CA1133813A - Analytical elements with improved reagent stability - Google Patents
Analytical elements with improved reagent stabilityInfo
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- CA1133813A CA1133813A CA309,175A CA309175A CA1133813A CA 1133813 A CA1133813 A CA 1133813A CA 309175 A CA309175 A CA 309175A CA 1133813 A CA1133813 A CA 1133813A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/525—Multi-layer analytical elements
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/62—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving uric acid
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Abstract
ANALYTICAL ELEMENTS WITH IMPROVED REAGENT STABILITY
Abstract Elements for the analysis of aqueous liquids are described. Deterioration of reagents, particularly peroxidase, during storage is reduced by inclusion in such elements of a copolymer derived from hydrophobic, addition-polymerizable monomers.
Abstract Elements for the analysis of aqueous liquids are described. Deterioration of reagents, particularly peroxidase, during storage is reduced by inclusion in such elements of a copolymer derived from hydrophobic, addition-polymerizable monomers.
Description
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 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-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 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
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 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-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 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 ,~,~
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 }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.
~, 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 _ 1~313 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.
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 (control)A 24 70 19 ~ 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 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.
-`- 1133813 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 *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 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.
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 }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.
~, 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 _ 1~313 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.
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 (control)A 24 70 19 ~ 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 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.
-`- 1133813 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 *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 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.
Claims (30)
1. In an element for the analysis of a predetermined analyte in an aqueous liquid, said element comprising:
a) a carrier permeable to said aqueous liquid and to said predetermined analyte and b) dispersed in said carrier, reagents capable of interacting with or facilitating interaction with said predetermined analyte or its reaction products to yield a detectable product, one of said reagents comprising peroxidase, the improvement comprising having dispersed in said carrier in addition to said reagents a polymer in an amount sufficient to impart stability to said peroxidase, said amount comprising from about 20 percent to about 50 percent of the total weight of said carrier material plus said polymer, said polymer comprising:
1) from about 80 to about 98 percent by weight of recurring units derived from one or more hydro-phobic, addition-polymerizable monomers selected from the group consisting of an alkyl acrylate, an alkyl methacrylate, styrene, and a substituted styrene, 2) from about 1 to about 20 percent by weight of recurring units derived from one or more anionic monomers selected from the group consisting of an acrylic acid, an acrylic acid salt, a methacrylic acid, a methacrylic acid salt, a sulfonic acid, a sulfonic acid salt, a sulfonate, a sulfate, a phosphate, and a phosphorate, and 3) from 0 to about 15 percent by weight of recurring units derived from one or more crosslinkable, ac-tive methylene group-containing monomers.
a) a carrier permeable to said aqueous liquid and to said predetermined analyte and b) dispersed in said carrier, reagents capable of interacting with or facilitating interaction with said predetermined analyte or its reaction products to yield a detectable product, one of said reagents comprising peroxidase, the improvement comprising having dispersed in said carrier in addition to said reagents a polymer in an amount sufficient to impart stability to said peroxidase, said amount comprising from about 20 percent to about 50 percent of the total weight of said carrier material plus said polymer, said polymer comprising:
1) from about 80 to about 98 percent by weight of recurring units derived from one or more hydro-phobic, addition-polymerizable monomers selected from the group consisting of an alkyl acrylate, an alkyl methacrylate, styrene, and a substituted styrene, 2) from about 1 to about 20 percent by weight of recurring units derived from one or more anionic monomers selected from the group consisting of an acrylic acid, an acrylic acid salt, a methacrylic acid, a methacrylic acid salt, a sulfonic acid, a sulfonic acid salt, a sulfonate, a sulfate, a phosphate, and a phosphorate, and 3) from 0 to about 15 percent by weight of recurring units derived from one or more crosslinkable, ac-tive methylene group-containing monomers.
2. An element as described in claim 1 wherein said reagents further comprise an enzyme capable of catalyzing the oxidation of said predetermined analyte to a peroxide and an indicator composition oxidizable by said peroxide in the presence of peroxidase to yield a detectable product.
