JPS6247552A - Multi-layered analyzing element - Google Patents

Abstract

PURPOSE:To prevent the decrease of analytical sensitivity by using a porous development layer contg. loose fibers, macromolecular polymer having a reactive group and at least one kinds of pulverous particles selected from pulverous silica particles, pulverous aluminum oxide particles and pulverous titanium oxide particles of which the surfaces are made hydrophobic. CONSTITUTION:The loose fibers, the macromolecular polymer having the reaction group and at least one kind of by pulverous particles selected from the pulverous silica particles, pulverous aluminum particles and pulverous titanium oxide particles of which the surfaces are made hydrophobic are incorporated into the porous development layer of a multi-layered analyzing element having a hydrophilic polymer layer and porous development layer on a base having liquid impermeability and light transmittivity. The multi-layered analyzing element having such porous development layer obviates a decrease of sensitivity in the result of analysis and does not require much energy for prepn. of a dispersion. The multi-layered analyzing element which eliminates an adverse influence such as ununiformity of the film thickness of the hydrophilic polymer layer is thus obtd.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a multilayer analytical element for analyzing specific components in a fluid sample, and more particularly to a multilayer analytical element having an improved porous spread layer. .

[Background of the Invention FA] In recent years, many methods for analyzing specific components in fluid samples have been developed. Particularly in the field of clinical chemistry, not only solution methods but also various types of dry methods have been developed. Among these, the dry multilayer analytical element disclosed in Japanese Patent Publication No. 53-21677 has attracted attention not only for its remarkable simplicity in handling the complicated operability of the solution method but also for its high accuracy.

The basic structure of this dry multilayer analytical element is as shown in the above-mentioned patent No. 11, a hydrophilic polymer layer on a water-impermeable transparent support,
and porous spread layers are laminated in sequence in the same direction.

The ease of operation and high precision characteristic of the multilayer analytical element are due to the porous spreading layer distributing the biological fluid sample at a constant capacity per unit area and supplying it to the hydrophilic polymer layer. This is because it has a metering function, so it can absorb weighing errors in pipetting operations that are common in analytical operations.

The above-mentioned Japanese Patent Publication No. 53-21677 discloses a plush polymer (non-fibrous) porous spreading layer.

Examples of this include plush polymers made of titanium dioxide and cellulose diacetate, and membrane filters. However, since plush polymers are made porous during the manufacturing process, it is extremely difficult to control the conditions to obtain the optimum porosity. Furthermore, in the case of a membrane filter, the hydrophilic polymer layer is in a semi-dry state and is pressed against the porous membrane, which may impair the uniformity of the thickness of the hydrophilic polymer layer.

Furthermore, JP-A No. 55-164356 discloses a woven fabric that has been subjected to hydrophilic treatment as a porous spreading layer, but this is also similar to the membrane filter disclosed in JP-B No. 53-21677 mentioned above. It is inevitable that the thickness of the hydrophilic polymer layer will become non-uniform due to pressure bonding.

On the other hand, a porous spreading layer made of loose fibers and a high molecular weight polymer having a reactive group, disclosed in JP-A-57-197466, is useful as it overcomes the above-mentioned drawbacks. However, as the fiber length increases, more energy is required to prepare a dispersion having the above composition.

Furthermore, according to the disclosure of JP-A-59-125060, the above-mentioned drawbacks are overcome by adding a white pigment to a porous spreading layer having a longer mm length and consisting of a reactive high molecular weight polymer. However, when titanium oxide is used as a white pigment, for example, the analysis target components include total protein, urea nitrogen, etc., which has the disadvantage of decreasing sensitivity.

The present inventors have made extensive studies to overcome the above-mentioned drawbacks, and as a result, have accomplished the present invention.

[Objective of the invention] A porosity that does not cause a decrease in sensitivity in analysis results, does not require a large amount of energy in preparing a dispersion, and does not have adverse effects such as non-uniformity of the thickness of the hydrophilic polymer layer. The object of the present invention is to provide a multilayer analysis element having a spreading layer.

[Structure of the Invention] The above 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, wherein the porous spreading layer is This is achieved by a multilayer analytical element containing at least one type of fine particles selected from silica fine particles, aluminum oxide fine particles, and titanium oxide fine particles whose surfaces are hydrophobized. Ru.

[Specific Structure of the Invention] The support used in the multilayer analytical element of the present invention is liquid-impermeable and light-transmissive (hereinafter referred to as the support of the present invention), and its type may be Any polymeric material such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene are suitable for this purpose. 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-mentioned support, it can be directly coated, 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 used in the multilayer analytical element of the present invention will be explained.

