Analytical element

An analytical element which comprises (a) a light-transmissive and liquid-impermeable support, (b) at least one reagent layer containing at least one reagent reactive with a component in a liquid sample and being constituted of a hydrophilic colloid, and (c) at least one developing layer of a fibrous structure being positioned on said reagent layer on the side opposite to said support for permitting the component in said liquid sample to permeate therethrough toward said reagent layer and having been formed by coating said reagent layer with a dispersion comprising an organic macromolecular polymer having reactive groups and a fibrous material.

This invention relates generally to analytical chemistry, particularly to 
an analytical element for analysis of a predetermined specific component 
in a liquid. More particularly, it pertains to a quantitative analytical 
element for assay of a specific component in a biological liquid sample. 
There have been developed a great number of methods for analysis of 
components in liquid samples. For example, there may be mentioned 
automatic quantitative analytical devices. These are frequently used and 
very useful especially in clinical test laboratories in hospitals and the 
like. Such automatic analytical devices employ techniques based on 
continuous system analysis, in which samples, diluents and analytical 
reagents are mixed together and conveyed to analytical devices, as 
disclosed in, for example, U.S. Pat. No. 2,797,149. 
Such continuous automatic analytical devices, however, are complicated and 
expensive, requiring skilled operators. In addition, repeated washing 
operations required to be performed after the analytical operations will 
consume much time as well as labor, and the wastewaters resulting from 
these operations will disadvantageously involve the problem of causing 
environmental pollution. 
On the other hand, as contrasted to the analytical system employing 
solutions as mentioned above, there is another analytical system employing 
dry chemistry. These are called as test papers or test strips and provided 
in the dry form, prepared by dipping an absorptive carrier such as a 
filter paper in an analytical reagent solution followed by drying, as 
disclosed in, for example, U.S. Pat. No. 3,050,373 or U.S. Pat. No. 
3,061,523. The test strip enables measurement by dipping in a liquid 
sample of analyte and then withdrawing to read the color change or density 
change on the test strip with the naked eye or by means of an instrument 
such as densitometer. 
These test strips are easy in handling and useful in giving instantly the 
test results. But, these test strips comprising reagents carried in 
absorptive carriers suffer from various vital defects, and therefore their 
applications are still limited to qualitative or semi-quantitative 
analysis. 
For overcoming these defects, there has been developed an analytical 
element as disclosed in U.S. Pat. No. 3,992,158. These elements have a 
reagent layer containing analytical reagents and a spreading layer 
comprising an isotropically porous, non-fibrous medium laminated on a 
transparent support. 
The developing layer disclosed in said Patent, however, has essentially 
only brittle strength. A high percentage of these films tend to break 
making it difficult to maintain a stable supply. Also, from aspects of 
preparation, it is required to control severely the conditions for 
coating, and constant void volume (porosity) can hardly be obtained if 
such conditions are not satisfied. 
The object of the present invention is to provide an analytical element 
having excellent quantitative characteristics without requiring skilled 
operational techniques. 
The present inventors have made extensive studies and were successful in 
overcoming the drawbacks as mentioned above by use of an analytical 
element having the following constitution. 
That is, the analytical element according to the present invention 
comprises a light-transmissive and liquid-impermeable support, at least 
one reagent layer containing at least one reagent reactive with a 
component in a liquid sample and being constituted of a hydrophilic 
colloid, and at least one developing layer having a fibrous structure 
positioned on the opposite side of said support to said reagent layer for 
permitting the component in said liquid sample to permeate therethrough to 
said reagent layer, said developing layer being formed by coating a 
dispersion comprising an organic macromolecular polymer having reactive 
groups and a fibrous material. 
In the following, the analytical element according to the present invention 
is described in further detail. 
First, the aforesaid liquid-impermeable and light-transmissive support of 
the analytical element according to the present invention (hereinafter 
abbreviated as the support according to the present invention) is not 
particularly limited, so long as it is impermeable to liquid and can 
transmit light. For example, various polymeric materials such as cellulose 
acetates, polyethyleneterephthalates, polycarbonates or polystyrenes are 
suitable for this purpose of use. The above support employed may have any 
desired thickness, but preferably a thickness of about 50 microns to 250 
microns. It is also possible to work freely one side of the support 
according to the present invention, which is the side for observation, 
depending on the intended purpose. When the aforesaid reagent layer 
according to the present invention is provided directly on the above 
support, it may directly be coated thereon. In some cases, adhesion 
between the reagent layer and the support may effectively be enhanced by 
application of a light-transmissive undercoating layer on the support. 
The above reagent layer according to the present invention contains 
reagents which carry out quantitative reactions with analytes to be 
analyzed in said layer, and it is used to permit the quantitative 
reactions to proceed within said layer. 
