Reduction of detectable species migration in elements for the analysis of liquids

An element for the analysis of liquids contains a radiation-transmissive, detectable species migration-inhibiting layer interposed between a porous radiation-blocking layer and a radiation-transmissive reagent layer. All three layers are permeable to a predetermined analyte. The reagent layer contains a composition that provides a detectable species such as a dye in proportion to the concentration of the analyte that diffuses into the reagent layer from the overlying porous radiation-blocking layer. The detectable species migration-inhibiting layer acts to reduce the migration of, for example, dye from the reagent layer into the porous radiation-blocking layer, where the optical density of the dye cannot easily be measured. Optionally, the above-described three layers can be carried on a radiation-transmissive support, and other layers such as spreading layers, registration layers, and subbing layers can also be present in the element.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to an improved element for the chemical 
analysis of liquids. More particularly it concerns multilayer elements 
that provide a means for determining the presence and/or concentration of 
a substance in a liquid by effecting the release or formation of a 
detectable species, such as a dye, within the element in quantities 
proportionate to the concentration of the substance in the liquid being 
analyzed. The present invention provides a means for inhibiting the 
migration or wandering of a substantial portion of this detectable species 
to areas or layers of the element in which the presence of the detectable 
species cannot easily be determined, either quantitatively or 
qualitatively. 
2. Description of Related Art 
It is often desirable or necessary to determine the presence and/or 
concentration of certain substances in liquids such as water, foodstuffs, 
and biological liquids. A variety of devices and methods have been 
employed for such analyses. 
Various inexpensive elements have been devised to facilitate rapid and 
convenient analyses under other than controlled laboratory conditions. 
Such elements often include a reagent for the analyte (the term analyte 
referring to the substance being analyzed for in the liquid sample). This 
reagent, upon contact with the analyte, causes the formation of a dye or 
brings about some other detectable change to indicate the presence of 
analyte in the liquid sample. One example of such an element is a pH test 
strip that comprises a paper or other absorbent material impregnated with 
an appropriate reagent or reagents. Simple elements of this type are most 
often employed when it is only necessary to make a quick visual 
determination of the presence of the analyte qualitatively or at best 
semi-quantitatively. 
More sophisticated elements are available for quantitative diagnostic 
analyses of biological liquids like blood or urine. When a liquid sample 
containing the analyte is brought into contact with these elements, they 
form the dye or other detectable change consistently and uniformly within 
the element in proportion to the concentration of the analyte in the 
liquid sample. Analyte concentration can then be determined, for example, 
by spectrophotometric measurement of the optical density of the dye formed 
in the element. 
Elements of this type are described in U.S. Pat. No. 3,992,158, issued Nov. 
16, 1976. These elements can consist of two or more desirably discrete 
layers that are superposed and in substantially continuous intimate 
contact with adjacent layers. One such multilayer element comprises a 
support layer having a reagent layer and an outermost spreading layer 
coated upon it. In this multilayer element, the spreading layer serves as 
a liquid sample permeable receiving and metering layer. That is, the 
liquid sample to be analyzed is placed on the spreading layer, which 
absorbs and transfers the liquid to the reagent layer. Preferably, as 
described in U.S. Pat. No. 3,992,158, the spreading layer is isotropically 
porous and transfers a uniform concentration (as measured across a per 
unit cross-sectional area of the spreading layer) of the analyte contained 
in the liquid sample to the underlying reagent layer. The reagent layer 
has certain reagents uniformly distributed therein. A detectable species 
such as a dye is formed within the reagent layer in an amount proportional 
to the concentration of analyte in the liquid. Typically, the reagent and 
support layers are radiation-transmissive so that a spectrophotometric 
measurement of the optical density of the dye formed in the reagent layer 
can be made with the element remaining intact. Additionally, the spreading 
layer may comprise a blushed polymer and a pigment to provide both uniform 
transfer of the liquid sample to the reagent layer and an opaque, 
reflective surface above the reagent layer to aid in a measurement of 
reflection density of the dye. With this element, however, some of the dye 
formed in the reagent layer may migrate or wander into the opaque 
spreading layer where it would not be detected during the dye-density 
measurement, thereby reducing the sensitivity and the accuracy of the 
analysis. 
