Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1~33813
It is well known to perform a quantitati~e or semi-
quantitative analysis of an aqueous liquid by contacting that
liquid with an analytical element containing a combination of
reagents capable of yielding a detectable product in proportion
to the concentration of a predetermined analyte in the aqueous
5 liquid. As used herein, "reagent" is intended to mean a material
that is interactive with the predetermined analyte, with a pre-
cursor of that analyte, or with any material produced during the
analysis of that analyte. Such interaction refers to chemical re-
activity, catalytic activity, or any other form of chemical or
10 physical interaction that can result in the ultimate production
of a change in the element that is detectable by suitable measure-
ment of radiant energy, usually in the visible light range of the
spectrum. The term "predetermined analyte" is meant to refer to
the substance whose concentration is intended to be measured
15 during the analysis.
One group of particularly useful analytical elements
utilizes an enzymatic assay wherein the predetermined analyte,
upon contact with the analytical element, reacts with oxygen in
the presence of a suitable enzyme contained in the element to
20 produce a peroxide in proportion to the concentration of the pre-
determined analyte in the aqueous liquid bein~ analyzed. A de-
tectable product is then yielded ~y the reaction of this peroxide
with an indicator composition in the presence of peroxidase, both
of which are also contained in the analytical element. The de-
25 tectable product is formed within the element in direct propor-
tion to the peroxide present and thus, also in propGrtiOn to the
concentration of the predetermined analyte in the aqueous liquid.
Elements and analyses of this type are described for example, in
U.S. Patent No. 3,992,158 and in DEOS 2,735,~90. Such elements
~.~,, ~
11338i3
u~ually contain the reagents described above, including peroxidas~
in a carrier that is permeable to aqueous liquids and to the pre-
determined analyte of choice. The carrier may be the main material
forming the element or ~ust one layer in a multi-layered element.
Its permeability assures that the aqueous liquid and predetermined
5 analyte will come into contact with each of the test reagents con-
tained in the carrier when such aqueous li~quid is brought into
contact with the carrier.
One of the advantages of such analytical elements is
that analyses can be performed quickly and reliably by persons
10 with little technical trainîng and no access to a "wet" chemical
laboratory. ~ physician, for înstance, might find it extremely
useful, as an aid to diagnoses, to store a supply of various
elements of this type for use in "on-the-spot" analyses of body
fluids such as urine or blood serum.
Vnfortunately, because of the carrier's necessary
permeability to aqueous liquids, the reagents contained in the
carrier may deteriorate during significant periods of storage and
thus deleteriously affect the accuracy and reliability of the
analysis. For example, exposure to air and moisture may ad-
20 versely affect the ability of peroxidase, contained in a test
element, to catalyze the oxidation of an indicator composition
by a peroxide, thus preventing the formation of detectable pro-
duct accurately and consis~ently in proportion to the concentra-
tion of predetermined analyte.
In order to improve the stability of reagents during
periods of storaye it has been thought desirable to construct the
carrier totally from a hydrophobic material to block moisture and
air from penetrating to the test reagents, such as in U.S. Patent
No. 3,630,~57. Unfortunately, such carriers are not sufficiently
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permeable to aqueous liquids and therefore would not be useful for
analyses which require that aqueous liquids be able to penetrate
to the reagents.
U S. Patent Nos. 3,212,855 and 3,598,704, suggest
adding water-soluble polymers or hydrophilic colloids to a bibu-
5 lous carrier to keep test reagents physically separated and tohelp preYent deterioration of such reagents because of the effects
of moisture. Such polymers may slow the rate of penetration of
moisture and air to test reagents. However, since they are water
soluble polymers, it is clear that moisture will eventually pene-
lO trate them. The protection they afford is therefore limited
U.S. Patent No. 3,616,251, suggests imbedding thereagents into the surface of a carrier comprising a hydrophobic
polymer by using an organic solvent, but the stated purpose is to
prevent the reagents from washing away when contacted with aqueous
15 liquids, and it appears that the reagents would still be sus-
ceptible to the deteriorative chemical effects of moistureand air.
A different problem may additionally occur when an
analytical element comprises a carrier p~rmeable to aqueous
liquids which is coated on a hydrophobic support material. It in-
20 volves unwanted curl. This is caused by absorption and evaporatDnof moisture in the carrier resulting in swelling and shrin~ing of
the carrier relative to the support material, which does not ab-
sorb or evaporate moisture. This problem is well known in the
photographic industry, where materials such as gelatin are coated
25 on hydrophobic support materials to form photographic films. U.S.
