REDUCTION OF DETECTABLE SPECIES MIGRATION IN ELEMENTS FOR THE ANALYSIS OF LIQUIDS

RALENTISSEMENT DE LA MIGRATION DES ESPECES IDENTIFIABLES A L'INTERIEUR D'ELEMENTS EMPLOYES POUR LE DOSAGE DES LIQUIDES

English Abstract

Abstract of the Disclosure
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.

Claims

We Claim:
1. An element for the analysis of liquids, said
element comprising a radiation-transmissive reagent layer
permeable to a predetermined analyte, which layer comprises
a composition that is interactive in the presence of said
analyte to provide a radiometrically detectable species, and
a porous radiation-blocking layer permeable to said analyte;
characterized by a radiation-transmissive,
detectable species migration-inhibiting layer interposed
between the reagent layer and the porous radiation-blocking
layer, said detectable species migration-inhibiting layer
being permeable to said analyte and inhibiting the migration
of said radiometrically detectable species to said porous
radiation-blocking layer upon contact of said element with the
liquid under analysis.
2. An element as described in Claim 1 wherein said
detectable species migration-inhibiting layer comprises an
immobilized antibody for said radiometrically detectable spe-
cies.
3. An element as described in Claim 1 wherein the
detectable species is a dye upon contact of said element with
the liquid under analysis.
4. An element as described in Claim 1 further com-
prising a spreading layer permeable to said analyte and wherein
the porous radiation-blocking layer, permeable to said analyte
is interposed between said reagent layer and said spreading
layer upon contact of said element with the liquid under analy-
sis.
21
2. An element as described in Claim 1 wherein said
detectable species migration inhibiting layer comprises an
immobilized antibody for said radiometrically detectable spe-
cies.
3, An element as described in Claim 1 wherein the
detectable species is a dye upon contact of said element with
the liquid under analysis.
4. An element as described in Claim 1 further com-
prising a spreading layer permeable to said analyte and wherein
the porous radiation-blocking layer, permeable to said Analyte
is interposed between said reagent layer and said spreading
layer upon contact of said element with the liquid under analy-
sis.
21

5. An element for the analysis of liquids, said ele-
ment comprising a radiation-transmissive support having thereon a
radiation-transmissive reagent layer permeable to a predetermined
analyte, which layer comprises a composition that is interactive
in the presence of said analyte to provide a dye, and an outer-
most radiation-blocking spreading layer permeable to said analyte
upon contact of said element with the liquid under analysis.
characterized by a radiation-transmissive, dye migration-
inhibiting layer interposed between the reagent layer and the
radiation-blocking spreading layer, said dye migration-inhibiting
layer being permeable to said analyte and inhibiting the migra-
tion of said dye to said radiation-blocking spreading layer upon
contact of said element with the liquid under analysis.
6. An element as described in Claim 5 which further
comprises at least one radiation-transmissive registration layer
interposed between said reagent layer and said support.
7. An element as described in Claim 5 which further
comprises a radiation-transmissive subbing layer interposed
between said reagent layer and said support.
8. An element as described in Claim 5 wherein the dye
migration-inhibiting layer comprises a mordant for said dye.
9. An element as described in Claim 5 wherein the dye
migration-inhibiting layer comprises a mordant for the dye, said
mordant having the structure
<IMG>
wherein <IMG> is <IMG> or <IMG>;
each of R1, R2 and R3, which may be the same or different,
is selected from alkyl, alkenyl, aralkyl, or aryl, each having
less than eight carbon atoms;
R4 is a ballasting group having more than eight carbon
atoms; and
X- is an acid anion.
22

10. An element as described in Claim 5 wherein the
dye migration-inhibiting layer comprises a mordant for the dye,
said mordant comprising a polymer having recurring units derived
from 70 to about 98 weight percent of one or a mixture of hydro-
phobic monomers and recurring units derived from about 2 to 30 weight
percent of at least one cationic monomer conforming to the structure:
<IMG>
wherein L is a linking group between Q and the atoms in
the chain of the polymer backbone;
n is 0 or 1;
X? is an acid anion; and
Q? is a linear or heterocyclic ammonium, phosphonium,
or sulfur-containing group having one of the following
structures:
<IMG>, <IMG>, <IMG>,
<IMG> <IMG>,
wherein <IMG> is <IMG> or <IMG>;
each of R1, R2, and R3, which may be the same or
different, is selected from alkyl; alkenyl, aralkyl, or aryl, each
having less than eight carbon atoms;
each of R5, R6, R7, and R8, which may be the same or
different, represent H or R1 as defined above; and
D is the atoms necessary to complete a heterocyclic
ring.
11. An element as described in Claim 5 wherein
the dye migration-inhibiting layer comprises a hydrophilic