Related elements are described in U.S. Pat. No. 4,042,335, of Clement, 
issued Aug. 16, 1977. A registration layer and an opaque or 
radiation-blocking layer are coated between the support layer and the 
reagent layer. During the analysis, a significant portion of the 
detectable species, e.g., a dye, formed in the reagent layer will diffuse 
through the radiation-blocking layer and into the registration layer, 
where the dye density will be measured. A mordant for the dye can be 
included in the registration layer to insure that the dye that has 
diffused into this layer will be fixed there for easy detection and will 
not be allowed to diffuse or migrate out of the registration layer. 
Elements such as this are suggested for use where it would not otherwise 
be practical to reliably measure the dye density within the reagent layer 
itself, for example, in analytical elements where other reagents and 
reaction products within the reagent layer also provide density, thus 
preventing any accurate spectrophotometric measurement of the optical 
density in this layer of only the dye. Such an element can provide a 
reliable analysis. However, it is obvious that a significant portion of 
the dye formed during the analysis can remain in the reagent layer or 
migrate into and remain in the radiation-blocking layer. The sensitivity 
and accuracy of the analytical element are thereby reduced, because the 
analyte-concentration determination must depend upon the measurement of 
the density of a smaller amount of dye than that which was actually 
formed. 
Other elements as described in U.S. Pat. No. 3,585,112 and U.S. Pat. No. 
3,917,453 disclose means for overcoming these problems. Both of these 
patents suggest the use of mordants in the reaction zone or layer to 
provide a degree of immobility to the indicator dye formed. These 
elements, like others of the prior art, however, are susceptible to the 
additional problem of the mordant interfering with the formation of the 
dye or interfering with any prerequisite reactions leading to the 
formation of the dye. Such interference can make the analysis completely 
unreliable. 
Accordingly, it is desirable to provide an analytical element that has all 
of the advantages of the elements described above, i.e., ease of use, low 
cost and quantitative results; and that also overcomes the problems 
inherent in prior art elements, such as reduced sensitivity and accuracy 
of results caused by (a) migration of detectable species into porous 
radiation-blocking layers and (b) interference with the formation or 
release of the detectable species by mordants used to inhibit such 
migrations. 
SUMMARY OF THE INVENTION 
The elements of the present invention have unexpectedly overcome the 
problems of prior art analytical elements, namely by providing for 
quantitative analyses which are highly accurate and sensitive. The present 
elements do so by inhibiting migration of the detectable species from the 
reagent layer to layers of the element where such species could not easily 
be measured, and by providing a means for avoiding interference with the 
reaction or reactions that result in detectable species formation in or 
release from the reagent layer. Elements according to this invention can 
be used for diagnostic purposes and include: a radiation-transmissive 
reagent layer, permeable to, and containing a composition interactive 
with, a predetermined analyte (or reaction product thereof) to provide a 
radiometrically detectable species; a porous radiation-blocking layer 
permeable to the analyte; and the improvement of having a 
radiation-transmissive, detectable species migration-inhibiting layer, 
permeable to the analyte and interposed between the reagent layer and the 
radiation-blocking layer. This layer prevents a substantial amount of the 
detectable species which may diffuse out of the reagent layer from 
entering the porous radiation-blocking layer where it is not practically 
measurable, by fixing such migrating detectable species within the 
detectable species migration-inhibiting layer, where it is easily 
detectable. Another advantage of the present invention is that the 
detectable species migration-inhibiting layer is separate from the reagent 
layer, so that it does not interfere with the analytical interaction(s) 
taking place in the reagent layer. 
Optionally, analytical elements of the present invention can be carried on 
a radiation-transmissive support, and other layers such as spreading 
layers, registration layers, and subbing layers can also be present in the 
element. Also, the porous radiation-blocking layer can itself function as 
a spreading layer in some embodiments of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The analytical elements of this invention are multi-layered, consisting of 
three or more desirably discrete layers that are superposed and in fluid 
contact with each other under conditions of use. These layers include a 
reagent layer, a porous radiation-blocking layer, and a detectable species 
migration-inhibiting layer. In certain embodiments of the invention the 
porous radiation-blocking layer can function also as a spreading layer, or 
there can be a separate spreading layer in addition to the porous 
radiation-blocking layer. In other embodiments the element can include a 
radiation-transmissive support layer in addition to the three layers 
described above. In still other embodiments additional 
radiation-transmissive layers, e.g., subbing layers or registration 
layers, can also be included in the analytical element. 