Typical examples of hydrophilic polymers used in the hydrophilic polymer layer include gelatin and its derivatives, polyvinyl alcohol, polyacrylamide, polyvinylpyrrolidone, hydroxyethylcellulose, agarose, carboxymethylcellulose sodium salt, etc. . Moreover, among the above synthetic polymers, it is possible to use them as copolymers.

Moreover, these above-mentioned hydrophilic polymers should be appropriately selected depending on the item.

For example, in the case of an analytical element for analyzing albumin or total protein, it is obvious that gelatin, which is a protein, and its derivatives cannot be used as the hydrophilic polymer.

In addition, when separating a portion of the reagent, especially if the reagent causes an undesirable reaction when mixed before using the analytical element, a solvent may be used, for example, as disclosed in JP-A-59-88097. It is possible to laminate two or more types of hydrophilic polymers having different solubility in water.

Furthermore, as disclosed in JP-A-59-88097, gelatin and polyvinylpyrrolidone are used, the first layer is gelatin (water is the solvent), the second layer is polyvinylpyrrolidone (lower alcohol solvent, such as ethanol, Butanol, etc.) can prevent unwanted mixing.

The hydrophilic polymer layer contains analytical reagents,
For example, it may contain an enzyme color former (coupler, hydrogen donor, etc.), a buffer, and the like. However, a part of the reagent may be separated by a hydrophilic polymer layer, as in the case of the previous one, or may be separated and placed in a porous spreading layer.

Furthermore, the hydrophilic polymer layer can also contain a surfactant. The surfactant used can be appropriately selected regardless of whether it is ionic or nonionic. Examples of ionic surfactants include alkylnaphthylsulfone MfM, commercially available from DuPont as Alkanol etc. These can be contained in various concentrations depending on the purpose, but generally about 0.01% of the hydrophilic polymer used as a binder.
From weight percent to about 10 weight ω% is used.

The multilayer analytical element of the present invention has a hydrophilic polymer layer on the support of the present invention, and a porous spreading layer of the present invention described in detail below is formed on the hydrophilic polymer layer. .

The porous spreading layer of the present invention is composed of loose fibers, a high molecular weight polymer having a reactive group, and at least one of silica particles whose surface has been made hydrophobic, aluminum oxide particles, and titanium oxide particles.

In pieces! Regarding polymers having I miscellaneous and reactive groups, the above-mentioned JP-A-57-197466 specification (
Those disclosed in detail in US Pat. No. 4,427,632 can be used. Specifically, natural cellulose IJAM1 and synthetic fibers sifted through a sieve with a certain mesh size are used, and those with a mesh size of 40 or more are used according to the Japanese Industrial Standards.

For example, natural cellulose includes filter paper powder commercially available from Toyo Roshi ■. You can obtain several types of these depending on the width of Furui's mesh. According to this, filter paper powder A ranges from 100 meshes to 20 meshes.
0 mesh on the same day, 200 meshes to 300 meshes on the same day, 300 meshes or more on the same day, 40 meshes on the same day to 100 meshes, and 200 meshes or more on the same day. Although any of these can be used, those having a mesh size of 200 or less are effectively used in the porous spreading layer according to the present invention.

Further, as the polymer having a reactive group, those disclosed in the above-mentioned JP-A-57-197466 can also be used. - Examples include styrene-glycidyl methacrylate copolymer (polymerization ratio 9:1), styrene-butyl methacrylate to glycidyl methacrylate copolymer (
Polymerization ratio 75:15:10), methyl acrylate-glycidyl methacrylate copolymer (polymerization ratio 8:2)
, styrene-aziridylethyl methacrylate copolymer (polymerization ratio 95:5), and the like.

The primary particles of each fine particle of titanium (Ti 02 ) preferably have an average particle size of about 100 mμ to about 3a+μ, more preferably about 601Ilμ to about 5 mμ.

It is preferable that the surface of these particles is hydrophobized using a silylating agent, a silane coupling agent, etc., and the surface is covered with an alkyl group.

The fine particles with a hydrophobized surface according to the present invention are made of silica without adsorbed water synthesized by a gas phase method, dimethyldichlorosilane and water vapor together with an inert carrier gas such as nitrogen at a temperature of about 400°C. It can be synthesized by heating. Details of this synthetic method are disclosed in French Patent No. 1,368,765.

