer is provided on the second side of the transparent support (depending on the case, via a layer such as an undercoat layer). The detection layer provided in the dry analytical element of the present invention is preferably a layer made of an aqueous binder, that is, a hydrophilic polymer that absorbs water and swells.

Hydrophilic polymers that may be used in the detection layer include: - natural or synthetic hydrophilic polymers having a swelling ratio of about 1.5 to 20, preferably about 2.5 to 15, at 30°C upon absorption of water; Polymer. Examples of such hydrophilic polymers include serrati (acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (phthalated gelatin, etc.)
, acarose, polyacrylamide, polyvinyl alcohol, polyhinylpyrrolidone, and the like.

The dry-smoked thickness of the detection layer is preferably in the range of about 1 μm to about tOOμI, more preferably about 3), tts to about 30μ. Furthermore, the detection layer contains surface active (
It may be cationic, amphoteric, or nonionic, or may contain a buffer.

An adhesive layer for adhering and laminating the developing layer may be provided on the second side of the detection layer, depending on the case, via the filter vortex layer and the reaction reagent layer. Preferably, the adhesive layer comprises a hydrophilic polymer capable of adhering the spreading layer when wetted with water or swollen with water. Examples of hydrophilic polymers that can be used in adhesive layers include:
Hydrophilic polymer similar to that used in the detection layer (-)
It will be done.

Among these, gelatin, gelatin derivatives, polyacrylamide and the like are preferred. The dry thickness of the adhesive layer generally ranges from about 0.5 .mu.m to about 20 .mu.m, preferably from about 1.mu.m to about 10 .mu.m. In addition to the water absorption layer, the adhesive layer may be provided on a desired layer in order to improve the adhesive force between other layers. The contact layer I is made of a hydrophilic polymer,
C can be set by applying an aqueous solution containing a surfactant and the like, which may be added as necessary, onto the detection layer, reaction reagent, etc. using a known method.

The reaction reagent layer of the dry analytical element of the present invention may contain a hydrophilic polymer, a buffer, light-shielding (reflective or absorbing) fine particles, etc., as necessary.

Examples of hydrophilic polymers that can be further included in the reaction reagent layer of the dry analytical element of the present invention include starch, cellulose, agarose, gelatin, and derivatives thereof (
(e.g., hydroxymethylation and hydroxypropylation, etc.), acrylamide polymers, copolymers of acrylamide and various vinyl monomers, polyvinyl alcohol, copolymers of vinylpyrrolone and various vinyl monomers, acrylate polymers, Examples include copolymers of Acrylay-1 with various vinyl monomers. Among the above hydrophilic polymers, polyvinyl alcohol, vinylpyrrolidone polymers, acrylamide polymers, and cellulose derivatives are preferred.

81 Examples of buffers that can be contained in the reaction reagent layer of the dry analytical element of the present invention include carbonates, borates, phosphates, and known gf agents such as Gooct's buffers. can be mentioned. These buffers are described in "Basic Experimental Methods for Proteins and Soluns, +" by Takeshi Horio et al., Nankodo, 1981.
>, etc., can be selected and used with reference to known documents.

The reaction reagent layer of the dry analytical element of the present invention may further contain light-shielding particles, which will be described later.

The light shielding layer is preferably a water-permeable layer in which light-reflecting fine particles are dispersed using a hydrophilic polymer having film-forming ability as a binder. The light-reflecting fine particles are a support that has light transparency and detectable changes (color change, color development, etc.) occurring in the detection layer. It blocks the color of the aqueous liquid dotted onto the developing layer, especially the red color of hemoglobin when the sample is whole blood, and also functions as a light-reflecting layer or a background layer.

Examples of light-reflecting fine particles include pigment fine particles j', for example, titanium dioxide fine particles (rutile type, anacousse type, or brutzite type) with particle diameters from about 0°1μ to about 12μ.
microcrystalline particles of μI), barium sulfate particles, aluminum particles, and the like. Among these, titanium dioxide fine particles and barium sulfate fine particles are preferred. Particularly preferred are anatase titanium dioxide fine particles.