In the present invention the layers are always arranged such that the 
detectable species migration-inhibiting layer is interposed between the 
porous radiation-blocking layer and the reagent layer. In those 
embodiments containing an additional layer to function as a spreading 
layer, the porous radiation-blocking layer is interposed between the 
spreading layer and the detectable species migration-inhibiting layer. In 
those embodiments containing a radiation-transmissive support layer, the 
reagent layer is interposed between the detectable species 
migration-inhibiting layer and the radiation-transmissive support layer. 
In those embodiments containing additional radiation-transmissive layers, 
such as subbing or registration layers, the additional subbing layers or 
registration layers are interposed between the reagent layer and the 
optional radiation-transmissive support layer. 
U.S. Pat. No. 3,992,158 and Clement, U.S. Pat. No. 4,042,335 issued Aug. 
16, 1977, both incorporated herein by reference, disclose reagent layers, 
porous radiation-blocking layers, support layers, subbing layers, 
registration layers, and preferred types of isotropically porous spreading 
layers, that are useful in the practice of the present invention. These 
materials also describe well known methods of preparing these layers to 
form individual multilayer elements and describe the use of such elements 
for various quantitative analyses. 
As used herein, the term, porous radiation-blocking layer, defines a layer 
that is permeable to a predetermined analyte (or reaction product thereof) 
dissolved or dispersed in a liquid, and that reflects, or optionally 
absorbs, detecting radiation, i.e., radiation used together with the 
elements of the invention to facilitate result detection of the particular 
detectable species which is provided by the reagent layer. In other words, 
the porous radiation-blocking layer will allow the predetermined analyte 
to pass through it, and it is used together with suitable detecting 
radiation to facilitate result detection in the analytical elements of the 
invention such as by reflection photometry. Because of the 
radiation-blocking properties of the porous radiation-blocking layer, the 
radiative properties, i.e., the particular emissive, transmissive, or 
absorptive properties, of any of the detectable species which migrates 
into this layer can be substantially masked or hidden. Therefore, 
detecting radiation used to determine the presence or absence of 
detectable species formed in the reagent layer may be unable to accurately 
detect that portion of the detectable species which, although provided in 
response to a given analyte, has migrated into the porous 
radiation-blocking layer. 
As noted above, the analytical elements of the present invention can 
optionally contain a separate spreading layer in addition to the porous 
radiation-blocking layer, or the porous radiation-blocking layer itself 
can also function as a spreading layer. Like the porous radiation-blocking 
layer, a spreading layer must be permeable to a predetermined analyte 
dissolved or dispersed in a liquid. When liquid containing the analyte is 
brought into contact with the outermost surface of a spreading layer, the 
spreading layer distributes the liquid within itself such that the 
concentration of the analyte provided at the surface of the spreading 
layer that faces the reagent layer of the element is regulated or 
controlled. Preferably, but not necessarily, the spreading layer is 
isotropically porous and delivers a uniform concentration of analyte to 
the reagent layer. In one embodiment of the present invention a separate 
spreading layer may be included in addition to the porous 
radiation-blocking layer, as noted above, and in such case the spreading 
layer may be either radiation-transmissive or radiation-blocking. 
Radiation-transmissive, as used herein, defines the ability to transmit 
detecting radiation used to determine the presence, optionally the 
absence, of the detectable species provided by the reagent layer. If 
desired, one or more interactive or reagent compositions may be 
incorporated in the spreading layer or separate porous radiation-blocking 
layer to interact with the analyte of choice, thereby forming an analyte 
reaction product which can undergo further interaction in the underlying 
reagent layer as described hereinafter. 
In one preferred embodiment of the present invention the porous 
radiation-blocking layer itself functions as an adequate spreading layer 
and comprises a blushed polymer and optionally a finely-divided 
particulate material such as a pigment. Layers of this type are discussed 
in detail in U.S. Pat. No. 3,992,158 and U.S. Pat. No. 4,042,335. Useful 
blushed polymers include cellulose acetate, amides, and the like. Useful 
particulate materials include pigments such as carbon, titanium dioxide, 
barium sulfate, and the like. 