The above-mentioned fine particles whose surface has been made hydrophobic can be easily obtained from Nippon Aerosil ■ under the following trade name, for example.

Product name - Surface treatment agent average particle size of secondary particles Aerodino FR972 ■ (AERO81116mμ dimethylcyclo R972)
Lucilan ■ Aerosil R972V ■ (AERO8I L 16 mtl dimethyl dichloro R972V) Lucilan 1.
[Has] 7Eronle R974 ■ (ΔERO31112111 dimethyl dichloro R974)
Lucilan 1. ■ Aerosil R976 (△ERO3I Lo 7mμ dimethyl dichloro R9
76) Lucilan ■ Aerosil R811 ■ (AERO81112mμ HexamethyldiR811)
Silazane Aerodino σR812 ■ (AERO8IL 7rAμ HexamethyldiR8
12) Silazane Aerosil R8
05 ■ (AERO8I L 12 mu Octi/L,
I-! J MeR805) t-
xysilane titanium oxide F805 (Titanium Oxide 21 mμ
Octyl Trime F 805)
Toxysilane ■ Particularly Aerosil R-805 can be preferably used for the purpose of the present invention.

In the composition of the porous spreading layer according to the present invention, the amount of the polymer having reactive groups with respect to the discrete fiber components is preferably about 40% by weight to about 10% by weight, more preferably about 35% by weight.
% to about 15% by weight. Further, the fine particles having a hydrophobic surface according to the present invention can be prepared by dispersing 1HIItffi.
It is preferably about 3% to about 18% by weight, more preferably about 5% to about 15% by weight relative to ω.

The porous spreading layer according to the present invention can contain a surfactant in addition to the above composition. Surfactants can be contained regardless of whether they are nonionic or ionic, but nonionic surfactants are useful coating aids because they dissolve well in both organic solvents and water, and they are also useful in biological applications. This is effective in improving the development speed when a target fluid sample is spotted on the porous development layer.

Examples of useful surfactants include: (1) Triton sodium rubirnafdylsulfonate, manufactured by DuPont), fluorotenside FT-248 (tetraedylammonium perfluorooctyl sulfonate, manufactured by Bayer),
Various nonionic and ionic surfactants can be mentioned, such as Florard FC-170G (perfluoroalkylpolyoxyethylene ethanol, manufactured by Minnesota Mining and Manufacturing Co., Ltd.), and these can be used in combination of one or two types. More than one species can be used.

The porous spreading layer according to the present invention can contain appropriate reagents as necessary.

For example, as in the multilayer analytical element for analyzing blood urea nitrogen disclosed in Japanese Patent Application Laid-Open No. 59-104552, N. It is possible to contain N-diethylaminoethylnaphthylamine etc. in a dispersed manner;
As disclosed in No. 9324, it is possible to contain a dispersed mixture of an enzyme and a protein that does not inhibit the activity of the enzyme.

Moreover, the separate [
t can also be contained in a form impregnated with reagents.

Various known methods can be used to prepare the dispersion of the porous spreading layer according to the present invention. For example, physical stirring, ultrasonic irradiation, etc. can be used alone or in combination.

In preparing the dispersion of the porous spreading layer according to the present invention, it is particularly desirable to use a dispersion method using ultrasonic irradiation in combination with a physical stirring method.

It goes without saying that the treatment time for these dispersions depends on the length of the dispersion liquid to be prepared and the energy used for dispersion, but under the same conditions, it is possible to add the microparticles with hydrophobized surfaces according to the present invention. In this case, the time required is about 1/20 to about 1/5 compared to the case without addition.

As described above, in conventional dispersion methods, applying energy for long-time dispersion often leads to undesired denaturation of the dispersion liquid.

The dispersion prepared as described above can be coated using any coating method used in the photographic industry, such as a slide hopper coating method, an extrusion coating method, a curtain coating method, etc.

The multilayer analytical element of the present invention can analyze various substances by selecting an electrochemical.

For example, glucose, blood urea nitrogen, cholesterol, triglyceride, uric acid, gorirubin, hemoglobin, creatine, creatinine, albumin, total protein, alkaline phosphatase, GOT, GPT, LD)-1
, CPK, etc.

In the multilayer analytical element of the present invention, acids, alkalis, salts, solidifying agents, resolving agents, surfactants, etc. may be appropriately contained in the porous layer and the hydrophilic polymer layer according to the purpose of the present invention. I can do it.

