Multi-layer chemical analytical materials

A multi-layer chemical analytical material comprising a support, a reagent layer on the support and a spreading layer on the reagent layer wherein the reagent layer is composed of a hydrophilic binder and fine hydrophobic particles dispersed in the hydrophilic binder, said hydrophobic particles containing a reagent capable of directly or indirectly reacting with the component being analyzed to produce a color change.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to an improved analytical material for use in the 
quantitative determination of a specific component contained in an aqueous 
liquid sample. More particularly, it is concerned with a multi-layer 
chemical analytical material comprising a support, a reagent layer and a 
spreading layer which is characterized in that the reagent layer is of an 
oil-in-water type dispersion wherein the reagent is contained in 
hydrophobic particles dispersed in a hydrophilic binder. 
2. Description of the Prior Art 
Multi-layer chemical analytical materials in sheet form which permit 
analysis by a dry operation are described, for example, in Japanese Patent 
Publication No. 33800/74 (corresponding to U.S. Pat. No. 3,630,957), 
Japanese Patent Application (OPI) Nos. 53888/74 (corresponding to U.S. 
Pat. No. 3,992,158), 137192/75 (corresponding to U.S. Pat. No. 3,983,005), 
40191/76 (corresponding to U.S. Pat. No. 4,042,335), 3488/77 
(corresponding to U.S. Pat. No. 4,006,403), 89796/78 (corresponding to 
U.S. Pat. No. 4,069,017), 131089/78 (corresponding to U.S. Pat. No. 
4,144,306), etc. Those analytical materials are of the construction that a 
reagent layer of a single or multi-layer construction is provided on a 
support and an isotropically porous spreading layer is provided on the 
reagent layer. On dropping an aqueous liquid sample on the spreading 
layer, the aqueous liquid sample penetrates into the reagent layer and 
undergoes a color-forming reaction, and this change in color density 
permits determination of the concentration of a certain component in the 
aqueous liquid sample. 
The quantitative determination of a certain component in an aqueous liquid 
can rarely be done with only one reagent and, consequently, a combination 
of several reagents is often used. For example, the quantitative 
determination of urea in blood is usually carried out not only by a direct 
determination using a single reagent but by a multi-stage reaction: urea 
is subjected to enzyme decomposition with a first reagent, urease, to form 
ammonia and the ammonia so formed is allowed to participate in the color 
formation by use of a second reagent, for example, a pH indicator or a 
mixed reagent of a diazonium salt and a coupler, whereby the quantitative 
determination of urea can finally be achieved. 
In some of the multi-layer analytical sheets wherein a plurality of 
reagents are used for the multi-stage reaction, all of the reagents can be 
incorporated in the reagent layer of the single layer construction whereas 
for other multi-layer analytical sheets it is preferred that the reagents 
are divided into the first reagent and the second reagent according to the 
order of reaction and separately incorporated in a plurality of reagent 
layers. 
The multi-layer analytical sheet as described in U.S. Pat. No. 3,011,874 
and Japanese Patent Application (OPI) No. 3488/77 is of a complicated 
multi-layer construction wherein a reagent layer is divided into two 
layers, one of the layers being composed of a hydraulic binder and the 
other being composed of a hydrophilic binder or protected by a film of a 
hydrophobic substance so as to prevent water from penetrating therein. 
That is, the quantitative determination of a certain component in an 
aqueous liquid sample is carried out by reaction in water followed by 
reaction in a non-aqueous medium. For the production of such a multi-layer 
analytical sheet, there is generally employed a method wherein coating and 
drying are successively carried out and at least two coating-drying steps 
are required. Where the hydrophilic binder layer is provided directly on 
the hydrophobic layer, the adhesion between the two layers is weak and 
they often separate from each other in a short time. In the practice of 
this method, therefore, the hydrophobic binder layer is first provided, at 
least one additional adhesive layer called an undercoating layer or 
intermediate layer is then provided on the hydrophobic binder layer, and 
the hydrophilic binder layer is finally coated on the adhesive layer. 
Thus, the method wherein the reagent layer is divided into the hydrophobic 
and hydrophilic layers gives rise to the drawback that the production 
process is markedly complicated. 