Reagent layers in the elements of this invention are 
radiation-transmissive, that is, they will transmit light in the range of 
the spectrum used to determine the presence and/or concentration of the 
detectable species provided by the reagent layer. Preferably, the reagent 
layer is uniformly permeable to the particular analyte to be measured. 
Within the reagent layer is distributed a material that can interact with 
the analyte or reaction product of the analyte. Such interaction causes 
the release of a preformed detectable species or the formation of such a 
detectable species within the reagent layer, preferably, in proportion to 
the concentration of the analyte in the liquid sample being analyzed. Such 
interaction is meant to refer to chemical activity, catalytic activity as 
in the formation of an enzyme-substrate complex, and any other form of 
chemical or physical interaction that can release, produce, or otherwise 
provide within the reagent layer a species that is radiometrically 
detectable, that is, by suitable measurement of light or other energy. 
Typically, the detectable species formed or released from the reagent 
layer is a dye which is radiometrically detectable by fluorometric or 
colorimetric, preferably colorimetric techniques. 
In addition, if necessary or desirable, appropriate buffer compositions may 
also be present in the reagent layer. Reagent layers of the present 
invention may also contain one or more hydrophilic colloids including 
natural colloids such as gelatin, agarose, polysaccharides, and the like; 
and/or synthetic resins such as poly(vinyl alcohol), poly(vinyl 
pyrrolidone), polyacrylamides, and the like. 
One application of the present invention comprises an element for the 
analysis of glucose in liquids wherein the interactive material in the 
reagent layer preferably comprises glucose oxidase, peroxidase, and an 
indicator composition. A useful indicator composition comprises 
4-aminoantipyrene hydrochloride and 7-hydroxy-1-naphthol. In the presence 
of glucose, the above interactive material effects the formation of a dye 
in proportion to the concentration of glucose in the sample being 
analyzed. This concentration can then be determined by 
spectrophotometrically measuring the optical density of the dye formed and 
performing an arithmetic calculation. Another embodiment of the present 
invention comprises an element for the analysis of calcium in liquids and 
includes a reagent layer containing an interactive material which is an 
indicator for calcium and forms a colored species in the presence of 
calcium, such as chlorophosphonazo III or arsenazo III. The use of 
arsenazo III as a calcium complexing agent is described in Anal. Chim. 
Acta., Vol. 53 (1971), p. 194-198. Other suitable indicators for calcium 
are known and may be found, for example, in Clinical Chemistry Principles 
and Technics, edited by Henry et. al., 2nd. ed., chapter 19, p. 648, 
published by Harper and Row (1974). Elements of the present invention are 
also useful in the analysis of many other substances in liquids in 
addition to calcium or glucose as noted above. 
As stated hereinabove, the elements of this invention can also include a 
radiation-transmissive support to support the other layers. Such a support 
transmits light in the range of the spectrum used to determine the 
presence and/or absence of detectable species provided by the reagent 
layer. In the case where the detectable species is a visibly colored 
material, e.g., a dye, this will allow the spectrophotometric measurement 
of the dye density to be performed through the support layer with all 
layers of the element still intact. A useful support layer can comprise 
cellulose acetate, polyethylene terephthalate, and the like. 
Other optional layers mentioned hereinabove include radiation-transmissive 
subbing and registration layers, which if used, are located between the 
reagent layer and the optional support layer. Subbing layers may also be 
included between other layers to provide the required adhesion and fluid 
contact between such layers. Such optional registration and subbing layers 
are known in the art and are described in U.S. Pat. No. 3,992,158, and in 
U.S. Pat. No. 4,042,335, both incorporated by reference hereinabove. 
