JPS63157999A - Analytic element for determination of enzymatic activity and production thereof - Google Patents

Abstract

PURPOSE:To shorten the time necessary for the determination of enzymatic activity with a dry analytic element containing a water-permeable liquid- developing layer and a reagent, by including a substrate, an oxidized coenzyme and an electron-transfer agent in the former layer and a pigment precursor in the latter layer. CONSTITUTION:The objective dry analytic element is composed of a water- permeable reagent layer placed opposite to the liquid-receiving surface of a liquid-developing layer. The developing layer contains the major parts of 3 components comprising a substrate, an oxidized coenzyme and an electron- transfer agent and the reagent layer contains the major part of a pigment precursor. Preferably, the developing layer contains a substrate for a lactate dehydrogenase.

Description

DETAILED DESCRIPTION OF THE INVENTION [Prior Art] A dry analytical element for measuring enzyme activity using a reaction rate method is known from JP-A-58-88097 and other publications. The dry analysis element disclosed in the above-mentioned published patent detects the production of reduced coenzyme and measures the concentration and activity of components in the liquid. In other words, the reduced coenzyme produced by the reaction between the product of a reaction involving an enzyme and the oxidized coenzyme, and the formazan dye produced from the electron-accepting dye precursor in the presence of an electron transfer agent are optically synthesized. To detect.

The above publication states that the substrate for the enzyme and the oxidized coenzyme may be contained in either the developing layer or the receiving layer, and the electron transfer agent and the dye precursor may also be contained in either the developing layer or the receiving layer. It is considered good. However, for the storage stability of the analytical element, it is preferable that the electron transfer agent and the dye precursor be contained in separate layers. However, when the electron transfer agent and the dye precursor are contained in different layers, these 4
It is not stated how the seed element is most advantageously arranged between the spreading layer and the receiving layer.

In one case, the receptor layer contains a substrate, an oxidized coenzyme, and a dye precursor, and only an electron transfer agent is contained in a developing layer; Regarding the case where a dye precursor is included in the developing layer, see JP-A-58
It is described in the Examples of No.-88097. However, when a dry analytical element was made with such a reagent arrangement, the reaction rate was slow and the measurement time was long when measuring enzyme activity.

In the above-mentioned published patent, in the example, a developing layer and two receptor layers are provided, a substrate and an oxidized coenzyme are contained in the developing layer, and an electron transfer agent and a dye precursor are contained in separate receptor layers. There is also a description of a case in which two receptor layers are provided, which complicates the manufacturing process and increases production costs.

[Technical Problems to be Solved] The present invention improves a dry analytical element for enzyme activity measurement that contains a substrate, an oxidized coenzyme, an electron transfer agent, and a dye precursor in a water-permeable layer, and prevents the reaction during enzyme activity measurement. The purpose of the present invention is to provide an analytical element for enzyme activity measurement that is fast and does not require long measurement time.

The present invention also improves a dry analytical element for measuring enzyme activity that includes a substrate, an oxidized coenzyme, an electron transfer agent, and a dye precursor in a water-permeable layer, and enables enzyme activity measurement to be performed at low production costs and with good storage stability. The purpose is to provide analysis elements for

The present invention also provides an improved method for manufacturing a dry analytical element for enzyme activity measurement having a water-permeable layer, which provides a fast reaction rate and does not require a long measurement time when measuring enzyme activity. The third purpose is to provide a manufacturing method.

[Means for solving technical problems] The above first and second objects have at least a water-permeable liquid developing layer and a reagent layer, and have a substrate, an oxidized cofermentation element, an electron transfer agent, and a dye precursor. A dry analytical element for measuring enzyme activity that contains the body in a water-permeable layer, including substrates, oxidized coenzymes,
This was accomplished with a dry analytical element containing the three main portions of the electron transfer agent in the developing layer and the main portion of the dye precursor in the reagent layer.