In accordance with the hitherto known methods for use in the quantitative 
determination of a certain component in an aqueous liquid sample wherein a 
plurality of reagents to be used are required to be divided into two or 
more groups, one group being reacted under hydrophilic circumstances and 
another group being reacted under non-aqueous circumstances, there has 
been employed a multi-layer analytical sheet of the complicated 
multi-layer construction in which the reagents are divided into a 
hydrophilic binder layer and the hydrophobic binder layer. On the other 
hand, the multi-layer analytical material of this invention is 
characterized in that the reagent layer is not divided into two layers and 
a single reagent layer composed of a hydrophilic binder and hydrophobic 
fine particles dispersed therein containing the reagent is employed. 
A multi-layer analytical material containing therein a reagent layer 
prepared by dissolving in an organic solvent a hydrophobic reagent which 
is insoluble or sparingly soluble in water and which is soluble in an 
organic solvent, and dispersing in a hydrophilic binder polymer the 
organic solvent solution of the reagent as fine particles is described in 
Example 4 of Japanese Patent Application (OPI) No. 26188/78 (corresponding 
to U.S. Pat. No. 4,089,747) and in Japanese Patent Application (OPI) No. 
73096/79 (corresponding to British Patent Publication No. 2 007 360A). 
However, in the reagent layer of such multi-layer analytical material, a 
complicated procedure is required for dispersing in the hydrophilic binder 
polymer the reagent which is insoluble or sparingly soluble in water such 
that the reagent which is insoluble or sparingly soluble in water is first 
dissolved in a solution of the organic solvent to prepare a mixed solution 
which is then dispersed as oily liquid fine particles. In contrast, in the 
multi-layer chemical analytical material of the present invention, fine 
particles containing the reagent functions not only to disperse in the 
hydrophilic binder polymer but also to prevent the permeation of water 
from the surface portion into the inside portion of the fine particles 
containing the reagent. 
SUMMARY OF THE INVENTION 
This invention provides: 
A multi-layer chemical analytical material comprising a light-transmitting 
and water-impermeable support, a reagent layer on the support and an 
aqueous liquid sample-spreading layer on the reagent layer wherein the 
reagent layer is composed of a hydrophilic binder and fine hydrophobic 
particles dispersed in the hydrophilic binder, the particles containing a 
regent capable of undergoing a direct or indirect reaction with the 
component to be analyzed to produce a color change; and 
A multi-layer chemical analytical material as described above wherein the 
hydrophilic binder contains a second reagent capable of reacting with the 
component to be determined.

DETAILED DESCRIPTION OF THE INVENTION 
One of the advantages of this invention is that the single layer 
construction of the reagent layer permits the coating step to be 
simplified to at least 1/2 that of the conventional multi-layer 
construction method as described previously. 
Another advantage is that in the multi-layer chemical analytical material 
as described above, the apparent detection limit may be increased. The 
reason for this is considered that in the multi-layer analytical material 
of this invention including the single reagent layer in which the 
hydrophobic fine particles containing the reagent are dispersed, as 
apparent from the geometric structure, the distance between the reagent 
contained in the hydrophobic fine particle and the reagent present in the 
hydrophilic binder is short and the contact area between them is markedly 
enlarged in comparison with the conventional multi-layer analytical sheet 
provided with a plurality of reagent layers, which allows various 
reactions to proceed more smoothly until a color is formed. 
Light-transmitting and water-impermeable supports for use in the 
multi-layer analytical material of this invention include plastic films 
such as polyethylene terephthalate, cellulose esters (e.g., cellulose 
diacetate, cellulose triacetate and cellulose acetate propionate), 
polycarbonate and polymethyl methacrylate, and glass plates; known 
transparent supports with a thickness of from about 50 .mu.m to about 2 mm 
can be used. 
Where the support is hydrophobic and the adhesion between the support and 
the hydrophilic binder of the reagent layer is insufficient, it may be 
subjected to processings to render the surface hydrophilic (e.g., 
ultraviolet ray irradiation, electron beam irradiation, flame processing, 
hydrolysis using alkali, etc.), or auxiliary processings, for example, the 
formation of an undercoating layer made up of a substance having 
appropriate adhesion to both the support and the hydrophilic binder of the 
reagent layer on the surface of the support and the formation of minute 
unevenness on the surface of the support to such an extent as not to 
greatly reduce the light transmission property (e.g., brushing and 
electrolytic etching). 