The detectable species migration-inhibiting layer of the present invention 
is interposed between the reagent layer and the porous radiation-blocking 
layer and is radiation-transmissive. The detectable species 
migration-inhibiting layer is permeable to the analyte, so that analyte 
can diffuse through it from the porous radiation-blocking layer and into 
the reagent layer. The detectable species migration-inhibiting layer 
functions such that a significant portion of any detectable species, e.g., 
a dye, migrating into it from the reagent layer is fixed in place or 
otherwise prevented from further migrating into the porous 
radiation-blocking layer (and further into the separate spreading layer, 
if one is present) wherein it cannot easily be measured. Detectable 
species migration-inhibiting layers of a preferred embodiment of the 
present invention comprise a hydrophilic colloid and a mordant for the 
particular detectable species formed in the reagent layer. Useful 
hydrophilic colloids include those mentioned hereinabove as useful in 
reagent layers of the described elements. Useful mordants are chosen 
according to the particular detectable species formed in the reagent 
layer. In the example of an element for the analysis of glucose in 
liquids, discussed above, one preferred mordant among others is a 
copolymer comprising recurring units of styrene; 
N-vinylbenzyl-N,N-dimethylbenzylammonium chloride; and divinyl benzene. It 
has been found that if the mordant is placed directly in the reagent 
layer, it often unexpectedly interferes with the reactions initiated by 
the presence of the analyte and prevents or significantly inhibits the 
formation or release of the detectable species. 
Other mordants useful in the present invention include compounds of the 
structure: 
##STR1## 
each of R.sup.1, R.sup.2 and R.sup.3, which may be the same or different, 
is selected from alkyl, alkenyl, aralkyl, or aryl having less than about 
eight carbon atoms, including cycloalkyls such as cyclohexyl, alkenyls 
such as allyl, aralkyls such as benzyl, and aryls such as phenyl and 
substituted phenyls; 
R.sup.4 is a ballasting group having more than about 8 carbon atoms such as 
alkyl, including substituted alkyl and alkyl having hetero atoms or groups 
within or appended to the alkyl chain, aralkyl, and aryl as defined above; 
and 
X.sup..crclbar. is an acid anion such as a halide ion, e.g., chloride or 
bromide; nitrate; methosulfate; p-toluenesulfonate; etc. 
One example of a useful mordant of Formula I above is a compound having the 
structure: 
##STR2## 
Other mordants useful in the invention are polymeric mordants including 
copolymers, e.g., terpolymers. A partial listing of representative useful 
polymeric mordants includes polymers having recurring units derived from 
70 to about 98 weight percent of one or a mixture of hydrophobic monomers, 
for example, styrene; and recurring units derived from about 2 to 30, 
preferably about 5 to 20 weight percent, of cationic monomers, such units 
typically, but not necessarily, conforming to the structure: 
##STR3## 
wherein L is a chemical linking group between Q and the atoms in the chain 
of the polymer backbone; 
n is 0 or 1; 
X.sup..crclbar. is an acid anion as defined above; and 
Q.sup..sym. is a linear or heterocyclic ammonium, phosphonium, or 
sulfur-containing group of the structure: 
##STR4## 
each of R.sup.1, R.sup.2, and R.sup.3, which may be the same or different, 
is as defined above; 
each of R.sup.5, R.sup.6, R.sup.7, and R.sup.8, which may be the same or 
different, represent H or R.sup.1 as defined above; and 
D is the atoms necessary to complete a heterocyclic ring. In addition to 
styrene other hydrophobic monomers useful as recurring units in these 
polymeric mordants include substituted styrenes, alkyl acrylates and 
methacrylates, difunctional monomers such as divinylbenzene and 
ethylenedimethacrylate, acrylamides, methacrylamides, and the like. 
A partial listing of representative cationic monomers useful in preparing 
these polymeric mordants includes: 
N-vinylbenzyl-N,N,N-trimethylammonium chloride, 
N-benzyl-N,N-dimethyl-N-vinylbenzylammonium chloride, 
N,n,n-trihexyl-N-vinylbenzylammonium chloride, 
N-(3-maleimidopropyl)-N,N,N-trimethylammonium chloride, 
N-benzyl-N-(3-maleimidopropyl)-N,N-dimethylammonium chloride, 
N-vinyloxycarbonylmethyl-N,N,N-trimethylammonium chloride, 
N-(3-acrylamido-3,3-dimethylpropyl)-N,N,N-trimethylammonium methosulfate, 
1,2-dimethyl-5-vinylpyridinium methosulfate, 
N-(2-hydroxy-3-methacryloyloxypropyl)-N,N,N-trimethylammonium chloride, 
N-(2-hydroxy-3-methacryloyloxypropyl)-N,N,N-trimethylammonium sulfate, 
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium iodide, 
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium p-toluenesulfonate, 
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium methosulfate, 
3-methyl-1-vinylimidazolium methosulfate, 
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium acetate, 
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium bromide, 
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium chloride, 
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium fluoride, 
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium nitrate, and 
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium phosphate. 