3. An element as described in claim 1 wherein said reagents further comprise a compound for adjusting pH.
4. An element as described in claim 1 wherein said carrier comprises a hydrophilic colloid selected from the group consisting of gelatin, a gelatin derivative, a cellulose derivative, a polysaccharide, an acrylamide, poly(vinyl alcohol), and poly(vinyl pyrrolidone).
5. An element as described in claim 1 which comprises more than one layer, said carrier comprising one of said layers.
6. An element as described in claim 1 wherein said carrier comprises a layer coated on a support material
7. An element as described in claim 1 wherein a subbing layer is coated on said carrier and a spreading-reflecting layer is coated on the subbing layer.
8. In an element for the analysis of a predetermined analyte in an aqueous liquid, said element comprising:
a) a carrier permeable to said aqueous liquid and to said predetermined analyte and b) dispersed in said carrier, reagents capable of interacting with or facilitating interaction with said predetermined analyte or its reaction products to yield a detectable product, one of said reagents comprising peroxidase, the improvement comprising having dispersed in said carrier in addition to said reagents a polymer comprising:
1) from about 80 to about 98 percent by weight of recurring units derived from one or more hydrophobic, addition-polymerizable monomers selected from the group consisting of an alkyl acrylate, an alkyl methacrylate, styrene, and a substituted styrene, 2) from about l to about 20 percent by weight of recurring units derived from one or more anionic monomers selected from the group consisting of an acrylic acid, an acrylic acid salt, a methyacrylic acid, a methyacrylic acid salt, a sulfonic acid, a sulfonic acid salt, a sulfonate, a sulfate, a phosphate, and a phosphorate, and 3) from 0 to about 15 percent by weight of recurring units derived from one or more crosslinkable, active methylene group-containing monomers, said polymer comprising from about 20 percent to about 50 percent of the total weight of said carrier plus said polymer.
a) a carrier permeable to said aqueous liquid and to said predetermined analyte and b) dispersed in said carrier, reagents capable of interacting with or facilitating interaction with said predetermined analyte or its reaction products to yield a detectable product, one of said reagents comprising peroxidase, the improvement comprising having dispersed in said carrier in addition to said reagents a polymer comprising:
1) from about 80 to about 98 percent by weight of recurring units derived from one or more hydrophobic, addition-polymerizable monomers selected from the group consisting of an alkyl acrylate, an alkyl methacrylate, styrene, and a substituted styrene, 2) from about l to about 20 percent by weight of recurring units derived from one or more anionic monomers selected from the group consisting of an acrylic acid, an acrylic acid salt, a methyacrylic acid, a methyacrylic acid salt, a sulfonic acid, a sulfonic acid salt, a sulfonate, a sulfate, a phosphate, and a phosphorate, and 3) from 0 to about 15 percent by weight of recurring units derived from one or more crosslinkable, active methylene group-containing monomers, said polymer comprising from about 20 percent to about 50 percent of the total weight of said carrier plus said polymer.
9. An element as described in claim 8 wherein said reagents further comprise an enzyme capable of catalyzing the oxidation of said predetermined analyte to a peroxide and an indicator composition oxidizable by said peroxide in the presence of peroxidase to yield a detectable product.
10. An element as described in claim 8 wherein said reagents further comprise a compound for adjusting pH.
11. An element as described in claim 8 wherein said carrier comprises a hydrophilic colloid selected from the group consisting of gelatin, a gelatin derivative, a cellulose derivative, a polysaccharide, an acrylamide, poly-(vinyl alcohol), and poly(vinyl pyrrolidone).
12. An element as described in claim 8 which com-prises more than one layer, said carrier comprising one of said layers.
13. An element as described in claim 8 wherein said carrier comprises a layer coated on a support material.
14. An element as described in claim 8 wherein a subbing layer is coated on said carrier and a spreading-reflecting layer is coated on the subbing layer.