The buffer used in the present invention should be selected in terms of its content, amount, and type as necessary. For example, in general, phosphate buffer (Na2HPO4NaOH), carbonate buffer (NaHCO3-Na2CO3>, borate buffer (Na2Bq0?-NaOH), TrisWm agent [Tris (Hydroxymethyl)aminomerun salt Pa], Gutto's buffer (e.g. CAPS-KOH, etc.)
Examples include.

Examples of the surfactant that can be used in the present invention include alkylphenoxypolyethoxyethanol [e.g., Triton X-100 (manufactured by Rohm and Haas)],
Typical examples include polyethylene oxide alkyl esters [eg, Emulgen-120 (manufactured by Kao Atlas Co., Ltd.)] and alkylphenoxyglycerin [eg, Surfactant 10G (manufactured by Oreen Corporation)].

The multilayer analytical element of the present invention can have various functional layers and layer configurations as desired. For example, reagent layers, reflective layers, subbing layers as described in U.S. Pat. No. 3,992,158, radiation blocking layers as described in U.S. Pat. No. 4,042,335, barrier layers as described in U.S. Pat. No. 4,066,403. , the registration layer described in U.S. Pat. No. 4,144.306, the migration prevention layer described in U.S. Pat. No. 4,166.093, the breakable body member described in U.S. Pat. It is possible to construct a multilayer analytical element tailored to the purpose of the invention.

The various layers mentioned above can be coated to any desired thickness by using slide hopper coating, extrusion coating, dip coating, etc., which are conventionally known in the photographic industry.

In addition, as for the layer structure, for example, Japanese Patent Application Laid-Open No. KI57-40191
No. (Special Publication No. 58-18628), U.S. Patent No. 4,11
It is possible to arbitrarily select the layer structure described in JP-A-58-131565, No. 0,079, and the like.

The multilayer analysis element crushed 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.

Fluid samples applicable to the multilayer analysis element of the present invention include:
Examples include biological fluid samples such as serum (including plasma and serum), lymph fluid, and urine. Also, the suspension of the fluid sample used is arbitrary, but preferably about 5μ! The amount is from 50 μl, more preferably from about 5 μy to about 20 μy.

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

Also, if necessary, 30°C to 50°C, preferably 35°C to 4°C.
After incubation at 0°C for 1 to 20 minutes, preferably 1 to 10 minutes, light in the range of 500 nm to 650 nm from the liquid-impermeable and light-transparent support side is applied as appropriate depending on the conditions. The reflected optical density may be measured through a filter that allows light of a specific wavelength to pass through, and the density may be determined from a calibration curve. Additionally, semi-quantitative measurements can be performed by creating color samples in advance and visually comparing them.

[Specific Example 1 of the Invention] Hereinafter, the present invention will be specifically explained with reference to Examples, but the embodiments of the present invention are not limited to these.

Example 1 A layer having the following composition was coated on a transparent undercoated polyethylene terephthalate support having a thickness of 180 microns.

(Hydrophilic polymer layer) 50.0 tl of deionized gelatin was dissolved in 250 l of distilled water, and 30 g of 1,7 dihydroxynaphthalene,
Dimedone 0669 and 5% Alkanol
Unit and glucose oxidase (manufactured by Toyobo ■)
79,000 units were added and dissolved.

Furthermore, add 1.5MK21”PO4 solution to 1.5MKH2P
1.5M phosphoric acid 1! buffered to p+-+-6,1o with O4 solution! 490 g of buffer solution was added, and 25 Q of 1% 1,2-ethane aqueous solution was added, and this coating solution was coated onto the above support using a blade coater having a gap of 375 microns and dried. .

(Porous development layer) A layer having the composition shown in Table 1 below.

However, in Table 1, *1) Cotton cellulose with 300 mesh or more made by Toyo Roshi @ J *2) Cotton cellulose with 40 to 100 mesh made by Toyo Uta Hook *3) Product manufactured by Rohm and Haas Name, octylphenoxypolyethoxyethanol *4) Average particle size 0.5p Main phrase Surface-untreated Nisamaji titanium *5) Made by Nippon Aerosil ■Product name: Hydrophilic silica AE
Non-hydrophobic version of RO8IR-R805 *6) Product name manufactured by Nippon Aerosil ■, hydrophilic butane oxide
T 1taniuIIIOxide T-805 not hydrophobized *7) Ultrasonic homogenizer USH-15ON25 type (
After coating the coating solution with the above composition onto the hydrophilic polymer layer using a blade coater with a gap of 375 microns,
It was dried and used as a multilayer analytical element sample.