The above reagent layer contains a hydrophilic colloid as a medium and is 
formed as a layer by being coated on a support. With such a constitution, 
as different from the prior art in which a carrier such as a filter paper 
is impregnated with reagents, reagents can be evenly contained in the 
layer, with an additional advantage that the content of reagents can 
freely be controlled. As a hydrophilic colloidal substance to be used in 
such a reagent layer according to the present invention, a natural or 
synthetic macromolecular substance is preferred. More preferably, there 
may be included gelatins, gelatin derivatives such as modified gelatins, 
polyvinyl alcohols, polyvinyl pyrrolidones, etc. Among them, a 
particularly preferred hydrophilic colloidal substance is a gelatin 
derivative such as gelatin. 
These hydrophilic colloidal substances preferably have a swelling degree of 
about 150 to 500%, and its film thickness, which can be selected as 
desired, is required to be at least about 5 microns. 
The reagents to be contained in the reagent layer formed as described above 
will of course be determined depending on the analytes to be analyzed in 
samples as well as on the analytical reaction selected for analysis of 
said analytes. When the analytical reaction selected is constituted of two 
or more reagents, these reagents may be contained by mixing together in 
the same reagent layer or alternatively contained in two or more separate 
layers. These constitutions may freely be selected so long as no 
detrimental effect is caused, partly because they are sometimes determined 
depending on the mechanism of the analytical reaction per se. 
On the other hand, it is possible to carry out the analytical reactions of 
two or more analytes in a sample in the same reagent layer. In this case, 
it is necessary to select the two or more kinds of analytical reactions so 
that they may not interfere with each other or have no influence on each 
other in measurement of the reaction products produced. 
The thus constituted reagent layer can generally be coated on the support 
according to the present invention in a manner of a coating, but, as 
mentioned above, there may also be provided various layers between the 
reagent layer and the support except for those which are not suitable for 
the purpose of the present invention. 
The developing layer of a fibrous structure according to the present 
invention is provided either as a single layer or a plurality of layers 
directly or indirectly on the reagent layer which has been previously 
provided on the support as described above. 
The developing layer of a fibrous structure is provided for the purposes 
mentioned below: 
(1) to uniformly distribute a predetermined volume of a liquid sample at a 
ratio of a constant volume per unit area in the reagent layer; 
(2) to remove substances or factors which interfere with the analytical 
reactions in the liquid sample; and 
(3) to perform background action to reflect the light to be measured 
transmitted through the support in carrying out, for example, 
spectro-photometric analysis. 
Thus, the fibrous developing layer according to the present invention can 
perform all of the three functions as mentioned above, but the three 
functions can also conveniently be separated into different layer 
according to the functions. 
Further, it is also possible to use a combination of a layer having two of 
the three functions and a layer having the other remaining function. 
The fibrous developing layer according to the present invention as 
described above may have a film thickness, which can freely be selected 
depending on the purpose, but preferably about 30 microns to about 600 
microns, more preferably about 50 microns to about 400 microns. 
And, the fibrous developing layer according to the present invention, as 
distinguished from the reticulate or fabric structure as disclosed in 
Japanese Patent Publication No. 6551/1978 or Japanese Provisional Patent 
Publication No. 164356/1980, has of course a free pore area which is 
substantially zero. 
As the materials for forming the fibrous developing layer according to the 
present invention, there may be included natural celluloses or derivatives 
thereof, synthetic fibers such as polyethylenes, polypropylenes, 
polyamides and others, said layers including those constituted of various 
fibers, irrespective of whether they are synthetic or natural, which are 
randomly three-dimensionally intertwined with each other. 
Further, as the materials for forming the above fibrous developing layer, 
there may be chosen either single species or plural species with any 
desired grain size, generally of 50 mesh to 325 mesh according to the JIS 
standard screen, preferably 100 to 320 mesh, more preferably 200 to 300 
mesh. 
The organic macromolecular polymer having reactive groups according to the 
present invention markedly enhances the film strength in the above fibrous 
developing layer through said reactive groups and also the adhesion 
strength through chemical bonding of said reactive groups with the layer 
beneath said layer maintains its appearance and structure against external 
physical force. 
The organic macromolecular polymer according to the present invention may 
have one or two or more kinds of reactive groups. If desired, heating or a 
catalyst may be employed for effecting chemical bonding between the 
reactive groups. The organic macromolecular polymer having reactive groups 
can be prepared, for example, by homopolymerization or copolymerization of 
monomers having reactive groups or precursors thereof. The macromolecular 
organic polymer having two or more kinds of reactive groups can be 
prepared, for example, by copolymerization of monomers having different 
kinds of reactive groups or precursors thereof. When monomers having 
precursors of the reactive groups are employed, they can be converted to 
organic macromolecular polymers having the reactive groups, for example, 
after formation of the organic macromolecular polymers, by treatment such 
as hydrolysis. In the present invention, the monomer unit having a 
reactive group may be contained in an amount of preferably about 0.1 to 
about 30% by weight based on said organic macromolecular polymer unit, 
particularly 0.5 to 20%. 