Patent No. 3,459,790, suggests the inclusion of certain hydro-
phobic polymers in gelatin layers of photographic films to al-
leviate this problem.
In light of the discussion abo~e, it would be desir-
13
able if an analytical element could be devised which comprises a
carrier that is permeable to aqueous liquids and contains reagents
such as peroxidase, but wherein the analytical element would not
suffer from the above-mentioned problem of instability of react-
ants, such as peroxidase, during periods of storage.
It has been found that certain copolymers, when dis-
persed in carriers permeable to aqueous liquids, can provide the
desirable effect of improved stability of reagents such as per-
oxidase. One would not predict that the stability of peroxidase
would be improved, because, although the copolymers of choice are
10 copolymers derived in large part from hydrophobic, addition-poly-
merizable monomers, they are merely dispersed within a carrier
permeable to aqueous liquids, and this carrier remains very per-
meable to aqueous liquids and to oxygen. One would expect that,
since moisture and oxygen can still penetrate the carrier andcome
15 into contact with the reagents, reagents such as peroxidase, which
could deteriorate during periods of storage in the prior art ele-
ments, would also deteriorate in the present elements However,
this is not in the present elements. However, this is not the
ca~e. Although the reason for this result is not understood at
20 this time, inclusion of the copolymers described hereinafter in
fact significantly improves reagent stability even though the
ability of the carrier to be permeated by moisture and oxygen is
not affected.
Brief Description of the Drawings
Figure 1 is a plot which il~ustrates the efect of
the addition of latex on the fresh response of leuco dye elements
Figure 2 is a series of plots which illustrate the
improved peroxidase stability of an element containing latex.
Accordingly, the invention provides an improved ele-
30 ment for the analysis of a predetermined analyte in an aqueous
liquid. The element co~:prises a carrier that is permeable to the
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predetermined analyte and to aqueous liquids. Alternatively, the
carrier may be one layer of a multi-layered element. Dispersed in
the carrier are reagents (one of which is peroxidase) which are
capable of interacting with or facilitating interaction with the
predetermined analyte or its reaction products to yield a detect-
5 able product. Also dispersed in the carrier is a copolymer com-
prising: from about 80 to about 98 percent by weight of recurring
units derived from one or more hydrophobic addition-polymerizable
monomers; from about 1 to about 20 percent by weight of recurring
units derived from one or more anionic monomers; and from 0 to
10 about 15 percent by weight of recurring units derived from one or
more cross-linkable, active methylene group-containing monomers.
The copolymer should normally comprise from about 20
to about 50 percent of the total weight of the carrier plus the
hydrophobic copolymer.
Preferred classes of these copolymers comprise: from
about 80 to about 98 percent by weight of recurring units derived
from one or more hydrophobic, addition-polymerizable monomers
such as alkyl acrylates, alkyl methacrylates, styrene, and sub-
stituted styrenes; from about 1 to about 20 percent by weight of
20 recurring units derived from one or more anionic monomers such as
acrylic acid, methacrylic acid, sulfonic acid group-containing
monomers, sulfonate group-containing monomers, sulfate group-con-
taining monomers, phosphate group-containing monomers, and phos-
phonate group-containing monomers including the salts of the
25 aforementioned acids, preferably the ammonium or alkali metal
salts thereof, preferred monomers being s~lfoal~yl acrylates,
sulfoalkyl methacrylates, sulfoalkylacrylamides, and sulfoalkyl-
methacrylamides, for example, as described in U.S. Patents
2,923,734 and 3,5Q6,707; and 0-15, preferably 2 to 10, weight
30 percent of recurring units derived from one or more crosslinkable,
acti~e methylene group-containing monomers such as described in
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,~,~
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U.S. Patents 3,459,79Q, 3,929,482, and 3~39~130, a preferred
monomer being 2-acetoacetoxyethyl methacrylate.
These preferred classes include, among others, the
following specific copolymers which have been found particularly
5 useful in the analytical elements of this invention:
Poly~methyl acrylate-co-3-acryloyoxypropane-
sulfonic acid, sodium salt-co-2-acetoacetoxyethyl
methacrylate) (weight ratio 88.75:4.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methyl-
propanesulfonic acid-co-2-acetoacetoxyethyl
methacrylate) (weiyht ratio 88.75:4.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methyl-
propanesulfonic acid-co-2-acetoacetoxyethyl
methacrylate) (weight ratio = 85:10:5);
Poly(n-butyl acrylate-co-3-acryloyloxypropane-
sulfonic acid, sodium salt-co-2-acetoacetoxyethyl
methacrylate~ (weight ratio 91:25:4.75:4.0);and
Poly(n-butyl acrylate-co-2-acrylamido-2-
methylpropane sulfonic acid-co-2-acetoacetoxyethyl
methacrylate) (weight ratio 85:10:5).5
The methods of making such copolymers are well known
to those skilled in the art and the nomers are either readily
available or their methods of synthesis are well known.