Examples of hydrophilic polymer binders that have peel-forming ability include, in addition to the hydrophilic polymers used in the production of the detection layer described above, weakly hydrophilic regenerated cellulose and cellulose acetate. In this case, gelatin, gelatin derivatives, and polyacrylamide caps are preferred. In addition, gelatin and gelatin derivatives may contain known hardening agents (
(crosslinking agent) may be added.

The light-shielding layer may be provided by applying an aqueous dispersion of light-shielding fine particles and a hydrophilic polymer onto the support directly or via another layer (especially an undercoat layer) by a known method, and drying. I can do it.

The light-shielding fine particles are evenly dispersed in the light-shielding layer. If necessary, light-shielding fine particles as described in 1. may also be included in the developing layer.

Preferably, the spreading layer is a spreading layer that performs a liquid metering function. Existing a 31-volume action on the liquid 1 means that the liquid sample dotted on the surface of the liquid sample is applied per unit area in the horizontal direction without substantially unevenly distributing the portion containing 1 to 1 in the liquid sample. It has the effect of spreading at an approximately constant rate.

Materials constituting the matrix of the spreading layer include filter paper, nonwoven fabric, woven fabric (e.g., float, plain weave such as boblin, etc.), knitted fabric (e.g., l-lico 1 knit, double 1 helicopter). I-edition, Milanese ≦ Hungou-), crow fiber Nito paper, and flash polymer. A three-dimensional structure made of a membrane filter 5 or polymer MIG 1 co-beads can be used. Among these, it is preferable to use a fibrous layer typified by woven fabrics and knitted fabrics in terms of the ability to hold reagents.

Details of these? B1-) is published in Japanese Unexamined Patent Publication No. 1643 in 1983.
No. 56, No. 57-66359 and No. 6Q-2227
Please refer to No. 69-.

The woven fabric or knitted fabric that can be used in the dry analysis element of the present invention can be treated with degreasing treatment such as washing with water to remove at least threads.
Preferably, oils and fats deposited during the production of woven or knitted fabrics are substantially removed.

When the above-mentioned fabric or fabric is used as a developing layer of an integrated multilayer analysis element, the fabric or fabric is further subjected to the physical activation process disclosed in Japanese Patent Application Laid-Open No. 57-66359 [JI! (preferably glow discharge treatment, corona discharge treatment, etc.) is applied to at least one side of the fabric;
The fabric or woven fabric is made hydrophilic by impregnating it with a hydrophilic polymer as disclosed in Publication No. 59, etc., or by the method disclosed in JP-A No. 60-222770 to make the fabric or fabric hydrophilic. ) can strengthen the adhesion with the layer.

integrated multilayer analysis element made of woven or woven fabric];
2- In order to adhere and laminate the bare 17n layer to the above-mentioned test, '1' layer, reaction reagent layer or adhesive layer, etc.
, 6-1356-Wa and Japanese Patent Application Laid-Open No. 57-66359, etc.

That is, water (or water containing a small amount of surfactant) is applied substantially uniformly to the water-absorbing layer or adhesive layer while it is still wet after being applied, or to the layer after drying to swell the layer. 1. Then, the wet or swollen layer of the fabric or knitted fabric is adhered and laminated by applying light pressure substantially uniformly to the wet or swollen layer.

When the spreading layer is made of Braju polymer or memphran filter, Japanese Patent Publication No. 5B-21677, etc., and when it is a three-dimensional lattice-like n53 precious material made of polymer microheath, Japanese Patent Application Laid-open No. 55-'l')0859. Publications, etc.
When it is made of filter paper or nonwoven fabric, it is disclosed in Japanese Patent Application Laid-open No. 57-1.4.
．． They can be provided according to the methods described in Japanese Patent No. 8250 and the like.