12. In an element for the analysis of liquids, said
element comprising a radiation-transmissive support having
thereon a radiation-transmissive reagent layer permeable
to a predetermined analyte, which layer comprises a composition
that is interactive in the presence of said analyte to provide
a dye, and an outermost radiation-blocking spreading layer
permeable to said analyte;
the improvement comprising a radiation-transmissive,
dye migration-inhibiting layer interposed between the reagent
layer and the radiation-blocking spreading layer, said dye
migration-inhibiting layer being permeable to said analyte
and comprising a hydrophilic colloid and a mordant for said
dye.
13. An element as described in Claim 12 wherein the
radiation-blocking spreading layer comprises a blushed polymer
and a pigment.
14. An element as described in Claim 12 wherein the
reagent layer comprises a hydrophilic colloid having siad
interactive composition distributed therein.
15. In an element for the analysis of liquids, said
element comprising a radiation-transmissive support having
thereon a radiation-transmissive reagnet layer permeable to a
predetermined analyte, which layer comprises a hydrophilic
colloid, said colloid having distributed therein a composition
that is interactive in the presence of said analyte to provide
a dye; and an outermost radiation-blocking spreading layer,
permeable to said analyte, comprising a finely-divided
particulate pigment and a blushed polymer;
24

the improvement comprising a radiation-transmissive
dye migration-inhibiting layer, interposed between the reagent
layer and the radiation-blocking spreading layer, said dye
migration-inhibiting layer being permeable to said analyte
and comprising a mordant for said dye and a hydrophilic colloid.
16. An element as described in Claim 15 wherein the
reagent layer is permeable to glucose and wherein the inter-
active composition in the reagent layer comprises glucose
oxidase, peroxidase, and an indicator composition comprising
a compound oxidizable in the presence of hydrogen peroxide and
peroxidase to effect formation of said dye.
17. An element as described in Claim 15 wherein
the reagent layer is permeable to calcium and wherein the
interactive composition in the reagent layer comprises an
indicator for calcium.

Description

llZ~
It ls orten deslrable or necessary to determlne
the presence and/or concentratlon o~ certain substances
in liqulds such as water, foodstufrs, and blologlcal llquids.
A varlety of de~ices and methods have been empl~yed rOr such
analyses.
Sophisticated elements are available for
qu~ntitative diagnostic analyses Or blological llquids llke
blood or ur~ne. When a llquid 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 concentratlon Or the
analyte in the liquid sample. Analyte concentration can then
be determined, for example, by spectrophotometric measurement
of the optical density Or the dye ~ormed in the element.
Elements of this type are described in U.S. Patent
No. 3,992,158. These elements can
consist of two or more desirably discrete layers that are
superposed and in substantially continuous intlmate contact with
ad~acent layers. One such multilayer element comprises a -
support layer havlng a reagent layer and an outermost spreading
layer coated upon it. In this multilayer element,
the liquid sample to be analyzed -~
ls placed on the spreading layer, which absorbs and transfers
the liquid to the reagent layer. Preferably, as described
in U.S. 3,992,158, the spreading layer is isotropically
porous and transrers a uni~orm 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. ~ detectable species,
such as ~ dye,ls ~ormed withln the reagent layer in an
amount proportional to the concentration Or analyte in the
,

39
s
llquid. Typlcally, the reagent and support layers are
radiation-transmisslve so that a ~pectrophotometric measurement
of the op~ical density Or the dye ~ormed ln the reagent ¦~1
layer can be made with the element remaining intact. 11
Additlonally, the spreadlng layer may comprlse a blushed ~¦
polymer and a pigment to provide both uniform transfer of
the liquid sample to the reagent layer and an opaque, reflectlve il
surface above the reagent layer to aid in a measurement of
reflection density of the dye. With this element, however,
some of the dye rormed in the reagent layer may migrate or
wander lnto 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. Patent No.
4,042,335. 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 ln the reagent layer ¦
will di~use through the radiation-blocking layer and into the -
re~istration layer, where the dye density will be measured.
A mordant for the dye can be included ln the reglstration
layer to insure that the dye that has dif~used 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 lt would not otherwise
be practlcal to reliably measure the dye denslty within the
reagent layer itself, ~or example, in analytical elements where
other reagents and reaction products withln the reagent layer
also provide density, thus preventing any accurate spectro- -
photomet~ic measurement of the optical density ln this layer
. .