The third purpose is to apply a developing layer using a composition containing a substrate, an oxidized coenzyme, and an electron transfer agent, and apply a composition containing a dye precursor in manufacturing the dry analytical element for enzyme activity measurement. This was achieved by applying a reagent layer using

[Aspects of the Invention] The present invention can be applied to various known dry analysis elements. The element may be multiple layers including a porous liquid spreading layer, a reactive reagent layer, as well as a support, a detection layer, a light shielding layer, an adhesive layer, a filtration layer, a water absorption layer, a subbing layer, and other layers known in the art. . As such an analysis element, U.S. Patent No. 3,992
, No. 158, No. 4゜042.335 and JP-A No. 1983
-164356 There are some disclosed in each specification.

When a light-transmitting support is used, the practical configuration of the dry analytical element of the present invention is (1) having a reagent layer on the support and a liquid spreading layer thereon.

(2) A device having a detection layer, a reagent layer, and a liquid development layer in this order on a support.

(3) A support having a reagent layer, a light reflection layer, and a liquid development layer in this order.

(4) A support having a detection layer, a reagent layer, a light reflection layer, and a liquid development layer in this order.

(5) A support having a detection layer, a light reflection layer, a reagent layer, and a liquid development layer in this order.

In (1) or (3) above, a water-absorbing layer may be provided between the support and the reagent layer. Preferred in the present invention is the above layer structure (1). In (1) to 3) above, a filtration layer may be provided between the reagent layer and the detection layer or liquid development layer. In (3) or 5) above, a filtration layer is further provided between the light reflection layer and the detection layer, the reagent layer or the liquid development layer, between the reagent layer and the detection layer, or between the reagent layer and the liquid development layer. You can.

The reaction reagent system included in the analytical element of the present invention consists of lactic acid or its salt as a substrate, NAD", an electron transfer agent, and an electron-accepting formazan dye precursor. Examples of the electron transfer agent include diaphorase, NN-
1ethylphenazoniu methosul
fate etc. can be used. Formazan dye precursors include INT or 2-(p-1odop
henyl)-3-(p-nitr.

phenyl)-5-phenyltetrazoli
um chloride + B T or 3.3' -
(3,3'-dimethoxy-4,4'-bi
phenyIene)-bis[2,5-diphen
yltetrazolium ehloridel, 3
．． 3'-(4,4'-biphenylene)-bi
s[2,5-diphenyltetrazolium
Nitrotetrazolium blue (NTB or NBT) or 3.3'-(3,3'-dimethoxy-4,4
'-b i pheny 1ene)-b is[2-
(p-ni tropheny I)-5-phe
ny 1tetrazolium ehloridel
is preferred.

Preferably, the porous liquid spreading layer is a spreading layer having a liquid metering function. Having a liquid metering effect means that the liquid sample that is dotted onto the surface of the sample is distributed in an approximately constant amount per unit area in the lateral (horizontal) direction without substantially unevenly distributing the liquid sample contained therein. It has the effect of expanding the ratio.

As the material constituting the spreading layer, filter paper, nonwoven fabric, woven fabric (for example, plain weave fabric), knitted fabric (for example, tricot fabric), glass fiber filter paper, etc. can be used. Among these, woven fabrics, knitted fabrics, etc. are preferred.

An adhesive layer for adhering and laminating the developing layer may be provided on layers such as the water absorption layer, detection layer, light guide layer, filtration layer, and reagent layer. The adhesive layer is preferably made of a hydrophilic polymer, such as gelatin, gelatin derivatives, polyacrylamide, etc., which can adhere the spreading layer when swollen with water.

The reagent layer of the dry analytical element of the present invention includes a hydrophilic polymer,
A buffering agent and the like can be included as necessary. Examples of hydrophilic polymers that can be included in the reagent layer include starch, cellulose, agarose, gelatin and derivatives thereof (such as phthalated gelatin), acrylamide polymers, and copolymers of acrylamide and various vinyl monomers. , methacrylamide polymers, copolymers of methacrylamide and various vinyl monomers, etc. can be used.