An example of one suitable polymeric mordant of the type described is that 
copolymer identified hereinbefore as useful in an element for the analysis 
of glucose in liquids. 
In addition to the use of mordants to formulate the detectable species 
migration-inhibiting layer used in the present invention, one can also 
employ as the migration-inhibiting material an antibody for the detectable 
species provided by the reagent layer. Such antibodies can be prepared by 
conventional immunological techniques and, of course, can vary widely 
depending on the particular material to be used as the detectable species 
in a given element of the invention. Typically, such antibodies are 
immobilized in the detectable species migration-inhibiting layer. 
Exemplary elements of this invention includes those illustrated in the 
accompanying drawings. In FIG. 1 is represented an analytical element 
composed of a reagent layer 12, a detectable species migration-inhibiting 
layer 14, a porous radiation-blocking layer 16, and, optionally, a 
spreading layer 18. All of these layers are in substantially continuous 
intimate contact with their adjacent layers. In an alternative embodiment 
of the invention, shown in FIG. 2, the analytical element is composed of a 
support 20 on which is coated a reagent layer 24, a detectable species 
migration-inhibiting layer 26, and a porous radiation-blocking layer 28, 
which in this case serves also as a spreading layer. Optionally, either or 
both subbing and registration layers 22 may also be included in the 
analytical element. All of these layers are in substantially continuous 
intimate contact with their adjacent layers. 
In the practice of this invention, a sample of a liquid to be analyzed is 
placed on the outermost surface layer of the element, which in the case of 
the element illustrated in FIG. 2 is the porous, radiation-blocking, 
spreading layer 28. Any predetermined analyte present in this liquid 
diffuses through the porous, radiation-blocking layer and the detectable 
species migration-inhibiting layer, and enters the reagent layer. There, 
interaction with the test reagents causes the release of or the formation 
of a detectable species such as a dye. This dye either remains in place or 
in part migrates out of the reagent layer, into the detectable species 
migration inhibiting layer, and also into any porous, 
radiation-transmissive layers underlying the reagent layer. All or most of 
the dye entering the detectable species migration-inhibiting layer is 
fixed in place and prevented from further migrating into the overlying 
porous radiation-blocking layer or layers. The reflective density of all 
dye in the detectable species migration-inhibiting layer, the reagent 
layer, and any other underlying radiation-transmissive layers is then 
determined while the element is still intact by measuring this density 
spectrophotometrically through all of these radiation-transmissive layers 
at the same time. 
The following examples are provided to further illustrate certain 
embodiments of the present invention. 
EXAMPLE 1 
Element For the Analysis Of Glucose 
Two elements for the analysis of glucose in liquids were prepared in the 
following manner: 
Polyethylene terephthalate film supports were coated with reagent layers 
comprising peroxidase at 10,200 U/m.sup.2, (the symbol U refers to 
international units, which are the well known and generally accepted units 
of measurement of enzyme activity), glucose oxidase at 24,400 U/m.sup.2, 
7-hydroxy-1-naphthol at 0.66 g/m.sup.2, and 4-aminoantipyrene 
hydrochloride at 0.86 g/m.sup.2. The reagent layer of control sample 1 
further comprised deionized gelatin at 21.5 g/m.sup.2. The reagent layer 
of sample 2 also comprised deionized gelatin, but at 19.4 g/m.sup.2. The 
second sample was then coated with a detectable species 
migration-inhibiting layer, in this case a dye migration-inhibiting layer 
comprising deionized gelatin at 2.1 g/m.sup.2 and the mordant, 
poly(styrene-co-N-vinylbenzyl-N,N-dimethylbenzylammonium 
chloride-co-divinyl benzene) (weight ratio 49.5:49.5:1.0) at 1.08 
g/m.sup.2. All gelatin-containing layers were buffered at pH 6.0 with a 
disodium phosphate-potassium phosphate buffer. Both samples were then 
overcoated with a subbing layer comprising n-isopropylacrylamide at 0.32 
g/m.sup.2 and a blushed-polymer, radiation-blocking, spreading layer 
comprising cellulose acetate at 9.4 g/m.sup.2 and titanium dioxide at 64.5 
g/m.sup.2. 