The reagent which can be incorporated can be selected depending upon the 
nature of the component to be determined. However, those described in 
Japanese Patent Application (OPI) No. 3488/77 (corresponding to U.S. Pat. 
No. 4,066,403) are preferred as the reagent to be incorporated in the 
chemical analytical material of the present invention. 
The hydrophobic fine particles of this invention are liquid or solid minute 
particles containing therein the reagent, which particles are permeable to 
a gas or a substance soluble in the hydrophobic solvent through diffusion 
or extraction, but not by water. 
Such hydrophobic fine particles containing the reagent can be prepared by 
the same method as in Japanese Patent Application (OPI) No. 13320/74 
(corresponding to U.S. Pat. No. 3,951,851) and exemplify oily minute 
particles prepared by adding a lipophilic binder polymer, tackifier, dye 
or pigment to oily matters prepared by dissolving the reagent in an 
organic solvent substantially incompatible with water; oily minute 
particles prepared by dissolving in an organic solvent substantially 
incompatible with water, a polymer soluble in an organic solvent, and 
dissolving or dispersing the reagent in the resulting solution; minute 
particles prepared by pulverizing a solid polymer containing the reagent; 
etc. In addition, those microcapsules prepared by encapsulating the above 
oily minute particles or minute particles prepared by pulverizing the 
solid polymer containing the reagent with a thin layer of a lipophilic 
polymer or a hydrophilic polymer can be used. 
Organic solvents substantially incompatible with water which are used for 
the preparation of the minute particles as described above include liquid 
plasticizers such as phthalic acid esters (e.g., dimethyl phthalate, 
diethyl phthalate, dibutyl phthalate, dicyclohexyl phthalate and dioctyl 
phthalate), adipic acid esters (e.g., diisodecyl adipate and dioctyl 
adipate), phosphoric acid esters (e.g., triethyl phosphate, tributyl 
phosphate, trioctyl phosphate and triphenyl phosphate); organic solvents 
such as toluene, xylene, mesitylene, alkylnaphthalenes, methyl acetate, 
ethyl acetate and butyl acetate; animal oils; vegetable oils and mineral 
oils. 
Organic solvent-soluble polymers which are used for the preparation of the 
minute particles such as described above include cellulose diacetate, 
cellulose triacetate, cellulose acetate propionate, ethyl cellulose, 
methyl methacrylate, polystyrene and polycarbonate of disphenol A. 
In this case, the organic solvent forms a solution, the reagent is 
dissolved or dispersed in the resulting solution, the solution or 
dispersion so obtained is added to an aqueous solution of a hydrophilic 
binder and emulsified or dispersed, and then a part or all of the organic 
solvent and water are removed by evaporation whereby there can be formed 
the reagent layer wherein the fine particles containing therein the 
reagent are dispersed in the hydrophilic binder. Further, microcapsules 
containing therein the reagent prepared by the same method as described in 
Japanese Patent Application (OPI) No. 13320/74 (corresponding to U.S. Pat. 
No. 3,951,851) can also be used as the fine particles. 
The thickness of the reagent layer is from about 0.5 .mu.m to about 50 
.mu.m and preferably from about 1 .mu.m to about 30 .mu.m. 
The size of the hydrophobic fine particles containing the reagent is from 
about 0.01 .mu.m to about 20 .mu.m and preferably from about 0.1 .mu.m to 
about 10 .mu.m. Each particle contains the major portion of the reagent in 
the inner portion thereof, exposing substantially no reagents on the 
surface thereof and does not allow water to permeate therein. 
The amount of the hydrophobic fine particles containing the reagent is from 
about 3% to about 90% and preferably about 5% to about 80% based on the 
total weight of the reagent layer. 
Hydrophilic binders for use in the reagent layer include water-soluble 
proteins such as gelatin, albumin and collagen; vegetable gums such as 
agar, sodium alginate and agarose; and water-soluble synthetic polymers 
such as an olefin-maleic anhydride copolymer, polyvinyl alcohol, polyvinyl 
pyrrolidone, polyacrylamide and poly(sodium benzenesulfonate). These 
binders are able to form a film containing the reagent and the dry film so 
formed readily allows an aqueous solution containing a component to be 
determined (aqueous liquid sample) to permeate therein. Of course, in the 
hydrophilic binder can be incorporated, in addition to the reagent 
incorporated in the hydrophobic fine particles, a reagent which reacts 
with the component to be determined, e.g., urease. 