Examples of buffers that can be contained in the reagent layer of the dry analytical element of the present invention include carbonate, borate, phosphate, and Good buffers. These buffers are described in "Basic Experimental Methods for Proteins and Enzymes J.
(Takeshi Horio et al., Nankodo, 1981) can be selected and used.

The light-shielding layer is used to measure the detectable changes (color change, color development, etc.) that occur in the detection layer, reagent layer, etc. from the light-transmitting support side. liquid color,
Particularly when the sample is whole blood, it blocks the red color of hemoglobin and functions as a light reflecting layer or background layer.

The light shielding layer is preferably a water-permeable layer in which light-reflecting fine particles such as titanium oxide and barium sulfate are dispersed using a hydrophilic polymer having film-forming ability as a binder. Preferable binders include gelatin, gelatin derivatives, polyacrylamide, and the like. The analysis element may contain particles of titanium oxide or the like in the development layer, reagent layer, detection layer, etc., if necessary.

This will be explained in more detail below using Examples.

[Example 1] An aqueous solution of the following composition (a) was applied onto a colorless transparent smooth film of polyethylene terephthalate I with a thickness of 180 μm and coated with gelatin so that the thickness after drying was 10 μm, and dried. (strongly colored layer).

(a) Gelatin 100g water
900g oxdylphenoxypolyethoxyethanol 2gNTB*
4g* 3.3'-(3,
3"-dimethoxy-4,4'-biphenylene)-bis[
2-(p-dithophenyl)-5-phenyltetrazolium clophyte] (pH 6.5 with dilute NaOH solution)
After moistening the above-mentioned coloring layer with about 30 g/m2 of water, a tricot knitted fabric made of polyethylene terephthalate spun yarn (36 gauge, 50D) was pressed and dried to form a spreading layer.

Apply 120cc/coating liquid of the following composition (b) to the above development layer.
It was coated at a ratio of m2 and dried to produce an integrated multilayer analytical element for measuring LDH activity.

(b) Nonylphenoxypolyethoxyethanol (n=40>' 0.3
g Octylphenoxypolyethoxyethanol (n=10) 0, 3 g
Polyacrylamide (molecular weight approximately 200,000) 10g water
190g tris(hydroxymethyl) aminomethane 1.9g lithium lactate 0.5g β-NAD""
0.2g diaphorase
4,000 units (pH adjusted to 8.5 with dilute NaOH solution) [Comparative Examples 2 and 3] The composition (a) of the reagent layer coating solution in Example 1 was changed to the following compositions (a-2) and (a-3). ), the composition (b) of the developing layer coating solution in Example 1 was changed to the following composition (b-2),
LD in the same manner as in Example 1 except that (b-3) was changed.
Prepare an analytical element for H activity measurement.

0hibe A) False■ Gelatin 100 100 Water
900 900 Octylphenoxypolyethoxyethanol 22 NTB* 20 Diaphorase 10,000 (Units other than diaphorase are g, diaphorase is international unit) 1000 900 Octylphenoxypolyethoxyethanol (n=40) 0.3 0. 3 octylphenoxy polyethoxyethanol (n=10) 0.3 0.3 polyacrylamide (molecular weight approximately 200,000) 10 10 Water
190 190 Tris(hydroxymethyl) Aminomethane 1.9 1.9 Lithium lactate 0.50 β-NAD''' 0.20 Diaphorase 0 40,000 (pH with dilute NaOH solution)
(adjusted to 8.5) [Comparative Example 4] An aqueous solution of the following composition (a-4) was dried to a thickness of 5 μm on a 180 μm thick polyethylene terephthalate colorless transparent smooth film coated with gelatin. It was applied and dried (coloring layer).