As previously stated, the useful copolymers described
above are dispersed in a carrier which may itself form the bulk
of the analytical element, or alternatively, the carrier may be
only one layer of a multi-layered element. In either case the
carrier comprises a material permeable to aqueous liquids and to
the predetermined analyte. The choice of a material is, of couxse,
variable and dependent on the intended use of the element and may
comprise naturally occurring hydrophilic substances like gelatin,
gelatin derivatives, hydrophilic cellulose derivatives, poly-
saccharides such as dextran, gum arabic, agarose and the like,
and also synthetic hydrophilic substances such as water-solubie
polyvinyl compounds like poly(vinyl alcohol, and poly~vinyl pyr-
rolidone), water-soluble acrylamide polymers, etc. The choice may
also depend partly on optical properties of the resultant carrier
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}813
if photometric sensing of the analytical result is intended.
If the carrier is one layer of a multi-layered elemen~
there may also be other layers such as spreading, reflecting,
blocking, subbing, supportt filtration, or registration layers as
described in U.S. Patent Nos. 3,992,158 and 4,042,335. These
5 patents also describe methods widely used to fabricate such multi-
layered elements. Particularly useful layers are a support layer
comprising a hydrophobic material like polyethylene terephthalate
onto which the carrier is coated and a spreading-reflecting layer
coated over the carrier and comprising a blushed polymer and a
10 pigment, for example~ cellulose acetate and titanium dioxide.
Furthermore, the element may incorporate multiple carrier layers.
In addition to the copolymer of choice, the carrier
has distributed within it one or more reagents necessary to inter-
act with, or facilitate interaction with, the predetermined anal-
15 yte or its reaction products to yield a detectable product. Inanalytical elements of this invention peroxidase is one of these
reagents. The choice of others depends on the analysis to be
performed. For example, other reagents might include a suitable
enzyme to catalyze the oxidation of the predetermined analyte to
20 yield a peroxide, an indicator composition capable of reacting
with peroxide in the presence of peroxidase to yield a detectable
product, a compound for controlling pH, etc. A number of useful
indicator compositions are described in ~EOX 2,735,690. Such
specific examples include an element for the analysis of glucose
25 which might contain such reagents as glucose oxidase, peroxidase,
7-hydroxy-1-napthol, 4-amino-antipyrene HCl, and a phosphate
buffer system at pH 6; and an element for the analysis of uric
acid which might contain such reagents as bis(vinylsulfonylmethyl)
ether, uricase, peroxidase, a leuco dye in a suitable solvent
30 such as a dispersion o~ 2(3,5-dimethoxy-4-hydroxyph~nyl) 4,5-bis-
(4-dimethylamino phenyl) imidazole in 2,4-di-n-pen-tylphenol, and
a borate buffer at pH 9.
~,
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The carrier will typically be prepaxed by coating a
~olution or dispersion of carrier material, copolymer, and rea-
gent~ on à surface from which the dried film or layer of carrier
can then be physically stripped. If the element is multi-layered,
multiple coatings can be made directly ~pon each other as des-
cribed in U.S. Patent No. 3,992,158. Particular reagents will beadded in the proportions already known in the art. The copolymer
of choice will usually be incorporated in proportions such that
the copolymer will comprise from about 20 percent to about 50 pe~
cent of the total weight of the carrier plus the copolymer.
As previously described, the elements of the present
inyention are used by contacting them with the aqueous liquid to
be analyzed and calculating the concentration of predetermined
analyte in the aqueous liquid from a spectrophotometric measure-
ment of the density of detectable product formed within the ele-
ment. ~ variety of different elements can be prepared in accor-
dance with the invention and can be adapted for use in carrying
out a wide variety of chemical analyses, not only in the field of
clinical chemistry but also in chemical research and in chemical
proce~s control applications.