With respect to the spreading layer of the chemical analytical material, in 
addition to the nonfibrous isotropically porous materials described in the 
patent specifications and references as illustrated hereinbefore, those 
fabrics which are made hydrophilic can be used as the aqueous liquid 
sample-spreading layer of the present multi-layer chemical analytical 
material. 
Examples of such nonfibrous isotropically porous materials are those 
wherein fine porous particles of a brush polymer (generally called a 
membrane filter), diatomaceous earth or fine crystalline materials (e.g., 
fine crystalline cellulose) are uniformly dispersed in the binder; porous 
materials wherein fine spherical beads of glass or a synthetic polymer are 
brought in point contact with each other and held there with a binder; and 
a brush polymer in which fine powders of TiO.sub.2, BaSO.sub.4 or the like 
are uniformly dispersed. (See James Flinn ed., Membrane Science and 
Technology, Plenun Press, New York (1970); P. Grabar & J. A. de Loureiro, 
Annales de L'institut Pasteur, 65, 159-189 (1939); and U.S. Pat. Nos. 
3,129,159 and 1,421,341.) 
Examples of such fabrics which are made hydrophilic are a fabric which is 
degreased by fully washing with water and then dried; and a fabric which 
is degreased by fully washing with water and then impregnated with a small 
amount of a hydrophilic polymer, a surfactant, a wetting agent, or a 
hydrophilic polymer having TiO.sub.2 or BaSO.sub.4 fine powders dispersed 
therein. The use of such a hydrophilic fabric as the aqueous liquid 
sample-spreading layer and the details of such fabrics are described in 
Japanese Patent Application No. 72047/79, filed June 8, 1979 
(corresponding to U.S. Pat. No. 4,292,272. 
Where the aqueous liquid sample-spreading layer is made up of the 
nonfibrous isotropically porous material, its thickness is from about 50 
.mu.m to about 500 .mu.m, preferably from about 80 .mu.m to about 300 
.mu.m. In the case of the fabric to be made hydrophilic, the thickness of 
the fabric which has been made hydrophilic and allowed to dry is from 
about 80 .mu.m to about 1 mm, preferably from about 100 .mu.m to about 400 
.mu.m. 
The aqueous liquid sample-spreading layer made up of the nonfibrous 
isotropically porous material can be made as described in Japanese Patent 
Application (OPI) Nos. 53888/74 (corresponding to U.S. Pat. No. 
3,992,158), 137192/75 (corresponding to U.S. Pat. No. 3,983,005), etc. For 
example, a solution or dispersion capable of forming a nonfibrous 
isotropically porous layer is coated on a reagent layer and dried, or a 
thin layer of the nonfibrous isotropically porous material is bonded to 
the reagent layer. In the case of the aqueous liquid sample-spreading 
layer made up of the fabric which has been made hydrophilic, the fabric 
can be bonded with the reagent layer. 
For bonding the nonfibrous isotropically porous material or the fabric 
which has been made hydrophilic onto the reagent layer, there can be 
employed a method in which while the reagent layer is wet or after wetting 
the dry surface of the reagent layer with water or water containing 
surfactant, the nonfibrous isotropically porous material or the fabric 
which has been made hydrophilic is brought in close contact with the 
reagent layer and bonded, if desired, by applying an appropriate pressure, 
by utilizing the characteristics of the hydrophilic binder polymer 
contained in the reagent layer. Another method utilizes an adhesive which 
is permeable in the aqueous liquid sample. In accordance with another 
method, an adhesive layer permeable in the aqueous liquid sample is 
provided on the reagent layer. 
On the multi-layer chemical analytical material of this invention can be 
provided, as neccessary, a second reagent layer (see U.S. Pat. No. 
3,992,158), a detecting layer (see U.S. Pat. No. 4,042,335) and a barrier 
layer (see U.S. Pat. Nos. 3,983,005 and 4,066,403). These layers are 
described in detail in the patent specifications as described 
hereinbefore. Referring to these descriptions they can be provided in this 
invention. 
In the multi-layer chemical analytical material of this invention, a 
radiation-blocking layer or a light reflection layer can be provided 
between the reagent layer and the aqueous liquid sample-spreading layer. 