(a-4) Gelatin 100g water
900g octylphenoxypolyethoxyethanol 3gNTB
4g (pH adjusted to 6.5 with dilute NaOH solution) An aqueous solution having the following composition (a-4') was applied onto the coloring layer so that the thickness after drying was 5 μm, and dried ( middle class).

(a-4') Gelatin 100g water
900g octylphenoxypolyethylesteranol 3g diaphorase
20,000 units (pH adjusted to 6.5 with dilute NaOH solution) After wetting the above intermediate layer with approximately 30 g/m2 of water, polyethylene terephthalate spun yarn (36
A tricot knitted fabric having a gauge of 50D> was pressed and dried to form a spread layer.

Apply 120ml of coating liquid of the following composition (b-4) to the above development layer.
It was coated at a rate of c/m2 and dried to produce an integrated multilayer analytical element for measuring LDH activity.

(b-4) Nonylphenoxypolyethoxyethanol (n=40) 0.3g Octylphenoxypolyethoxyethanol (n=10) 0.3g polyacrylamide (molecular weight approximately 200,000) 10g water
190g tris(hydroxymethyl) aminomethane 1.9g lithium lactate 0.5g β-NAD″1
0.2g (pH 8 with dilute NaOH solution)
．． 5) The analytical elements of Example 1 and Comparative Example were adjusted to 4.
After forced aging at 5° C. for 3 days, the reflected optical density immediately after spotting 7% human serum was as shown in Table 1.

Table 1 It can be seen that the LDH analytical element of the present invention has a lower background concentration than the analytical elements of Comparative Examples 3 and 4.

10 μβ of a commercially controlled serum (Monitrol IX), which has been assayed to have an LDH activity of 120 units per p (37°C), was applied to the developing layer of each analytical element, and kept at a constant temperature of 37°C with moisture evaporation sufficiently prevented. Place it on a heating plate and heat it at a wavelength of 540 nm.
The reflected optical density was measured after 2 minutes and 5 minutes. The results of determining the change in reflected optical density between 2 minutes and 5 minutes are as follows:
It was as shown in Table 2.

Table 2 From Table 2, it can be seen that the LDH analytical element of the present invention has a faster color development rate than the analytical elements of Comparative Examples 2 and 3. It is almost the same as Comparative Example 4.

Combining the results shown in Tables 1 and 2, the analytical elements of Comparative Examples 3 and 4 have large background concentrations after the forced aging test, and the analytical elements of Comparative Example 2 have a background concentration equivalent to that of the present invention. However, it is concluded that the analytical element of the present invention is the most superior because the coloring reaction rate is low.

Claims (1)

[Claims] 1) having at least a water-permeable liquid spreading layer and a reagent layer;
The reagent layer is located on the opposite side of the liquid-receiving surface of the liquid developing layer, and is a dry analytical element for measuring enzyme activity that contains a substrate, an oxidized coenzyme, an electron transfer agent, and a dye precursor in the water-permeable layer. A dry analytical element characterized in that a developing layer contains three main parts: a substrate, an oxidized coenzyme, and an electron transfer agent, and a reagent layer contains a main part of a dye precursor. 2) The dry analytical element according to claim 1, wherein the developing layer contains a substrate for lactate dehydrogenase. 3) having at least a water-permeable liquid spreading layer and a reagent layer;
The reagent layer is located on the side opposite to the liquid-receiving surface of the liquid developing layer, and is a dry analytical element for measuring enzyme activity that contains a substrate, an oxidized coenzyme, an electron transfer agent, and a dye precursor in the water-permeable layer. A manufacturing method, which comprises applying a developing layer using a composition containing a substrate, an oxidized coenzyme, and an electron transfer agent, and applying a reagent layer using a composition containing a dye precursor. 4) The method according to claim 3, characterized in that the spreading layer is coated using a composition containing a substrate, an oxidized coenzyme, and an electron transfer agent.