15. In an element for the analysis of a predeter-mined analyte in an aqueous liquid, said element comprising:
a) a carrier permeable to said aqueous liquid and to said predetermined analyte and b) dispersed in said carrier, reagents capable of interacting with or facilitating interaction with said predetermined analyte or its reaction products to yield a detectable product, one of said reagents comprising peroxidase, the improvement comprising having dispersed in said carrier in addition to said reagents a polymer in an amount suffi-cient to impart stability to said peroxidase, said amount comprising from about 20 percent to about 50 percent of the total weight of said carrier material plus said polymer, said polymer selected from the group consisting of:
Poly(methyl acrylate-co-3-acryloxyloxypropanesulfonic 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) (weight ratio 88.75:4.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methyl ?
propanesulfonlc acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio = 85:10:5);
Poly(n-butyl acrylate-co-3-acryloyloxypropane ?
sulfonlc 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).
a) a carrier permeable to said aqueous liquid and to said predetermined analyte and b) dispersed in said carrier, reagents capable of interacting with or facilitating interaction with said predetermined analyte or its reaction products to yield a detectable product, one of said reagents comprising peroxidase, the improvement comprising having dispersed in said carrier in addition to said reagents a polymer in an amount suffi-cient to impart stability to said peroxidase, said amount comprising from about 20 percent to about 50 percent of the total weight of said carrier material plus said polymer, said polymer selected from the group consisting of:
Poly(methyl acrylate-co-3-acryloxyloxypropanesulfonic 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) (weight ratio 88.75:4.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methyl ?
propanesulfonlc acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio = 85:10:5);
Poly(n-butyl acrylate-co-3-acryloyloxypropane ?
sulfonlc 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).
16. In an element for the analysis of a predetermined analyte in an aqueous liquid, said element comprising:
a) a carrier permeable to said aqueous liquid and to said predetermined analyte and b) dispersed in said carrier, reagents capable of interacting with or facilitating interaction with said predetermined analyte or its reaction pro-ducts to yield a detectable product, one of said reagents comprising peroxidase, the improvement comprising having dispersed in said carrier in addition to said reagents a polymer selected from the group consisting of Poly(methyl acrylate-co-3-acryloyloxypropane ?
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) (weight ratlo 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), said polymer comprising from about 20 percent to about 50 percent of the total weight of said carrier plus said polymer.
a) a carrier permeable to said aqueous liquid and to said predetermined analyte and b) dispersed in said carrier, reagents capable of interacting with or facilitating interaction with said predetermined analyte or its reaction pro-ducts to yield a detectable product, one of said reagents comprising peroxidase, the improvement comprising having dispersed in said carrier in addition to said reagents a polymer selected from the group consisting of Poly(methyl acrylate-co-3-acryloyloxypropane ?
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) (weight ratlo 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), said polymer comprising from about 20 percent to about 50 percent of the total weight of said carrier plus said polymer.
17. An element as described in claim 15 wherein said reagents further comprise an enzyme capable of catalyzing the oxidation of said predetermined analyte to a peroxide and an indicator composition oxidizable by said peroxide in the presence of peroxidase to yield a detectable product.
18. An element as described in claim 15 wherein said reagents further comprise a compound for adjusting pH.
19. An element as described in claim 15 wherein said carrier comprises a hydrophilic colloid selected from the group consisting of gelatin, a gelatin derivative, a cellulose derivative, a polysaccharide, an acrylamide, poly(vinyl alcohol), and poly(vinyl pyrrolidone).
20. An element as described in claim 15 which comprises more than one layer, said carrier comprising one of said layers.
21. An element as described in claim 15 wherein said carrier comprises a layer coated on a support material.
22. An element as described in claim 15 wherein a subbing layer is coated on said carrier and a spreading-reflecting layer is coated on the subbing layer.