Additionally, between the aqueous liquid sample-spreading layer and the 
reagent layer or the radiation-blocking layer or the light reflection 
layer can be provided an adhesive layer for which the aqueous liquid 
sample is permeable, for the purpose of firmly bonding the aqueous liquid 
sample-spreading layer. The radiation-blocking layer or light reflection 
layer and adhesive layer are described in detail in the patent 
specifications as described hereinbefore (e.g., U.S. Pat. Nos. 3,992,158 
and 4,042,335). Referring to these descriptions they can be provided in 
this invention. 
As the radiation-blocking layer or light reflection layer, a layer made up 
of a hydrophilic binder polymer and a fine white powder, such as a 
TiO.sub.2 fine powder and a BaSO.sub.4 fine powder, dispersed therein and 
having a thickness of from about 1 .mu.m to about 50 .mu.m, preferably 
from about 2 .mu.m to about 20 .mu.m; a layer made up of a hydrophilic 
binder polymer and a fine powder of a material having a white or pale 
metal luster, such as aluminum, dispersed therein and having a thickness 
of from about 2 .mu.m to about 50 .mu.m, preferably from about 2 .mu.m to 
about 20 .mu.m; or an aqueous liquid sample-permeable porous metal thin 
layer made up of a white or pale metal, such as aluminum, and having a 
thickness of from about 5 nm to about 100 nm, preferably from about 5 nm 
to about 50 nm can be provided. 
As the adhesive layer, a layer made up of the same polymer as the aqueous 
liquid sample-permeable hydrophilic polymer used as a binder in the 
reagent layer, radiation-blocking layer or light reflection layer and 
having a thickness of from about 0.5 .mu.m to about 10 .mu.m, preferably 
from about 0.7 .mu.m to about 5 .mu.m can be used. 
In bonding the aqueous liquid sample-spreading layer on the adhesive layer 
composed of the hydrophilic polymer, the aqueous solution of the 
hydrophilic polymer is coated on the reagent layer, radiation-blocking 
layer or light reflection layer and then while it is still wet or after it 
is dried, the surface is wetted with water or water containing a 
surfactant, the nonfibrous isotropically porous material or the fabric 
which has been made hydrophilic is brought into contact with the surface 
of the adhesive layer and bonded by applying an appropriate pressure. 
Alternatively, a solution or dispersion capable of forming a nonfibrous 
isotropically porous layer may be coated on the adhesive layer to obtain 
the multi-layer chemical analytical material wherein the aqueous liquid 
sample-spreading layer is firmly bonded. 
Hereinafter the multi-layer chemical analytical material of this invention 
and the hitherto known multi-layer analytical sheet will be compared with 
reference to the accompanying drawings. 
FIG. 1 is an illustrative view of a conventional multi-layer analytical 
sheet for use in the quantitative determination of the amount of urea in 
serum wherein an ammonia gas-permeable and water-impermeable hydrophobic 
second reagent layer 22 composed of a hydrophobic binder and an indicator 
contained therein, the indicator undergoing discoloration by the action of 
ammonia gas is provided on a water-impermeable transparent support 1, and 
a hydrophilic first reagent layer 21 composed of gelatin and urease and a 
buffering agent dispersed therein is provided on the hydrophobic second 
reagent layer 22, and furthermore a nonfibrous isotropically porous 
spreading layer 3 for spreading an aqueous liquid sample is provided on 
the first reagent layer 21. 
On attaching a droplet of serum on the spreading layer 3 from the direction 
of A, the serum uniformly extends so that the volume per unit area is 
nearly uniform and reaches the first reagent layer 21 where the urea is 
decomposed by urease. The ammonia so formed is gasified and permeates the 
second reagent layer 22 where the pH indicator undergoes discoloration 
depending upon the amount of ammonia. The degree of discoloration is 
examined from the direction of B and the amount of urea is quantitatively 
determined. 
FIG. 2 is an illustrative view of an embodiment of the multi-layer 
analytical material of this invention wherein a single reagent layer 23 
composed of a hydrophilic binder and hydrophobic fine particles 20 
dispersed therein is provided on a light-transmitting and 
water-impermeable support 1, and porous spreading layer 4 is provided on 
the reagent layer 23. In more detail, a hydrophobic oil or hydrophobic 
binder containing the indicator is emulsified and dispersed in a 
hydrophilic binder aqueous solution containing an enzyme system to give a 
dispersion and the dispersion so prepared is coated to provide the reagent 
layer 23. The determination of urea in serum is carried out in the same 
manner as explained for the conventional one of FIG. 1. 