39
Of only the dye. Such an elemen~ can provlde a reliable analysis. $
However, lt is obvlous that a signi~lcant portion of the dye
formed during the analys~s can remaln in ~he reagent layer
or mi6rate into and re~,ain in the radiatlon-blocklng layer.
The sensitivity and accuracy o~ the analytical element are
thereby reduced, because the analyte-concentration determination
must depend upon the measurement of the denslty of a smaller
amount of dye than that which was actually formed. I
Other elements as described ln U.S. Patent No. ~i
10 3,585,112 and U.S. Patent No. 3j917,453 disclose means ~or
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 interferlng with any prerequis~te
reactions leading to the formation of the dye. Such inter- ~¦
ference can make the analysis completely unreliable.
Accordingly, lt is deslrable 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 ln 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.
The elements of the present invention have -
overcome the problems Or prior art analytical elements
by providing for quantitative analyses which are highly accurate
!
-3-

and sensltlv~ sbe pr~ent ~le~nts do ~o ~y 1nhibltingmigration of the detectable specles rrom the reagent layer
~o layers Or the element where such species could not easlly
be measured, and by providing a means for avoiding lnterference
with the reaction or reactions that result in detectable
specles ~ormation in,or release ~rom,the reagent layer.
Elements according to thls invention can be used for diagnostlc
purposes and include: a radiation-~ransmissive reagent layer,
permeable to, and contalning 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
lmprovement 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
o~ the detectable species which may dif~use out of the
reagent layer from entering the porous radlation-blocking layer
where it is not practically measurable, by fixing such
migrating detectable species within the detectable species
misration-inhibiting layer, where it ls easlly detectable. ~i
Another advantage of the present lnvention is that the
detectable species migratlon-inhibiting layer is separate
` ~rom the reagent layer, so that it does not interfere wlth
the analytical interaction(s) taking place ln the reagent
layer.
Optionally, analytical elements of the present
lnvention can be carried on a radiatlon-transmissive supportS l ;
and other layers such as spreading layers, registration layers,
and subbing layers can also be present in the element. Also~
:: ,

the porous radlatlon-blocklng layer can l~elr runction ~s a
spreadlng layer in some embodlments Or the present ~nvent10n.
In the accompanylng drawln~s,each of Fig. l and
Fig. 2 is an enlarged sectional vlew lllustratlng a preferred
embodiment of an analytical element Or this lnvention.
The analytlcal elements o~ thls invent1on are mult1- -
layered, consisting o~ three or more deslrably discrete layers
that are superposed and in rluid contact with each other under
condltions o~ use. These layers include a reagent laye~, a
porous radiation-blocklng layer, and a detectable species
migration-inhibiting layer. In certain embodiments of the
lnventio~ the porous radiation-blocking layer can ~unction
also as a spreading layer, or there can be a separate spreading
layer ln 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,addltional
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 ls interposed between the porous radiation-blocking
layer and the reagent layer. In those embodiments containing
an additional layer to ~unction 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
; -5- -
- ,- . - . . . ' ,, ~ ' :. , , ~
: . . . . .
....