As the monomers having the reactive groups as described above, there may be 
mentioned monomers having epoxy groups, monomers having aziridyl groups, 
monomers having formyl groups, monomers having hydroxymethyl groups, 
monomers having isocyanate groups, monomers having thiol groups and 
monomers having carbamoyl groups. 
As a monomers having an epoxy group, there may be mentioned, for example, 
glycidyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 
4-vinylcyclohexane monoepoxide, etc. A monomer having an aziridyl group 
may be exemplified by aziridylethyl methacrylate, 1-ethylenesulfonyl 
aziridine, 1-ethylenecarbonyl aziridine, aziridylethyl acrylate, etc. 
Typical examples of a monomer having a formyl group are acrolein and 
methacrolein. A monomer having a hydroxymethyl group may include, for 
example, N-methylol-acrylamide, N-methylol-methacrylamide, 
N-methylol-diacetoneacrylamide, and the like. Typical examples of a 
monomer having an isocyanate group are vinyl isocyanate and allyl 
isocyanate. Examples of a monomer having a thiol group are vinyl thiol, 
p-thiol styrene, m-thiol styrene, vinyl benzyl thiol and acetyl 
derivatives of these. As a monomer having a carbamoyl group, there may be 
included, for example, acrylamide, methacrylamide, maleinamide, diacetone 
acrylamide, etc. 
As other monomers to be copolymerized with the monomers having reactive 
groups, there may be selected any monomer, so long as the resultant 
organic macromolecular polymer satisfies the conditions of liquid 
impermeability and non-swellability. 
Among the monomers having various reactive groups as mentioned above, 
typical examples of the monomers having an epoxy group, aziridyl group, 
hydroxylmethyl group or carbamoyl group may be inclusive of those 
mentioned hereinabove. Examples of monomers having other reactive groups 
are as follows. As a monomer having carboxyl group, there may be mentioned 
acrylic acid, methacrylic acid, itaconic acid, maleic acid, itaconic acid 
half-ester, maleic acid half-ester, etc. A monomer having an amino group 
may be exemplified by aminostyrene, N,N-dimethylaminoethyl acrylate, 
N,N-dimethylaminoethyl methacrylate. Typical examples of a monomer having 
a methoxy group are methoxyethyl acrylate, ethoxyethyl acrylate, 
methoxyethyl methacrylate, ethoxyethyl methacrylate, and the like. As a 
monomer having --COOC.sub.4 H.sub.9 (t) group, there may be included 
tert-butyl acrylate, tert-butyl methacrylate. Examples of a monomer having 
a ureido group are ureidoethyl acrylate, ureidoethyl methacrylate, 
ureidovinyl ether (e.g., those represented by the formula CH.sub.2 
.dbd.CHONRCONHR', wherein R represents a hydrogen atom or a methyl and R' 
a hydrogen atom or a lower alkyl such as methyl or ethyl). As a monomer 
having a hydroxyl group, there may be mentioned 2-hydroxyethyl acrylate, 
2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, 2-hydroxypropyl 
acrylate, etc. A monomer having a haloethylsulfonyl group may be 
exemplified by chloroethylsulfonylethyl methacrylate, 
bromoethylsulfonylethyl methacrylate, etc. A typical example of a monomer 
having a vinylsulfonyl group is vinylsulfonylethyl methacrylate. Examples 
of a monomer having an active methylene containing group are acryloyl 
acetone and methacryloyl acetone. As a monomer having a 
carboxymethoxymethyl group, there may be mentioned, for example, 
N-carboxymethoxymethyl-acrylamide and 
N-carboxymethoxymethyl-methacrylamide. 
Examples of other preferable monomers to be copolymerized with the monomers 
having reactive groups as described above are set forth below. 
##STR1## 
wherein each of R.sup.1 and R.sup.2, which can be the same or different, 
represents a non-interfering substituent such as a hydrogen atom, a 
halogen atom, or a substituted or unsubstituted, amino-free alkyl or aryl 
group having 1 to 10 carbon atoms and R.sup.3 represents a hydrogen atom, 
a halogen atom, or a substituted or unsubstituted, amino-free aliphatic or 
aromatic group having 1 to 10 carbon atoms. As aliphatic or aromatic 
groups, there may be included alkyl groups, alkoxy groups, aryl groups and 
aryloxy groups. Typical examples of the monomers represented by the 
formula (I) are styrene, vinyltoluene, vinylbenzyl chloride, 
t-butylstyrene, etc. 
(II) 
EQU CHR.sup.6 .dbd.CR.sup.4 --COOR.sup.5 
wherein R.sup.6 has the same meaning as R.sup.1 in the formula (I), R.sup.4 
represents a hydrogen atom or a methyl and R.sup.5 represents an aryl, an 
alkyl, an alkaryl or aralkyl group, each having 1 to 10 carbon atoms. 
(III) Polymerizable unsaturated nitrile monomers such as acrylonitrile and 
methacrylonitrile. 