From the above comparison, it can be seen that the multi-layer analytical 
material of this invention is advantageous in its ease of production in 
comparison with the conventional analytical sheet. 
The following examples are given to illustrate this invention in greater 
detail. 
COMATIVE EXAMPLE 
Multi-Layer Chemical Analytical Material for Analysis of Urea Aqueous 
Solution 
In 6 g of dibutyl phthalate was dissolved 20 mg of a pH indicator, 
bromothymol blue, to prepare a hydrophobic reagent solution. Then, 20 mg 
of urease and 200 mg of disodium ethylenediaminetetraacetate (EDTA 2Na) 
were dissolved in 15 g of a 10% by weight aqueous solution of gelatin to 
prepare a hydrophilic reagent solution. 
The hydrophobic reagent solution was emulsified and dispersed in the 
hydrophilic reagent solution by a conventional method using a supersonic 
emulsifier to form an oil-in-water (O/W) type emulsion. The size of the 
hydrophobic particles containing the bromothymol blue was 5 .mu.m or less. 
The dispersion thus obtained was coated on a 170 .mu.m thick transparent 
polyethylene terephthalate (PET) film which had been undercoated to form a 
reagent layer. The thickness of the layer after drying was about 20 .mu.m, 
and microscopic analysis confirmed that the reagent layer was made up of 
fine oil particles dispersed in the gelatin phase. 
After wetting the surface of the reagent layer with water, a membrane 
filter (Microfilter FM 500 produced by Fuji Photo Film Co., Ltd.) was 
laminated on and bonded together with the reagent layer by pressing under 
humid conditions to provide thereon a porous spreading layer. A 
multi-layer chemical analytical material for the quantitative 
determination of an urea aqueous solution was thus obtained. 
After 10 .mu.l of an urea aqueous solution was dropped on the spreading 
layer and then heated at 37.degree. C. for 5 minutes, the optical density 
(OD) was measured with light having a wavelength of 600 nm by use of a 
reflection optical densitometer. At concentrations of the urea aqueous 
solution of 30 mg/dl, 60 mg/dl and 100 mg/dl, the corresponding OD values 
were respectively 0.35, 0.53 and 0.78. 
The calibration curve obtained from the above values is nearly a straight 
line. Therefore, it can be seen that the urea content of an urea aqueous 
solution can be quantitatively determined by use of the multi-layer 
chemical analytical material as obtained above. 
EXAMPLE 1 
Multi-Layer Chemical Analytical Material for Quantitative Analysis of Serum 
Urea 
20 mg of bromothymol blue and 200 mg of ethyl cellulose (a product of 
Hercules Inc.) were dissolved in a mixed solvent of 6 ml of methyl acetate 
and 2 ml of dibutyl phthalate to prepare a hydrophobic reagent-polymer 
solution. Then, 20 mg of urease and 200 mg of EDTA 2Na were dissolved in 
15 g of a 10% by weight aqueous solution of gelatin to prepare a 
hydrophilic reagent-binder aqueous solution. 
The hydrophobic reagent-polymer solution was added to the hydrophilic 
reagent-binder aqueous solution, and the hydrophobic reagent-polymer 
solution was emulsified and dispersed in the hydrophilic-binder aqueous 
solution by a conventional method using a supersonic emulsifier. The 
dispersion was stirred for 30 minutes at 55.degree. C. whereby the organic 
solvent (methyl acetate) was evaporated off to prepare a dispersion having 
solid particles of ethyl cellulose (polymer) containing bromothymol blue 
(pH indicator) dispersed in the hydrophilic reagent-binder aqueous 
solution. The size of the solid particles of the polymer containing 
bromothymol blue was about 5 .mu.m or less. 
The dispersion thus obtained was coated on a 170 .mu.m thick transparent 
PET film which has been undercoated with gelatin, and then dried to form a 
reagent layer in which the solid fine particles of the polymer were 
dispersed. The thickness of the reagent layer after drying was about 20 
.mu.m, and microscopic analysis confirmed that the reagent layer was made 
up of the solid fine particles of the polymer dispersed in gelatin. 