suppcrt layer, the reagent layer ls lnterposed between the
deeectable specles migration~inhibitlng layer and the radiatlon-
tr~nsmissive support layer. In those embodiments contalning
additional radiation-transmlssive layers, such as subblng or
re~istration layers, the additional subbing layers or registra-
tion layers are interposed between the reagent layer and the
optional radlation-transmissive support layer.
U.S. Patent No. 3,992,158 and U.S. Patent No.
4,042,335 disclose reagent layers, porous radlation-
blocking layers, support layers, subbing layers, registratlon
layers, and preferred types of isotropically porous spreading
layers, that are useful in the practlce of the present
invention. These materials also describe well known methods
Or preparing these layers to form individual multilayer
elements and describe the use of such elements for various
quantitative analyses. -
As used herein3 the term '~orous radiatlon-blocklng
layer" defines a layer that is permeable to a predetermined
analyte (or reaction product thereof) dissolved or dispersed
~0 in a liquid, and that reflects, or optionally absorbs, detecting
radiation. "Detecting radiation" is radiation used together with the
elements of the invention to facilitate result detection of
the particular detectable species which is provided by the ¦
reasent 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 racilitate result detection in the analytical ele~ents Or
the invention,suGh as by reflection photometry. Because of
the radiation-blocking properties o~ the porous radiatlon-
blocking layer, the radiative properties, i.e.~ the partlcular ~ -
emissive,;transmisslve, or absorptive properties, of any Or
' '

t~e detec~able specles w~ic~ ml~rates lnto ~hls layer can be
I
substantially masked or hidden. Therefore, de~ectlng radlation
used to determine the presence or absence Or detectable
species formed in the rea~ent layer may be unable to detect
accurately that portlon Or the detectable species which, although l
provided in response to a &lven analyte, has ml~rated lnto I
the porous radiation~blockln6 layer.
As noted above, the analytical elements of the
present invention can optionally contain a separate spreading
layer ~n addition to the porous radiation-blocking layer~ or
the porous radiation-blockln~ layer itsel~ can also function -
as a spreading layer. Llke the porous radlation-blocking
layer, a spreading layer must be permeable to a predetermlned
analyte dissolved or dispersed ln a liquid. When liquid
containing the analyte is brought into contact with the
outermost surface Or a spreadlng layer, the spreading layer
distributes the liquid within the spreading layer such that the concentra- l
tion of the analyte provided at the surface of the spreading layer ~ -
that races the reagent layer of the element ls regulated or 1,
controlled. Pre~erably, but not necessarily, the spreading
layer is isotropically porous and delivers a uniform concentra- -
tion of analyte to the reagent layer. In one embodi~ent of
the presen~ invention a separate spreading layer may be -
lncluded ln 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-
transmi5sive",as used herein, derines the ability to transmit
` detecting radiation used to determine the presence and/or concen-
tration, optionally the absence, o~ the detectable species provided by the -
reagent layer. Ir desired, one or more interactlve or reagent
compositions may be incorporated ln the spreading layer or ~ ` -
separate porous radiation-blocking layer to interact with the
snalyte o~ choice, thereby rorming an analyte reaction product
_7~
. ~ , ,, ' ' ~`' ' . ,'.
- ~
.

whlch c~n under~o further lnteractlon in the underlying rea~ent
layer as described herelnarter.
In one preferred embodlment Or the present invention
the porous radlation-blocking layer ltself fur,ctlons as an
adequate spreading layer a~d comprises a blushed polymer
and optlonally a finely-divided particulate material such as
a pi~ment. Layers of this type are dlscussed in detail
ln U.S~ Patents 3,992,158 and 4,042,335.
Useful blushed polymers include cellulose acetate, amides, ¦
and the like. Useful particulate materials lnclude pigments
such as carbon, titanium dioxlde, barlum sulfate, and the
like.
Reagent layers in the elements of this invention
are radlation-transmissive. 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 o~
the analyte. Such lnteraction causes the release of a
preformed detectable species or the ~ormation of such a
detectable species within the reagent layer, preferably, in
proportion to the concentration of the analyte in the liquid
sample bein6 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 radio-
metrically detectable, that is, by suitable measurement of
llght or other energy~ Typically, the detectable species
rormed or released from the reagent layer is a dye which is
radiometrically detectable by rluorometric or colorimetric~
pre~erably colorimetri~ techniques. -
.
-8-
',` "` ' , " , .