(IV) Interparticle crosslinking monomers having two addition-polymerizable 
groups such as divinylbenzene, N,N-methylene-bis(acrylamide), ethylene 
diacrylate and ethylene dimethacrylate. 
By copolymerization of a suitable combination of these monomers with the 
aforesaid monomers having reactive groups, it is possible to constitute 
the polymer particle units according to the present invention. The 
monomers of formulae (I), (II) and (III) may be contained in amounts of 0 
to 99.5% by weight based on said organic macromolecular polymer units; and 
the monomer represented by formula (IV) may be contained in an amount of 0 
to 10% by weight, preferably 0 to 5% by weight based on said organic 
macromolecular polymer unit.

Among the organic macromolecular polymers according to the present 
invention, typical examples of those having one kind of reactive group are 
shown below, by which the present invention is not limited. The numerals 
in the brackets affixed to each exemplary compound indicate the weight 
percents of monomers employed in polymerization. 
EXEMPLARY COMPOUNDS 
(1-1) Poly(styrene-co-glycidyl methacrylate) [90/10] 
(1-2) Poly(styrene-co-methyl acrylate-co-glycidyl methacrylate) [80/15/5] 
(1-3) Poly(styrene-co-n-butyl methacrylate-co-glycidiyl methacrylate) 
[75/15/10] 
(1-4) Poly(styrene-co-vinylbenzyl chloride-co-glycidyl methacrylate) 
[80/10/10] 
(1-5) Poly(styrene-co-divinylbenzene-co-glycidyl acylate) [90/2/8] 
(1-6) Poly(p-vinyltoluene-co-glycidyl methacrylate) [90/10] 
(1-7) Poly(methylmethacrylate-co-glycidyl methacrylate) [80/20] 
(1-8) Poly(styrene-co-N,N-dimethylaminoethyl methacrylate) [95/5] 
(1-9) Poly(styrene-co-aziridylethyl methacrylate) [95/5] 
(1-10) Poly(styrene-co-methyl acrylate-co-acrolein) [90/5/5] 
(1-11) Poly(styrene-co-acrylamide) [95/5] 
(1-12) Poly(styrene-co-vinylthiol) [95/5] 
(1-13) Poly(styrene-co-methylolacrylamide) [95/5] 
(1-14) Poly(styrene-co-t-butylacrylate-co-glycidyl methacrylate) [90/5/5] 
(1-15) Poly(styrene-co-vinylisocyanate) [95/5] 
(1-16) Poly(methylacrylate-co-styrene-co-N-methylolacrylamide) [50/35/15] 
(1-17) Poly(styrene-co-N,N-dimethylaminoethyl methacrylate) [90/10] 
(1-18) Poly(styrene-co-acrylic acid) [97/3] 
(1-19) Poly(styrene-co-acrylamide) [97/3] 
(1-20) Poly(p-vinyltoluene-co-tert-butyl acrylate) [95/5] 
(1-21) Poly(methyl acrylate-co-methacrylamide) [95/5] 
(1-22) Poly(styrene-co-N-methylol acrylamide) [95/5] 
(1-23) Poly(p-vinylbenzylchloride-co-N-methylol acrylamide) [96/4] 
(1-24) Poly(styrene-co-itaconic acid) [98/2] 
(1-25) Poly(styrene-co-tert-butyl acrylate) [92/8] 
(1-26) Poly(methyl acrylate-co-styrene-co-acrolein) [30/65/5] 
(1-27) Poly(methyl methacrylate-co-styrene-co-2-hydroxyethyl methacrylate) 
[25/70/5] 
(1-28) Poly(styrene-co-vinylsulfonylethyl acrylate) [80/20] 
(1-29) Poly(styrene-co-N,N-diethylaminomethyl acrylate) [97.5/2.5] 
(1-30) Poly(styrene-co-methyl acrylate-co-acetoacetoxyethyl acrylate) 
[90/5/5] 
(1-31) Poly(styrene-co-methacrylic acid) [95/5] 
Further, as another embodiment of the present invention, examples of 
organic macromolecular polymers containing two or more kinds of monomer 
units having different reactive groups are shown below, by which the 
present invention is not limited. 