After wetting the surface of the reagent layer with water, a membrane 
filter (Microfilter FM 500 produced by Fuji Photo Film Co., Ltd.) was 
laminated on and bonded together with the reagent layer by pressing under 
humid conditions to provide thereon a porous spreading layer. A 
multi-layer chemical analytical material for the quantitative 
determination of serum urea was thus obtained. 
Each of buffer aqueous solutions having different pH values ranging between 
6 and 8 was applied onto the porous spreading layer of the thus obtained 
multi-layer chemical analytical material, and then allowed to stand at 
37.degree. C. for 5 minutes. No color change of the pH indicator in the 
reagent layer of each analytical material was observed. This indicates 
that the solid fine particles of the polymer containing the pH indicator 
in the multi-layer chemical analytical material were water-impermeable and 
that the pH indicator (bromothymol blue) was not present in a 
substantially exposed state on the surfaces of the solid fine particles of 
the polymer. 
10 .mu.l of each of synthetic serums containing urea having different 
concentrations in a phosphorous acid buffer aqueous solution having a pH 
value of 7.0 was measured out by means of a micro pipette, and applied 
onto the porous spreading layer of the multi-layer chemical analytical 
material, followed by allowing the material to stand at 37.degree. C. for 
5 minutes. The optical density (OD) was measured with light having a 
wavelength of 600 nm by use of a reflection optical densitometer. At 
concentrations of the urea in the synthetic serum of 30 mg/dl, 60 mg/dl 
and 100 mg/dl, the corresponding OD values were respectively 0.38, 0.57 
and 0.83. 
The calibration curve obtained from the above values is nearly a straight 
line. Therefore, it can be seen that the urea content in the serum can be 
quantitatively determined by use of the multi-layer chemical analytical 
material as obtained above. 
EXAMPLE 2 
Multi-Layer Chemical Analytical Material for Analysis of Ammonia Water 
A mixture of 5 mg of 4-(2,4-dinitrobenzyl)-1-propylquinolium chlorate 
(indicator) and 200 mg of ethyl cellulose (N-100 produced by Hercules Co.) 
was dissolved in a mixed solvent of 5 ml of ethyl acetate and 3 ml of 
toluene to prepare a hydrophobic reagent solution. As a hydrophilic binder 
solution, 20 g of a 10% by weight aqueous solution of gelatin was 
prepared. The hydrophobic reagent solution was emulsified and dispersed in 
the hydrophilic reagent solution by a conventional method and by 
continuing the stirring at 50.degree. C. for 30 minutes, the solvents 
(ethyl acetate and toluene) were evaporated to produce a dispersion 
wherein microcapsules in which hydrophobic solid particles containing the 
indicator were encapsulated with a thin film of ethyl cellulose were 
dispersed in the hydrophilic binder (gelatin) aqueous solution. 
The thus obtained solution was coated on a transparent PET film and dried 
in the same manner as in Example 1, and subsequently the same porous 
spreading layer as described in Example 1 was laminated thereon to provide 
a multi-layer chemical analytical material. 
Then, each of buffer aqueous solutions having different pH values ranging 
between 6 and 8 was applied onto the porous spreading layer of the thus 
obtained multi-layer chemical analytical material. No color change of the 
pH indicator was observed. This indicates that the hydrophobic fine 
particles (microcapsules) containing the pH indicator in the multi-layer 
chemical analytical material were water-impermeable. 
By using ammonia water having different concentrations of 0.02%, 0.04%, 
0.08% and 0.10%, the quantitative property was confirmed. 
EXAMPLE 3 
Multi-Layer Chemical Analytical Material for Quantitative Determination of 
Serum Urea 
A solution of 20 mg of bromothymol blue and 200 mg of polystyrene (Sebian A 
produced by Daicel Ltd.) in a mixed solvent of 5 g of toluene and 3 g of 
dibutyl phthalate was emulsified and dispersed in 20 g of a 10% by weight 
aqueous solution of gelatin. Then, by continuing the stirring at 
50.degree. C. for 30 minutes, the toluene was evaporated to produce a 
dispersion wherein microcapsules in which hydrophobic liquid particles 
containing the indicator were encapsulated with a thin film of polystyrene 
were dispersed in the hydrophilic binder (gelatin) aqueous solution. To 
this solution was further added a solution prepared by dissolving 20 mg of 
urease and 200 mg EDTA 2Na in 2 ml of water. The resulting solution was 
coated on a transparent PET film and dried in the same manner as in 
Example 1 to provide an about 15 .mu.m thick reagent layer containing 
hydrophobic fine particles (microcapsules) which contained the reagent. 