In additlon, lf nec~sary or deslrable, appropr1ate
bu~er composltions may al~o be present ln the reagent l~yer.
Re~6ent layers Or the present lnventlon may also contaln
one or more ~ydrophilic colloid~ lncluding natural c~lloids
suc~ as 6elatln, agarose, polysaccharldes~ and the llke; and/or
synthetic resins such as poly~lnyl alcohol)~ poly(vinyl
pyrrolidone), polyacrylamides~ and the llke.
One appllcation Or the present invention comprlses
an element ror the analysls of ~lucose ln llqulds,wherein
the interactlve material ln the reagent layer preferably
comprises ~lucose oxidase, perox~dase~ and an indlcator jl
composition. A useful indicator c~mposltion comprlses 4- ¦~
amlnoantipyrene hydrochloride and 7~hydroxy-1-naphthol. In i
the presence of glucose, the above interactive material
effects the ~ormatlon o~ a dye in proport~on to the concen-
tratlon Or glucose in the sample belng analyzed. Thls concen-
tration can then be determlned by spectrophotometrlcally
measurin~ the optlcal density o~ the dye rormed and performing
an arithmetic calculation. Another embodiment Or the present
lnvention comprises an element ror the analysis of calcium in
llquids and includes a reagent layer containing an interactive -
material which is an indicator ~or calcium and ~orms a ,l
~olored species ln the presence Or calc~um9 such as chloro- ~l
phosphona~ III(i.e. sodium 3,6-bis(4-chloro-2-phosphonophenyla~o) 4, S~;~ ;
dihydroxy-2, 7-naphthalene di~ulfonate) or arsenazo III -~
(i.e. 1,8-dihydroxy naphthalene-3.6-disulfonic acid-2, 7-bis
l(azo-2)~phenylarsonic acidJ). The use of arsenazo III as
a calcium complexing agent is described in Anal. Chim. Acta.,
vol. 53 (1971), pp~ 194-198~ Other suitable indicator for
calcium are known and may be found, for example, in Clinical
Chemistry Princi~les and Technics,edited by Henry et al, 2nd.ed.~
.
.
_ g_

Elements Or the presenS inventlon are also useful ln the
analysis of many other substances in liquids in additiOn to
calcium or glucose as noted above.
As stated hereinabove, the elements of thls invention
can also lnclude a radiatlon-transmlsslve support to support
the other layers. Such a support transmits llght in the
-9a-

other layers. Such a support transmits light in the
range Or the spectrum used to determlne the presence and/or
absence of detectable specles provided by the reagent layer.
In the case where the detectable species ls a vlslbly colored
material, e.g., a dye, this will allow the spectrophotometric
measurement of the dye density to be per~ormed through the
support layer with all layers of the element stlll lntact.
A useful support layer can comprise cellulose acetate~
polyethylene terephthalate, and the like.
Other optional layers mentioned herelnabo~e lnclude
radiation-transmissive subbing and registratlon layers, whlch
lf used, are located between the reagent layer and the optlonal -
support layer. Subbing layers may also be lncluded between
other layers to provide the requlred adheslon and rluid contact
between such layers. Such optlonal re~istration and subbing
layers are known ln the art and are described in U.S. Patent
No. 3,992,158, and in U.S. Patent No. 4,042,335. ;
~:,
The detectable species migration-inhibiting layer
20 of the present invention is interposed between the reagent i -~
layer and the porous radiation-blocking layer and is radiation-
transmissive. The detectable species migratlon-inhibiting
layer is permeable to the analyte, so that analyte can diffuse
through it from the porous radiation-blocklng layer and into ,
the reagent layer. The detectable species migration-lnhibiting
layer functions such that a significant portion of any
detectable species, e.g., a dye, migrating into it from the
reagent layer ~s fixed in place or otherwise prevented from
further migrating into the porous radiation-blocking layer (and
1 30 ~urther into the separate spreading layer~ ir one is presen
wherein it cannot easily be measured. Detectable species
migration-inhibit~ng layers o~ a pre~erred embodIment Or th~
present i~vention comprise a hydrophilic collold and a mordant
--1 0--
. .