EXEMPLARY COMPOUNDS 
(2-1) Poly(styrene-co-glycidyl methacrylate-co-N,N-dimethylaminoethyl 
methacrylate) [90/5/5] 
(2-2) Poly(styrene-co-methacrylic acid-co-acrylamide) [95/2/3] 
(2-3) Poly(styrene-co-N-methylol acrylamide-co-methoxyethyl acrylate) 
[90/5/5] 
(2-4) Poly(p-vinyltoluene-co-N-methylol acrylamide-co-acrylic acid) 
[90/8/2] 
(2-5) Poly(methyl methacrylate-co-glycidyl methacrylate-co-t-butyl 
acrylate) [80/10/10] 
(2-6) Poly(styrene-co-p-vinylbenzyl chloride-co-acrylic acid-co-ethyl 
acrylate) [75/10/5/10] 
(2-7) Poly(styrene-co-methacrolein-co-2-hydroxyethyl methacrylate) [90/5/5] 
(2-8) Poly(styrene-co-acrolein-co-acetoacetoxyethyl methacrylate) [85/5/10] 
(2-9) Poly(styrene-co-N,N-dimethylaminoethyl acrylate-co-vinylsulfonylethyl 
methacrylate) [90/5/5] 
(2-10) Poly(p-vinyltoluene-co-aminostyrene-co-vinylsulfonylethyl 
methacrylate) [85/10/5] 
The above organic macromolecular polymer containing these reactive groups 
can be produced by various conventional polymerization methods. 
Typical addition polymerization methods may include solution polymerization 
(appropriate precipitation operation and, in some cases, crushing and 
particle classification operations are used for formation of said 
heat-stable particle units), suspension polymerization (sometimes referred 
to as pearl polymerization), emulsion polymerization, dispersion 
polymerization and precipitation polymerization. Preferably, suspension 
polymerization and emulsion polymerization are employed. 
In the following, Synthetic examples of the exemplary compounds of the 
present invention are shown, but the present invention is not limited 
thereby. 
SYNTHESIS EXAMPLE 1 
Synthesis of the exemplary compound (1-1) 
A mixture of monomers and a polymerization initiator comprising 90 parts of 
styrene, 10 parts of glycidyl methacrylate, and 3 parts of 
2,2'-azobis(2,4-dimethylvaleronitrile) was added into 700 ml of an aqueous 
solution of 3% by weight of tricalcium phosphate and 0.04% by weight of 
sodium dodecylbenzene sulfonate based on the above monomers, while 
stirring the mixture at a stirring speed of 5000 r.p.m. by means of a 
TK-homojetter (produced by Tokushu Kika Kogyo). After completion of the 
addition, the mixture was further continued to be stirred for about 30 
minutes until the particle size became about 20 microns as observed by a 
microscope, whereupon the mixture was transferred into a four-necked flask 
equipped with a conventional stirrer (anchor type), a cooling tube, a 
nitrogen gas inlet tube and a thermometer. The stirring speed was changed 
to 200 r.p.m., and polymerization was carried out at 60.degree. C. under a 
nitrogen gas stream for 8 hours to complete the reaction. Then, the 
contents were cooled to room temperature and tricalcium phosphate was 
removed by decomposition with a dilute aqueous hydrochloric acid solution. 
The residual mixture was washed repeatedly with water and thereafter the 
polymer was separated by filtration, followed by drying, to obtain the 
polymer of the exemplary compound (1-1). 
SYNTHESIS EXAMPLE 2 
Synthesis of the exemplary compound (1-1) 
Into a 1000 ml four-necked flask equipped with a thermometer, a stirring 
means, a cooling tube and a nitrogen inlet tube, there were changed 500 ml 
of degassed distilled water, 5 ml of Trax H-45 (trade name, surfactant, 
produced by Nippon Oil and Fat Co., Ltd., effective ingredient 30%), 135 g 
of styrene and 15 g of glycidyl methacrylate, and the mixture was stirred 
at a stirring speed of 50 r.p.m. while under the flows of nitrogen and 
cooling water. Then, the inner temperature in the flask was elevated to 
60.degree. C., and aqueous solutions of 1.3 g of potassium persulfate and 
0.865 g of sodium metabisulfite each dissolved in 20 ml of degassed water 
were added at the same time. The reaction was carried out for 6 hours 
while maintaining the stirring speed at 250 r.p.m. and the inner 
temperature in the flask at 60.degree. C. Then, the reaction mixture was 
cooled to room temperature and the reaction product filtered to give a 
latex of the exemplary compound (1-1) having a viscosity of 5.5 cps 
(measured by B-type viscometer) with a solid content of 20%. 
SYNTHESIS EXAMPLE 3 
Synthesis of the exemplary compound (1-3) 
A mixture of monomers and a polymerization initiator comprising 75 parts of 
styrene, 15 parts of n-butyl methacrylate, 10 parts of glycidyl 
methacrylate and 3 parts of 2,2'-azobis(2,4-dimethylvaleronitrile) was 
added to 700 ml of an aqueous solution of 2% by weight of tricalcium 
phosphate and 0.02% by weight of sodium dodecylbenzene sulfonate based on 
the above monomers, while stirring the mixture at a stirring speed of 2000 
r.p.m. by means of a TK-homojetter. After completion of the addition, the 
mixture was further continued to be stirred for 30 minutes until the 
particle sizes of the droplets of the monomer mixture became about 100 
microns, whereupon the reaction and the procedures as described in 
Synthetic example 1 were repeated, to provide the polymer of the exemplary 
compound (1-3). 