The following examples are provided to further illus-
trate specific embodiments of the invention and their advantages
over analytical elements of the prior art. In these examples
"Copolymer No. 1" refers to
Poly(methyl acrylate-co-3-acryloyloxypropane-
sulfonic acid, sodium salt-co-2-acetoacetoxyethyl
methacrylate) (weight ratio 88.75:4.75:6.5).
and "Copolymer No. 2" refers to
Poly(methyl acrylate-co-2-acrylamido-2-methyl-
propanesulfonic acid-co-2-acetoacetoxyethyl
methacrylate (weight ratio 88.75:4.75:6.5).
Example 1 - Improved Reagent Stability In Elements For Glucose
Analysis
Several multilayer elements fGr the analysis of glu-
cose in aqueous liquids were prepared, differing in formulations
_g _
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as indicated:
Element A -- Control (contains none of Copolymer No. 1)
A polyethylene terephthalate film support was coated
with a carrier comprising deionized gelatin (21.5 g/m ), glycerol
(2.15 g/m2), glucose oxidase (10,000 U/m2), peroxidase (10,000
U/m ), 7-hydroxy-1-naphthol (0.66 g/m2), 4-amino-antipyrine HC:l
(0.86 g/m ), and a phosphate buffer system (pH 6.0). A subbing
layer and a blushed polymer spreading layer comprising cellulose
acetate (6.6 g/m2) and Tio2 (46,0 g/m2) were then applied.
_ ement B -- (Contains Copolymer No~ 1)
Same as Element A except that in place of glycerol
Polymer No. 1 (21.5 g/m2) was added.
Element C -- (Contains less gelatin and more~Copoly-
_ r No. 1 than Element B)
Same as Element B except that the amount of dionized
gelatin in the carrier was 16 g/m2 and the amount of Copolymer
No. 1 was 24.2 g/m .
The elements were stored at room temperature (26C)
and 50~ RH. Density readings at a wavelength of 540 nm were then
taken of the background (i.e. dry element) and also of elements
spotted with standard solutions containing 800 mg% gluco~e. Results,
shown in Table 1, indicate that the elements containing Copolymer
No. 1 exhibited significantly less decrease in the density in a
dry element (background) and in the density in an element spo~ed
with glucose solution after storage for 16 and 24 weeks than the
elements containing none of Copolymer No. 1. This indicates
significantly improved reagent stability in the elements con-
taining Copolymer No. 1.
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1~38~3
Table 1
E fect of Copolymer No. 1 on Elements For Glucose Assay
Storage % Deterioration
ElementTime (~ Change in Density From Fresh Element)
(weeks)Dry Element Glucose-Spotted Element
(control)A 16 46 20
B 16 11 5
C 16 0 4
(control)A 24 70 19
B 24 26 0
~ C 24 9 2
Example 2 - Comparison Of Two Copolym-ers
Two elements (D and E) were prepared as in Example 1
except that the carrier of element D contained 16.0 g/m of de-
ionîzed gelatin and 16.0 9/m2 of Copolymer No. 1, and element E
contained 16 g/m2 of deionized gelatin 16.0 g/m2 of Copolymer No.
2. Also, the buffer system used in the elements was 3,3-dimethyl
glutaric acid (1.96 g/m2) at pH 5Ø
Density readings were taken of the dry background and
of elements spotted with a standard solution containing600m~ glu-
cose after keeping for 24 weeks at room temperature (26C and 50%
RH), refrigerated (6C, 50~ RH) and frozen (-23C, 50~ RH).
As shown in Table 2, these copolymers behaved es-
sentially the same regarding stability and sensitivity to the
glucose standard. ~Note that the background density readings were
made in millivolts, which are inversely proportional to density
units.)
Table 2
_omparison Of Copolymers Nos. 1 and 2
Element Temp. C/~ RH Background D
m~G~ucose-Spotted Element
D26/50 727 1.406
6/50 750 1.368
-23/50 776 1.393
E26/50 713 1.407
6/50 737 1.377
-23/50 776 1.341
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Example 3 ImiproveiddKeeping in Elements Fox The Determination Of
Several multilayer elements for the analysis of uric
acid in aqueous liquids were prepared according to the following:
A polyethylene terephthalate film support was coated
with a carrier comprising deionized gelatin (10.8 g/m2), bis(vinyl-
sulfonylmethyl ether) (0.129 g/m2), peroxidase (6500 U/m2), uricase
(215 U/m2), a dispePsion of 2(3,5-dimethoxy-4-hydroxyphenyl)-4,5-
bis(4-dimethylamino phenyl) imidazole (.14 g/m2) in 2,4-di-n-amyl-
phenol (1.35 g/m2), and borate buffer at pH 9.