for the partlcular detectable specles rormed in the reagent
layer. Userul hydrophllic collolds lnclude those menkioned
hereinabove as use~ul in rea~ent layers of the described
elements. Use~ul mordants are chosen accordlng to the
particular detectable specles formed in the reagent layer.
In the example Or an element for the analysis of glucose ln
liquids, dlscussed above, one pre~erred mordant among others
is a copolymer comprlsing recurring units Or styrene, N- ~!
vinylbenzyl-N,N-dimethylbenzylammonium chloride~ and divinyl
benzene. It has been found that lf the mordant is placed
directly in the reagent layer, it often unexpectedly lnter-
reres with the reactions initiated by the presence of the
analyte and prevents or significantly lnhlbits the forma~ion
or release Or the detectable species.
O~her mordants use~ul in the present lnvention
include compounds of the structure:
R4
I. Rl_~_R3 X~
R2 ,
wherein -Z~- is -N~- or -P~-;
each o~ Rl, R2 and R3, which may be the same or
di~erent, ls selected ~rom alkyl, alkenyl, aralkyl, or aryl, each
having less than 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 ls a ballasting group havlng more than ei~h~
carbon atoms such as alkyl, including substituted alkyl
30 and alkyl havlng hetero atoms or groups within or appended to
the alkyllchaing aralkyl, and aryl as deflned above; and T

¦ X~ is an ~cid anion such as a hallde lon, e.g.,
¦ chloride or bromide; nitra~; methosulrate; p-toluenesulronate;
etc.
One example of a u~eful mordant of Formula I above
ls a compound having the structure:
II. CloH
H13C6-N ~C6H13 X~ '
C6H13
¦ Other mordants useful in the invention are polymeric
mordants lncluding copolymers, e~g., terpolymers. A partial
listing Or representatlve use~ul polymeric mordants includes
polymers having recurring unlts derived from 70 to about 98 :
weight percent Or one or a mixture of hydrophobic monomers,
: for example, styrene; and recurring units derived ~rom about 2
to 30 weight percent, preferably about 5 to 20 weight percent, of at least one ~ :
cationic monomer, such units typicPlly, but not necessarily, confo~
to the structure:
R7 R~
.
~ III. CH-C- ;~
'~ X4
wherein L is a chemlcal linking group between Q~ nd the atoms in
the chain of the polymer backbone; ~.
n is 0 or li :
X is an acid anion as deflned above; and
Q~ is 2 linear or heterocycllc ammonium, phosphonium,
- or sulrur-conta1ning group Or the structure:
IVa. Rl_z~_R3, IVb. fD ~ IVc. ~D-~
R2 ~Z -CH, ~Z =CH,
~.
: -12- .
. . .
, -- , :
:

~2~
Rl S0 ,1
q ~ ., .
IYd. SW- J IVe. ~ N-H
R5 R6 jl
whereln -Z~- is -N~- or -P~-;
each of Rl, R2, and R3, which may be the same or
dirrer~nt~ is as defined above;
each of R5, R6, R7, and R8, which may be the same or
di~ferent, represent H or R as defined above; and
D is the atoms necessary to complete a heterocyclic
ring. In addition to styrene other hydrophoblc monomers
useful as recurring units in these polymer~c mordants include .
substituted styrenes~ alkyl acrylates and methacrylates~ di-
runctional monomers such as divinylbenzene and ethylenedi-
methacrylate J acrylamides, methacrylamides, and the like.
A partial listing Or representative cationic
monomers useful ln 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, 1l '
N-(3-maleimidopropyl)-N,N,N-trimethylammonium chloride~ l
N-benzyl-N-(3-maleimidopropyl)-N,N-dimethyla~monium chloride, ¦ :
N-vinyloxycarbonylmethyl-N,N,N-trimethylammonium chloride, .
N-(3-acrylamido-3,3-dimethylpropyl)-N,N,N-trimethylammonium ,
methosulfate~
1,2-dimethyl-5-vinylpyridinium methosul~ate,
N-(2-hydroxy-3-methacryloyloxypropyl)-N,N,N-trimethylammonium
chloride,
N-(2-hydroxy-3-methacryloyloxypropyl)-N,N,N-trimethylamrnonlum
sulfate, -
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium iodide, : :1
N-(2-methacryloyloxyethyl)-N~N,N-trlmethylammonium p-toluene- -
sul~onate,
N-(2-methacryloyloxyethyl)-N,N,N-trimethylammonium methosulfate,
-13-
,` . . '~ '~.