The above organic macromolecular polymer containing the reactive groups 
according to the present invention have a glass transition temperature 
(hereinafer abbreviated as T.sub.g), typically of 30.degree. C. or higher, 
preferably a T.sub.g of 40.degree. C. or higher. The term "T.sub.g " 
mentioned in the present specification means a temperature at which the 
polymer undergoes change in state from a glassy state to a rubbery state, 
and may be contemplated as an index for heat stability of the polymer. The 
T.sub.g of a polymer can be measured according to the method as described 
in, for example, "Techniques and Methods of Polymer Evaluation" Vol. 1, 
Marcel Dekker, Inc., N.Y. (1966). 
Various methods can be employed in applying said organic macromolecular 
polymer in the above dispersion for forming the fibrous developing layer 
of the present invention. For example, said organic macromolecular polymer 
may be dissolved in the above dispersion, or alternatively said organic 
macromolecular polymer may preferably be dispersed in a liquid carrier 
which does not dissolve said polymer (e.g. in the form of fine powders or 
a latex). 
The above organic macromolecular polymer may be used in an amount which can 
widely be selected, but preferably in an amount such that a substantial 
portion of the interstitial volume formed by three-dimensional 
intertwining of fibers may not be filled therewith, namely 50% by weight 
to 0.005% by weight, preferably 30% by weight to 0.05% by weight based on 
the weight of fibers. 
Further, either one kind or two or more kinds of the above organic 
macromolecular polymer may be employed and it is also useful to use a 
hydrophilic collodial substance as described above in combination. 
The fibrous developing layer of the present invention can be prepared by 
coating according various methods. As an example, the following steps may 
be mentioned. That is, the fibers of the present invention are dispersed 
in a liquid carrier which does not dissolve said fibers, then the organic 
macromolecular polymer having reactive groups as described above is added 
thereto to prepare a dispersion of said fibers, and after application of 
the stable dispersion onto a support, the liquid carrier is removed while 
forming said fibrous structure. 
A dispersion useful for preparation of the fibrous developing layer is 
required to be stable during a time sufficient to apply the dispersion 
onto a support. 
To form such stable dispersions, a wide variety of techniques can be used 
individually or in combination. One useful technique comprises the 
addition of a surfactant to the liquid carrier to facilitate distribution 
and stabilization of the fibers in the dispersion. 
Representative surfactants which can be employed include Triton.RTM.X-100 
(octylphenoxy polyethoxyethanol from Rohm and Hass) and Surfactant 
10G.RTM. (nonylphenoxy polyglycidol from Olin Corp.). 
The above surfactant may be employed in an amount which can be selected 
from a wide range, but generally 30% by weight to 0.005% by weight, 
preferably 20% by weight to 0.05% by weight, based on the weight of the 
fibrous material. As alternative techniques, there are procedures such as 
sonication treatments, physical blending and agitation treatments and pH 
adjustments. These techniques can be more useful when combined with the 
technique as described above. 
As the liquid carrier in the aforesaid dispersion, there may be employed an 
aqueous liquid. But other liquid carriers such as organic liquids may also 
be available, provided that said fibers are insoluble in the carrier so 
that their fibrous structural character can be retained. 
Representative liquid carriers other than water may include water-miscible 
organic solvents, mixtures of water with water-miscible organic solvents 
and appropriate water-immiscible organic solvents. Examples of 
water-miscible organic solvents are lower alcohols (namely, alcohols with 
alkyl groups having 1 to 4 carbon atoms), acetone and tetrahydrofuran. As 
the water-immiscible solvents, there may be mentioned lower alkyl esters 
such as ethyl acetate, halogenated organic solvents such as halogenated 
hydrocarbons (e.g., chloroform, methylene chloride and carbon 
tetrachloride), and hydrocarbon solvents such as aromatic hydrocarbons 
(e.g. benzene, toluene and xylene) and aliphatic hydrocarbons (e.g. 
hexane, decalin, etc.). 
The analytical elements according to the present invention containing the 
aforesaid fibrous developing layer can have any one of a variety of 
different configurations. It may have one or more of the fibrous 
developing layers of the present invention, or alternatively a suitable 
combination of the fibrous developing layer with any of a variety of 
functional layer, reagent containing layers and members, as exemplified by 
the reagent layer, the filtration layer, the reflection layer and the 
subbing layer as disclosed in U.S. Pat. No. 3,992,158, the 
radiation-blocking layer as disclosed in U.S. Pat. No. 4,042,335, the 
barrier layer as disclosed in U.S. Pat. No. 4,066,403, the registration 
layer as disclosed in U.S. Pat. No. 4,144,306, the migration-inhibition 
layer as disclosed in U.S. Pat. No. 4,166,093, the scintillation layer as 
disclosed in U.S. Pat. No. 4,127,499, the scavenger layer as disclosed in 
Japanese Provisional Patent Publication No. 90859/1980 and the destructive 
pad-shaped member as disclosed in U.S. Pat. No. 4,110,079, to constitute 
the analytical element adapted to accommodate the object of the present 
invention. 