The multilayer analytical element sample of the present invention prepared as described above (
1) to (3) and comparative multilayer analytical element samples (1) to (6)
After dropping 10μ2W4 of glucose aqueous solution at 131 degrees to each and incubating at 37℃ for 7 minutes,
Using a 13 degree meter PDA, -65 (Konishi Roku Photo Industry side),
The reflection density was measured using a 54 snm filter, and a calibration curve was created from the glucose concentration and the reflection degree by linear regression.

Furthermore, the same operation was performed using human serum, and mouth = 10
After examining the simultaneous reproducibility, we investigated the variation in the color development area, that is, the developed area, of this multilayer analytical element.

The results are shown in Table 2 below.

However, in Table 2, in the straight line where the test m line is y = ax + b, a represents its slope, the intersection with the bl and ty axes, x is the sample concentration (g/dffi), and y is the anti-tJ4 concentration (D2)
represents. Further, r is a correlation coefficient of X1y and is expressed by the following formula. ' However, x measured value by Koni assay (g/dffi) x:
Average value of ×(y) Reflection density by the analytical element y:y This average value Also, in the analysis results, the × in the upper row is the average value, the SD of the middle f= is the standard deviation, and the CV in the lower row is expressed by the following formula. Simultaneous reproducibility.

5C) CV (simultaneous reproducibility) --x 100 (%) As shown in the margin below, multilayer analytical element samples (1) to <3) of the present invention
The comparative multilayer analytical element sample using short fibers (under the same manufacturing conditions as 1) has the same sensitivity and simultaneous reproducibility, and has the same variation in coloration area. Comparative multilayer analytical element samples containing titanium oxide fine particles, silica fine particles, or titanium oxide fine particles (
3), (4), and (5) have lower sensitivity and poor simultaneous reproducibility. In addition, a comparative multilayer analysis element test using only long fibers was conducted.
(2) has poor simultaneous reproducibility and large variations in coloring area. Furthermore, a comparative multilayer analysis element sample (6
) is there enough simultaneous reproducibility? There is a large variation in color area.

Example-2 A layer of the following composition was coated onto a transparent 180 micron thick primed polyethylene terephthalate support.

(Hydrophilic polymer layer) Acrylamide/methyl methacrylate copolymer (polymerization ratio 9/1) 10% aqueous solution 359, Emulgen 120 (
Product name, nonionic surfactant, manufactured by Kao Atlas ■) 50
Add 5i% aqueous solution and 245Q citric acid, stir and dissolve, then add 30% aqueous sodium hydroxide solution to DH3.7
It was adjusted to 50 d with distilled water. This coating solution was coated onto the support using a blade coater with a gap of 250 microns and dried.

(Porous development layer) A layer having the composition shown in Table 3 below.

A coating solution having the above composition was applied onto the hydrophilic polymer layer using a blade coater with an r1 gap of 375 microns and dried to obtain a multilayer analytical element sample.

10μ of various levels of human serum albumin aqueous solutions were applied to this multilayer analytical element sample! After dripping and incubating for 7 minutes at 37°C, Sakura Korai Densitometer PDA-65 (
Reflection density was measured using a 545 nm filter at Konishi Roku Photo Industry ■).

Furthermore, the albumin concentration of the albumin aqueous solution was determined according to a standard method, and the albumin concentration (Mdf) was added to X INB.
, the reflection density (D) was taken and plotted on the y-axis, and a calibration m-line was created by -order regression analysis.

The results are shown in Table-4. However, a%b%r has the same meaning as in Table-2.

As is clear from Table 2 in the margin below, #comparative multilayer analytical element samples (7) and (8), which use silica fine particles with the same particle size but a hydrophilic surface, use silica fine particles with a hydrophobic surface. Compared to the multilayer analysis cord samples (4) and (5) of the present invention used, the sensitivity was significantly lowered.

Patent applicant: Konishiroku Photo Industry Co., Ltd. Representative Patent Attorney Miyahito Ichinose □, “J

Claims (1)

[Claims] In a multilayer analytical element having a hydrophilic polymer layer and a porous spreading layer on a liquid-impermeable and light-transmitting support, the porous spreading layer is made of a polymer having discrete fibers and reactive groups. A multilayer analytical element comprising a polymer and at least one type of fine particles selected from silica fine particles, aluminum oxide fine particles, and titanium oxide fine particles whose surfaces have been made hydrophobic.