8~3
3-methyl-1-vinyl1mldazol1um methosul~te~
I N-(2-methzcryloyloxyethyl)-N~N~N-trlmethylammonium acetate,
¦ N-~-methacryloyloxyethyl)-N~N~N trlmethylammonlum bromide,
N-(2-methacryloyloxyethyl)-N~N~N~tr~methylammonium chlorlde,
N-(2-methacryloyloxyethyl)-N,N,N-trimethylamrnonium rluorlde,
N-(2-methacryloyloxyethyl)-N~N,N-trimethylammonium nltrate, and
N-~2-methacryloyloxyethyl)-N,N,N-trlmethylammonlum phosphate.
An example of one suitable polymeric morda~t Or khe
type desc~ibed ls that copolymer identified hereinbefore as
use~ul ln an element for the analysis of glucose ln liquids.
In addition to the use o~ mordants to ~ormulate the
detectable species migration~inhlbitlng layer used ln the
present lnvention, one can also employ as the migratlon-
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 partlcular material to be used
as the detectable species in a given element of the invention.
Typically, such antibodies are immobillzed in the detectable
species migration-inhibiting layer.
Exemplary elements of this invention includes those
illustrated in the accompanying drawings. Ih Figure 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. A11 of these layers are in substantially continuous
lntimate contact with their ad~acent layers. In an alternative
embodiment of the invention, shown ln Figure 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-blocklng layer 28, which in this case
serves al~o as a spreading layer. Optionally, either or both
subbing and registration layers 22 may also be included in the
-14-
`
. .
.

an~lytlcal element. All Or the~e layers are in substantially
-continuous ln~lmate contact wlth thelr adJacent layers.
In the practlce o~ this inventlon, a sample of a
llquid to be analyzed is placed on the outermost surface
layer Or the element 9 which in the case Or the element lllus-
trated in ~lgure 2 is the porous~ radlatlon-blocking, spreading
layer 28. Any predetermined analyte present ln thls llquld
di~uses through the porous, radlat~on-blocklng layer 28 and the ,,
detectable species mlgration-lnhibiting layer 26, and enters the f,,
reasent layer 24. There, lnteraction wlth the test reagents causes
the release of or the formatlon Or a detectable specles such
as a dye. This dye either remains ln place or in part
mi~rates out of the reagent layer 24, lnto the detectable
species mlgration inhibiting layer 26, and also into any porous, ,
radiation-transmlssive layers underlying the reagent layer. -
All or most o~ the dye enterlng the detectable species
mi~ration-inhibiting layer 26 ls fixed in place and prevented
~rom ~urther migrating into the overlylng porous radiation-
blocking layer or layers 28. ~he reflective density of all dye
in th~ detectable species migration-inhibiting layer 26, the
reagent layer 24, 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 tlme. ,l
The following examples are provided to further ~¦
illustrate certain embodiments o~ the present invention.
Example 1 - Element For The Analysis 0~ Glucose
Two elements ~or the analysis of glucose in
llquids were prepared in the following manner:
-15- ;

Polyethylene t~r~phthalate fllm supports were coated
wlth reagent layers comprlsln~ peroxidase at 10,200 U/m2,
(the symbol U refers to internatlonal unlts~ whlch are the
well known and generally accepted unlts of measurement of
enzyme activity), glucos2 oxldase at 24g400 U/m2, 7 hydroxy-
l-naphthol at 0.66 g/m , and 4-amlnoantipyrene hydrochloride l .
at o.86 g/m2. The rea6ent layer Or control sample 1 ~urther
comprised deionized gelatin at 21.5 g/m . The reagent layer
o~ sample 2 also comprised deionized gelatln, but at 19.4 g/m2.
The second sample was then coated with a detectable species l
migration-inhibitlng layer, ln this case a dye migration- ¦
inhibiting layer comprising deionized gelatin at 2.1 g/m2
and the mordant, poly(styrene-co-N-vinylbenzyl-N,N-dimethyl-
ben ylammonium chloride-co-divinyl benzene) (weight ratlo
49.5:49.5:1.0) at 1.08 g/m2. All gelatin-containing layers -
were buffered at pH 6.o with a disodium phosphate-potassium ~
phosphate buffer. Both samples were then overcoated with ¦ .
a subbing layer comprising n-isopropylacrylamide at 0.32 g/m2
and a blushed-polymer, radiatlon-blocking, spreading layer l .
comprising cellulose acetate at 9.4 g/m and titanium dioxide ¦
at 64.5 g/m2.
The two resulting elements were then contacted at
the outermost surface of their spreading layers with 10 ~1
samples of glucose standards contalning various concentrations
of glucose. After 7 minutes of contact at 37C the reflection
densities Or the dye rormed were measured spectrophotometrlcallY
using a photomultipller unit and a Wratten 65 rilter. The
following Table I illustrates the resu.~.ts, the control sample .
being representative Or elements Or the prior art.
-16-
! !
1` .
.