Further, the analytical element having the fibrous developing layer 
according to the present invention can be subjected to the so called 
"calendering treatment" by passing through a pair of pressure rollers 
thereby to increase the flatness of the surface of said developing layer 
and obtain more favorable effect in optical reflection. 
The analytical element of the present invention having the constitution as 
described above can accomplish its object by supplying a liquid sample 
through the fibrous developing layer and observing the analytical reaction 
in the reagent layer from the side of the transparent support. 
The amount of a liquid sample to be applied to the analytical element of 
the present invention can be determined as desired, but preferably in the 
range of from about 50 .mu.l to about 5 .mu.l, more preferably from about 
20 .mu.l to about 5 .mu.l. Usually, about 10 .mu.l of a liquid sample is 
preferably used. 
The analytical reaction to be used for the analytical element of the 
present invention may optionally be determined depending on the intended 
purpose. For example, it may be employed for analysis of a biological 
liquid sample, namely for analysis of components in blood or urine. 
These can easily be constituted by suitable selection of the analytical 
reagents so as to be available for analysis of, for example, glucose, urea 
nitrogen, ammonia, uric acid, cholesterol triglyceride, creatine, 
creatinine, bilirubin as well as many other components. 
The analytical element having the fibrous developing layer of this 
invention can be coated by the dip coating method, the air knife method, 
the curtain coating method or the extrusion coating method with the use of 
a hopper as disclosed in U.S. Pat. No. 2,681,294. If desired, it is also 
possible to use the method as disclosed in U.S. Pat. No. 2,761,791 and 
U.K. Pat. No. 837,095 for simultaneous coating of two or more layers. 
Elements of the present invention can be adapted for use not only in the 
field of clinical chemistry, but in other fields of chemical analysis. In 
addition, by utilizing the function of holding a certain amount of liquid 
within a certain area of the film, the element of the present invention 
can be associated with other functional layers (e.g., layers of 
photographic elements). 
Analytical elements of the present invention are very advantageous for use 
in clinical testing of body fluids, such as blood, blood serum, lymph and 
urine. In particular, blood serum is conventionally used in analysis of 
blood. But the analytical element can be conveniently applicable for 
analysis of any of whole blood, blood serum and blood plasma. 
When whole blood is used, a radiation-blocking layer or other reflecting 
layer may be provided, if necessary, in order to avoid interference of 
detecting radiation by the blood cells. Of course, if it is desired to 
observe directly the color of blood cells directly, such as in a 
haemoglobin analysis, no such reflecting layer is necessary. 
After the analytical result is obtained as a detectable change, by use of 
the analytical element of the present invention, it is measured by 
reflection spectrophotometry, transmission spectrophotometry, fluorescence 
spectrophotometry or scintillation counting, corresponding the various 
detectable changes. The thus obtained values of measurement can be 
utilized for determination of the unknown quantity of analyte with 
reference to the calibration curve previously prepared. 
Also, by utilization of the reactive groups possessed by the organic 
macromolecular polymer contained in the fibrous developing layer of the 
present invention, application of the present analytical element for 
immunoassay is possible. 
The analytical element of the present invention, having the constitution as 
described in detail above, is substantially free from generation of 
irregular concentrations of reagents or chromatographic phenomenon, and 
therefore it can be used for quantitative analysis of a liquid sample, 
especially components in a biological liquid sample, easily and rapidly by 
means of a conventional spectrophotometer. 
Further, formation of a fibrous developing layer in the analytical element 
of the present invention can be effected by simple coating and drying 
thereof, under the conditions of coating and drying which are not 
specifically limited, to a practical advantage of very easy preparation. 
The present invention is illustrated in further detail by referring to the 
following Examples, by which the present invention is not limited at all. 
EXAMPLE 1 
On a transparent poly(ethyleneterephthalate) support of a film thickness of 
about 180 microns, there was coated 216 mg/dm.sup.2 of deionized gelatin 
to a thickness of about 20 microns of dried film. On the above gelatin 
layer, there were further coated the fibrous dispersions having the 
compositions as shown in Table 1 to form Samples 1, 2, 3, 4, 5 and 6, 
respectively. 
TABLE 1 
__________________________________________________________________________ 
Fibrous dispersion 
Sample No. 
Dispersion No. 