If the hydrophilic polymer binder in the aforementioned detection layer or adhesive layer is gelatin or a Serachi derivative, the gelatin (, ): derivative) remains in the form of an undried gel after the layer is applied!
It is also possible to adopt a method of adhering the material #''I constituting the developed layer 2 between the layers and layering it on top.

The reaction reagent in the fluid development layer is a self-developing substrate. When further containing a hydrophilic polymer, a buffering agent, a light-shielding agent, etc., if necessary, apply a coating solution containing the items listed in - from above the developing layer or spray it and dry it. It can be used.

In addition, when the 14C spreading layer is made of fibers, knitted fabrics, paper, non-woven fabrics, etc., a method can be used in which the spreading layer is immersed in a solution containing the fS drug, etc., and then dried. In addition, in the case of laminating the developed layers as described above to form a wood-shaped multilayer analysis element, dry the developed layers without soaking as described above. It is also possible to use a method of laminating and integrating the layers of the pond.
In the case of a layer formed by coating, for example a layer made of plush polymer, etc., a solution or solution of a reagent, etc. may be mixed with the coating stream of this layer. When a layer contains a reagent, etc., how many reagents 1η
You may use different methods to It is also possible to divide the test into several times in several reagent towns.

The present invention will be explained in more detail below with reference to Examples.

[Example] An aqueous solution having the following composition (:1) was applied onto a colorless transparent smooth film of polyethylene derephthalate 1 to a thickness of 180 μm that was undercoated with gelatin so that the thickness after drying was 10 μm, and dried. do.

<a,> Gelatin 300g surfactant
5g (Olin 5urfac
t, ant IOG) Bolico (styrene-N-methylmolporinium methylstyrene-dihinylhensen) Polymerization ratio 55:43:2 ]5χ latex solution 280g water
2], 50 g
Na, Ol (adjust pH to 7.0 with solution.

-] 6 Next, an aqueous solution having the following composition (1-+) is applied to the "gelatin layer 1" - so that the thickness after drying is 5 μ[0] and dried.

(1 item) Gelatin 200g Surface viscosity agent
5g (Olin Su+(ac
ta, nt 10G) α-cucursicus 7
1,000,000 TO water 2
Adjust p i-1 to 70 with 600 g dilute Na○H solution.

Next, water was uniformly supplied to the entire surface of the gelatin layer at a rate of about 30 g/layer to moisten it. Laminate (1: 1) under gentle pressure and dry.

Next, water t'?; rri having the following composition (()) was applied almost uniformly to this cloth at a rate of 200 cc/m2, and the cloth was combed to produce an integrated multilayer analytical element for measuring amylase.

varani), lophenyl α-D-mal), pentaoside 60gNa, CI
9g water
1700g potassium phosphate
Adjust pH to 70 with 60 g dilute Na 011 solution.

For comparison, the following composition (
An analytical element for measuring amylase was prepared by using a solution of the following composition (Co) in place of the solution of composition (c) for the solution of b).

(b') Ceradine 200g surfactant
5g (Olin 5urfac
tant IOG) water
Adjust the pH to 70 using 2600g dilute Na○1■ solution.

Palani 1 ~ lophenyl α-D-mal I pentaoside 60 g α-glucosigose 2] 50,000 T UNaCI
9g water
1.700 g potassium phosphate
60g cold Na○■
．． Adjust to 0.

Table 1 shows the results of measuring the background densities (wavelength: 400 mμ) of the analytical elements of Examples of the present invention and for comparison using a reflection densitometer.

Table 1 As is clear from Table 1, the analytical element of the present invention has a very low background concentration compared to the analytical element for comparison.

Claims (1)

[Claims] It has at least two water-permeable layers on a light-transparent support, and at least one of the water-permeable layers contains trisaccharide, disaccharide or monosaccharide bound to para-nitrophenol in the presence of amylase. Another water-permeable layer comprising a self-developing oligosaccharide substrate capable of dissociating sugars (and optionally tetrasaccharides) and provided between the water-permeable layer comprising the self-developing oligosaccharide substrate and the support. Multilayer dry liquid analysis element characterized by containing glucosidase in the sexual layer