.. ... ,,_. . `--
.,,
~C
~C~
~ H
o o o o u~
U ~J ~D O ~D O
. ~ ~ ~Y; ~
CC ~ a
~ a~ ~ c ~ ~
C
.,~
.,, ~ .
.~ E c
R O ta Q E~
,~ U~ ~ U~
,C ~ ~ ~ ~r o
HH C`l ~¢ 1~ --~ ~ ~ ~J
51:1l ~ ~ ~ ~ O ;~
.:1 C: ~1 U~ Q~ 1
O O ~O~ ~ O
~ I~ ~C
U W
~: a
~ o '
o C aJ C::
~ o3
Q~
u~
1~ ~ C~l O O O O
~ ~ I N
~`
I
-17-
~ .
, :
.

Example 2 - Element For The Analysis or Calclum
Two elements, one wlth and the other without a
detectable species migration-inhlbiting layer contalning a
mordant, were prepared according to the followlng:
A tereph~halate r~ lm support was coated with a reagent
layer comprising gelatin (4.3 g/m2), Triton X-lOOTM (0.17 g/m2)~
chlorophosphonazo III (0.21 g/m2), bis(vinylsulfonylmethyl)
ether (0.04 g/m2) and 0.1 M 3,3-dimethylglutaric acid, pH 5.4; a
detectable species (dye) migration-inhibiting layer comprising
gelatin (4.3 g/m2), and poly(styrene-co-N-vinylbenzyl-N,N-dimeth-
ylbenzylammonium chloride-co-divinylbenzene) (2.15 g/m2); a sub-
bing layer comprising (poly-N-isopropylacrylamide) (0.32 g/m2);
and a blushed-polymer, radiation-blocking spreading layer com-
prising TiO2 (50.4 g/m2), cellulose acetate (7.0 g/m2) and Triton
X-405TM ~1.4 g/m2). Triton X-lOOTM and Triton X-405TM are alkyl-
pheno~y polyethoxy ethanols, commercially available ~rom the Rohm
and Haas Company.
A second control element ~outside the scope Or the
present invention) was prep~red in the sa~e manner except
without a detectable species migration-lnhibiting layer between
the spreadlng layer and reagent layer.
The elements were evaluated as in Example l, using
calcium standards containing 1 to 5 mM of calcium and readlng
the reflection densities at 670 nm. Table II s~ows ~he
improved results obtained with the element containing the
detectable species migration-inhiblting layer, in thls case a
dye migration-inhibiting layer.
The results o~ Examples l and 2 above indicate that a
signi~icantly ~igher dye density was consistently measured
wlth ~he element containing a detectable species mlgratlon-
lnhiblting layer. The control element, having no such layer,
allowed signi~lcant amounts o~ the dye to mlgrate lnto the
blushed~polymer~ radiatlon-blocking, spreadlng layer where lt
coul~ not b~ det~cted.
-18-

- - -- --- - -
~ c~c
C C
a 1~ '
~ o o
I CO U~ O ~ ~ ~
c ~ ~ ~ m
c E ~ a O o O O O O
h ~ ~ J
: Q~ ~1 m u~ Q~
:~
CQ~ C
~ ,1~
~ E_~
R O I~ ~ C~
,C Q~ I .,~
C ~ U~ U~ Q~
H H aa ~
O O ~ ~ ~ ~ ~
., ~ U ~ U~
~ ~ ~~ ~0 o o o o o o
E~ :~ c U c : .
:~ ~ ~ ~ ,~
O -~ ~:
o c
u E E ~ ~:
~1 0 . ~ .:
~ u
c~ ~ o ,~
~ ...
~ u --
L) C
~ O : `
~ , ~ ;
~ :~
-19- -

Example 3
Example No. 2 was repeated, except that the ~eagent
layer contained as a calcium indicator 0.48 G/M2
arsenazo III, rather than chorophosphonazo III. The reagent
layer was buffered to a ph of 5.6. The resulting element
demonstated a dye density comparable to that of the test
element of example 2.
-20