The two resulting elements were then contacted at the outermost surface of 
their spreading layers with 10 .mu.l samples of glucose standards 
containing various concentrations of glucose. After 7 minutes of contact 
at 37.degree. C. the reflection densities of the dye formed were measured 
spectrophotometrically using a photomultiplier unit and a Wratten 65 
filter. The following Table I illustrates the results, the control sample 
being representative of elements of the prior art. 
TABLE I 
______________________________________ 
Effect of Dye Migration-Inhibiting Layer on 
Measurement of Density of Dye Formed in Element 
For Glucose Assay 
Measured Measured Dye Density 
Actual Glucose 
Dye Density in Sample Containing 
Concentration 
in Control Dye Migration-Inhibiting 
(mg/dl) Sample (D.sub.R) 
Layer (D.sub.R) 
______________________________________ 
100 0.44 0.60 
200 0.80 1.00 
400 1.23 1.65 
800 1.84 2.03 
______________________________________ 
EXAMPLE 2 
Element For The Analysis Of Calcium 
Two elements, one with and the other without a detectable species 
migration-inhibiting layer containing a mordant, were prepared according 
to the following: 
A terephthalate film support was coated with a reagent layer comprising 
gelatin (4.3 g/m.sup.2), Triton X-100 (0.17 g/m.sup.2), chorophosphonazo 
III (0.21 g/m.sup.2), bis(vinylsulfonylmethyl) ether (0.04 g/m.sup.2) and 
0.1 M 3,3-dimethylglutaric acid, pH 5.4; a dye migration-inhibiting layer 
comprising gelatin (4.3 g/m.sup.2), and 
poly(styrene-co-N-vinylbenzyl-N,N-dimethylbenzyl ammonium 
chloride-co-divinylbenzene) (2.15 g/m.sup.2); a subbing layer comprising 
(poly-N-isopropylacrylamide) (0.32 g/m.sup.2); and a blushed-polymer, 
radiation-blocking spreading layer comprising TiO.sub.2 (50.4 g/m.sup.2), 
cellulose acetate (7.0 g/m.sup.2) and Triton X-405 (1.4 g/m.sup.2). 
A second control element (outside the scope of the present invention) was 
prepared in the same manner except without a detectable species 
migration-inhibiting layer between the spreading layer and reagent layer. 
The elements were evaluated as in Example 1, using calcium standards 
containing 1 to 5 mM of calcium and reading the reflection densities at 
670 nm. Table II shows the improved results obtained with the element 
containing the detectable species migration-inhibiting layer, in this case 
a dye migration-inhibiting layer. 
The results of Examples 1 and 2 above indicate that a significantly higher 
dye density was consistently measured with the element containing a 
detectable species migration-inhibiting layer. The control element, having 
no such layer, allowed significant amounts of the dye to migrate into the 
blushed-polymer, radiation-blocking, spreading layer where it could not be 
detected. 
TABLE II 
______________________________________ 
Effect of Dye Migration-Inhibiting Layer on 
Measurement of Density of Dye Formed in Element 
For Calcium Assay 
Measured Measured Dye Density 
Actual Calcium 
Dye Density in Sample Containing 
Concentration 
in Control Dye Migration-Inhibiting 
(mM) Sample (D.sub.R) 
Layer (D.sub.R) 
______________________________________ 
0 0.246 0.28 
1 0.267 0.75 
2 0.269 0.80 
3 0.279 0.82 
4 0.269 0.84 
5 0.262 0.84 
______________________________________ 
EXAMPLE 3 
Example No. 2 was repeated, except that the reagent layer contained as a 
calcium indicator 0.48 g/m.sup.2 arsenazo III, rather than 
chlorophosphonazo III. The reagent layer was buffered to a pH of 5.6. The 
resulting element demonstrated a dye density comparable to that of the 
test element of Example 2. 
The invention has been described in detail with particular reference to 
certain preferred embodiments thereof, but it will be understood that 
variations and modifications can be effected within the spirit and scope 
of the invention.