In addition, the carrier layers of Elements G and H
contained 5.4 g/m2 and 10.8 g~m2 of Copolymer No. 1, respectively.
Element F contained no such copolymer.
Above the carrier layer was coated a gelatin layer
comprising deionized gelatin (5.4 g/m ), and bis(vinyl sulfonyl
methyl)ether (0.065 g/m2) in borate buffer at pH 9, a subbing
layer comprising poly(n-isopropyl acrylamide) (.3 g/m2) and a
spreadinq layer comprising TiO2 (46.0 g/m2) and cellulose acetate
(6.6 g/m ).
The elements were then evaluated for changes in
density of dry background and density after spotting with a 15.0
mg% uric acid solution after incubation for 0, 8, and 24 weeks at
26C /50% RH.
As can be seen in Table 3, inclusion of Copolymer No.
l in the carrier layer o~ the uric acid element produces sub-
stantially less change in dry background and response over time,
thereby increasing the useful life of the element significantly.
Essentially the same results were obtained when Co-
polymer No. 2 was used in the uric acid element instead of Copo~
mer No. l.
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Ta~le 3
Effect of Copolymer No. 1 On Elements For Uric Acid Assay
Storage % Deterioration
5 Element Time (~ Change In Density From Fresh Element)
reeks) Dry Element Uric Acid-Spotted Element
(control)F 8 14 *
G 8 7 *
10 H 8 4 *
(control)F 24 16 59
G 24 ll 32
H 24 8 30
*Data not available.
Example 4
Multilayer analytical elements for the determination
of triglycerides in body fluids were prepared as follows:
A polyethylene terephthalate support was coated with
a chemistry layer comprising deionized gelatin (5.4 g/m2), dime-
done (0.33 g/m ), Triton X-100TM(0.39 g/m2), 2-(3,5-dimethoxy-4-
hydroxyphenyl)-4,5-bis(4-dimethylaminophenyl) imidazole (0.4 g/m~
in (2,4-di-n-amylphenol)(4.0 g/m2), ~bis(vinylsulfonylmethyl)~
ether (BVSME) (0.03 g/m ), MgC12 (0.01 g/m ), ATP (1.35 g/m2),
peroxidase (6997 ~/m2), glycerol kinase (323 U/m2), -glycero-
phospha~e oxidase (2831 U/m2), Copolymer No. 1 (5.38 g/m2) and
0.05 M phosphate buffer, pH 7.0; a gel pad comprising deionized
gelatin (5.4 g/m ), Triton X-100T (0.06 g/m ), BVSME (0.03 g/m~,
Copolymer No. 1 (5.4 g/m2) and 0.05 M phosphate buffer, pH 7.0;
an enzyme layer comprising Lipase M ~3.24 g/m2), poly ~,-isopro-
pylacrylamide-co-2-(methacryloyloxy) ethyltrime~hylammonium
methosulfate-co-2-hydroxyethyl acrylate~ (weight ratio 70:20:lQ)
(1.08 g/m ), and Triton X-100TM (0.08 g/m ); a subbing layer
comprising poly-n-isopropylacrylamide; and a spreading layer
comprising TiO2 (50.0 g/m ), cellulose acetate (7.0 g/m ),
Triton X-100T (5.5 g/m2).
A second element was prepared in the same manner,
except no ~opolymer No. 1 was included in either the chemistry
layer or gel pad. The freshly prepared elements were each tested
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1133813
with solutions containing 0-300 mg/dl txiglyceride. Figure 1
shows that higher densities are obtained with this dye system
when a copolymer such as Copolymer No. 1 is included.
Example 5
Elements, prepared as described in Example 4 above,
were incubated for 4 weeks at 26C and 50~ RH and 8 weeks at 6C
and 50% RH. The elements were subsequently tested with 1) Cordis,
a commercial preparation of triglycerides ~3.52 mM or 340 mg/dl);
2) glycerol (~3.52 mM in 7% albumin and 0.155 M NaCl); 3) a-gly-
cerophosphate (7.04 mM in 7% albumin/saline); 4) H202 (3~52 mM in
water).
Results, shown in Figure 2 (a, b, c, d~ indicate that
the addition of Copolymer No. 1 greatly improves peroxidase sta-
bility in this element. As shown in Curve B, however, glycerol
kinase stability is not improved by Copolymer No. 1 at 26C and
50% RH.
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