On the reagent layer was coated a 5% aqueous solution of gelatin containing 
35% of fine titanium dioxide particles to provide thereon a 17 .mu.m thick 
radiation-blocking layer. Furthermore, on the radiation-blocking layer was 
laminated the same porous spreading layer as in Example 1 to provide a 
multi-layer chemical analytical material. 
On applying buffer aqueous solutions having different pH values ranging 
between 6 and 8 onto the multi-layer analytical material as produced 
above, no color change of the indicator was observed. This indicates that 
the hydrophobic fine particles containing the reagent were 
water-impermeable. 
Synthetic serums with different concentrations of urea were prepared by 
adding urea to a phosphoric acid buffer aqueous solution containing 7% of 
serum protein and having a pH value of 7.0. These serums were tested on 
the multi-layer chemical analytical material as obtained above, in the 
same manner as in Example 1. The results are shown in the table below. 
______________________________________ 
Urea Reflection Optical Density 
(mg/dl) (wavelength 600 nm) 
______________________________________ 
0 0.21 
25 0.39 
50 0.55 
75 0.74 
100 0.92 
______________________________________ 
The above results indicate that the urea content of the synthetic serum and 
the reflection optical density vary nearly linearly. It, therefore, can be 
seen that the multi-layer chemical analytical material permits the 
quantitative determination of the urea content of aqueous liquid samples 
containing urea. 
EXAMPLE 4 
Multi-Layer Chemical Analytical Material for Quantitative Analysis of Blood 
Urea 
One surface of a broadcloth (a product of Nisshin Spinning Co., Ltd.) woven 
at a No. 60 cotton count yarn was made hydrophilic by impregnating it with 
a 1% aqueous solution of gelatin to prepare a fabric having a gelatin 
content of about 2.5% at drying. Separately, a reagent layer and a 
radiation-blocking layer were provided on a transparent PET film and dried 
in the same manners as those employed in Example 1 and Example 3, 
respectively. Thereafter, the radiation-blocking layer was wetted and 
swollen with a 0.2% aqueous solution of a nonionic surface active agent 
(polyoxyethylene isooctylphenyl ether), and the above-described fabric 
which had been made hydrophilic was laminated on and bonded together with 
the thus treated radiation-blocking layer by lightly pressing in such a 
manner that the surface of the fabric which had been impregnated with the 
gelatin aqueous solution was brought into contact with the 
radiation-blocking layer. In this respect, the fabric which had been made 
hydrophilic functions as the spreading layer for the quantitative 
determination of an aqueous liquid sample. 
Quantitative determination of blood urea could be made in the same manner 
as in Example 1 using fresh blood (whole blood) by the use of the thus 
prepared multi-layer chemical analytical material for the quantitiative 
analysis of blood urea. 
EXAMPLE 5 
Multi-Layer Chemical Analytical Material for Analysis of Ammonia Water 
The same procedure as in Example 2 were repeated except that the cotton 
fabric prepared in the same manner as in Example 4 was used in place of 
the membrane filter and laminated on the radiation-blocking layer in the 
same manner as in Example 4, whereby a multi-layer chemical analytical 
material was prepared. 
By applying 10 .mu.l of ammonia water having different concentrations of 
0.02%, 0.04%, 0.08% and 0.10% onto the porous spreading layer (cotton 
fabric having been made hydrophilic) of the multi-layer chemical 
analytical material, the quantitative property was confirmed. 
EXAMPLE 6 
Multi-Layer Chemical Analytical Material for Quantitative Determination of 
Serum Urea 
The same procedures as in Example 3 were repeated except that the cotton 
fabric prepared in the same manner as in Example 4 was used in place of 
the membrane filter and laminated on the radiation-blocking layer in the 
same manner as in Example 4, whereby a multi-layer chemical analytical 
material was prepared. 
The test was carried out in the same manner as in Example 3 using buffer 
aqueous solutions having different pH values ranging between 6 and 8 and 
synthetic serums whereby the same results as in Example 3 were obtained. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent to one skilled in the 
art that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.