Fiber 
Liquid carrier 
Organic polymer 
Surfactant 
__________________________________________________________________________ 
Sample-1 
Dispersion-1 
F-1, 5 g 
Xylene, 14 ml 
Exemplary com- 
G 
pound (1-1) *(1) 
0.5 g 
0.75 g 
Sample-2 
Dispersion-2 
F-1, 5g 
Toluene, 15 ml 
Exemplary com- 
H 
pound (1-12) *(1) 
0.2 g 
0.5 g 
Sample-3 
Dispersion-3 
F-1, 5 g 
Acetone, 14 ml 
Exemplary com- 
G 
pound (2-1) *(1) 
0.3 g 
0.25 g 
Sample-4 
Dispersion-4 
F-2, 5 g 
Xylene, 19 ml 
Exemplary com- 
-- 
pound (1-10) *(1) 
0.5 g 
Sample-5 
Dispersion-5 
F-1, 5 g 
Water *(2), 20 ml 
Exemplary com- 
G 
pound (1-1) *(2) 
0.4 g 
0.3 g 
Sample-6 
Dispersion-6 
F-1, 5 g 
Water *(2), 20 ml 
Exemplary com- 
G 
pound (2-1) *(2) 
0.4 g 
0.5 g 
__________________________________________________________________________ 
In the above Table: 
F1 is filter paper powder C (produced by Toyo Roshi); 
F2 is filter paper powder A (produced by Toyo Roshi); 
G is octylphenoxypolyethoxyethanol; 
H is nonylphenoxyglycidol. 
*(1) used as a solution 
*(2) used as a 15% latex/water dispersion 
On the other hand, as a comparative sample, a filter paper (No. 7 produced 
by Toyo Roshi Co.) was directly adhered on a transparent 
poly(ethyleneterephthalate) support with a thickness of 180 .mu.m to 
provide Comparative sample (I), and the same filter paper after 
impregnated with 5% aqueous gelatin solution followed by drying was 
adhered directly on the same support to provide Comparative sample (II). 
On these Samples 1, 2, 3, 4, 5, 6 and Comparative samples (I) and (II), 10 
.mu.l of an aqueous solution of a red dye Brilliant Scarlet 3R was added 
dropwise, and 7 minutes later, spot diameters were measured from the side 
of the support. 
Also, reflection density (by Sakura Photoelectric Densitometer PAD-60 
Model, produced by Konishiroku Photo Industry Co., Ltd.) was measured from 
the side of the support using a green (.lambda..sub.max =546 nm) filter to 
determine the difference .DELTA.D between the maximum density and the 
minimum density within the spot. Measurements were conducted ten times for 
each Sample and each Comparative sample to determine an average value, 
maximum and minimum values of spot diameter and also the maximum .DELTA.D 
within the ten measurements. 
The results are given in the following Table 2. 
TABLE 2 
______________________________________ 
Spot diameter (mm) 
Maximum Minimum Average .DELTA.D 
______________________________________ 
Comparative 
16.1 10.3 13.3 0.21 
sample (I) 
Comparative 
16.4 9.1 12.7 0.24 
sample (II) 
Sample of the 
10.5 10.0 10.1 0.02 
invention (1) 
Sample of the 
10.1 10.0 10.0 0.01 
invention (2) 
Sample of the 
11.1 10.5 11.1 0.04 
invention (3) 
Sample of the 
10.1 10.0 10.1 0.02 
invention (4) 
Sample of the 
10.2 10.1 10.2 0.03 
invention (5) 
Sample of the 
10.1 10.0 10.1 0.02 
invention (6) 
______________________________________ 
As shown in the above Table 2, the Samples of the present invention vary 
little in spot diameter and the density distribution in the coloring 
region is very small. 
On the other hand, in Comparative samples, spot diameters are greatly 
varied. In addition, the density is also greatly varied within the 
coloring region. 
EXAMPLE 2 
On a transparent poly(ethyleneterephthalate) support having a thickness of 
180 microns which had been subjected to undercoating, a reagent layer for 
assay of glucose comprising the following composition was coated: 
A reagent layer for assay of glucose, containing the following components, 
adjusted with 5% aqueous sodium hydroxide solution to pH 7.0: 
______________________________________ 
Glucose oxidase 240 U/dm.sup.2 
4-Aminoantipyrine hydrochloride 
0.0086 g/dm.sup.2 
1,7-Dihydroxynaphthalene 
0.0065 g/dm.sup.2 
Peroxidase 180 U/dm.sup.2 
5,5-Dimethyl-1,3-cyclohexadione 
0.0022 g/dm.sup.2 
6-Amino-4,5-dihydroxy-2-methyl 
0.0002 g/dm.sup.2 
pyrimidine 
3,3-Dimethylglutaric acid 
0.0196 g/dm.sup.2 
Deionized gelatin 0.196 g/dm.sup.2 
______________________________________ 
On the above reagent layer, the dispersion-I of Example 1 was coated and 
dried to prepare Sample-7, and similarly the dispersion-6 of Example 1 
coated and dried to prepare Sample-8. On these analytical elements, there 
were added dropwise aqueous solutions of various concentrations of 100 
mg/dl, 150 mg/dl, 200 mg/dl, 250 mg/dl, 300 mg/dl, 350 mg/dl and 
artificial serum containing equal amounts of glucose. After incubation at 
37.degree. C. for 7 minutes, the densities of reddish brown dye formed 
were measured at wavelength of 490 nm, whereby it was found that the 
glucose density was proportional to the reflection density.