23. In an element for the analysis of glucose in aqueous liquids, said element comprising:
a) a carrier permeable to said aqueous liquid and to glucose and b) dispersed in said carrier, reagents comprising glucose oxidase, peroxidase, 7-hydroxy-1-naphthol, 4-amino-antipyrine HCl, and a phosphate buffer system (pH 6), the improvement comprising having dispersed in said carrier in addition to said reagents a polymer in an amount suffi-cient to impart stability to said peroxidase, said amount comprising from about 20 percent to about 50 percent of the total weight of said carrier material plus said polymer, said polymer being selected from the group consisting of:
Poly(methyl acrylate-co-3-acryloyloxypropanesulfonic acid, sodium salt-co-2-acetoacetoxyethyl methacrylate) (weight ratio 88.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 88.75:4.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 85:10:5);
Poly(n-butyl acrylate-co-3-acryloyloxypropanesulfonic acid, sodium salt-co-2-acetoacetoxyethyl methacrylate) (weight ratio 91.25:4.75:4.0); and Poly(n-butyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 85:10:5).
a) a carrier permeable to said aqueous liquid and to glucose and b) dispersed in said carrier, reagents comprising glucose oxidase, peroxidase, 7-hydroxy-1-naphthol, 4-amino-antipyrine HCl, and a phosphate buffer system (pH 6), the improvement comprising having dispersed in said carrier in addition to said reagents a polymer in an amount suffi-cient to impart stability to said peroxidase, said amount comprising from about 20 percent to about 50 percent of the total weight of said carrier material plus said polymer, said polymer being selected from the group consisting of:
Poly(methyl acrylate-co-3-acryloyloxypropanesulfonic acid, sodium salt-co-2-acetoacetoxyethyl methacrylate) (weight ratio 88.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 88.75:4.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 85:10:5);
Poly(n-butyl acrylate-co-3-acryloyloxypropanesulfonic acid, sodium salt-co-2-acetoacetoxyethyl methacrylate) (weight ratio 91.25:4.75:4.0); and Poly(n-butyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 85:10:5).
24. An element as described in claim 23 wherein said carrier comprises a hydrophilic colloid selected from the group consisting of gelatin, a gelatin derivative, a cellulose derivative, a polysaccharide, an acrylamide, poly-(vinyl alcohol), and poly(vinyl pyrrolidone).
25. An element as described in claim 23 wherein said polymer comprises from about 20 percent to about 50 per-cent of the total weight of said carrier plus said polymer.
26. An element as described in claim 23 wherein the carrier is a layer comprising deionized gelatin coated on a polyethylene terephthalate film support and said carrier is overcoated with a subbing layer and a blushed-polymer, spreading-reflecting layer comprising cellulose acetate and titanium dioxide.
27. In an element for the analytis of uric acid in aqueous liquids, said element comprising:
a) a carrier permeable to said aqueous liquid and to uric acid and b) dispersed in said carrier reagents comprising bis-(vinylsulfonylmethyl)ether, uricase, peroxidase, a dispersion of 2(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-dimethylamino phenyl)imidazole in 2,4-di-n-pentylphenol, and a borate buffer (pH 9), the improvement comprising having dispersed in said carrier in additlon to said reagents a polymer in an amount suffl-cient to impart stability to said peroxidase, said amount comprising from about 20 percent to about 50 percent of the total weight of said carrier material plu8 said polymer, said polymer being selected from the group consistlng of:
Poly(methyl acrylate-co-3-acryloyloxypropanesulfonic acid, sodium salt-co-2-acetoacetoxyethyl methacrylate) (weight ratio 88.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 88.75:4.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 85:10:5);
Poly(n-butyl acrylate-co-3-acryloyloxypropanesulfonic acid, sodium salt-co-2-acetoacetoxyethyl methacrylate) (weight ratio 91.25:4.75:4.0); and Poly(n-butyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 85:10:5).
a) a carrier permeable to said aqueous liquid and to uric acid and b) dispersed in said carrier reagents comprising bis-(vinylsulfonylmethyl)ether, uricase, peroxidase, a dispersion of 2(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-dimethylamino phenyl)imidazole in 2,4-di-n-pentylphenol, and a borate buffer (pH 9), the improvement comprising having dispersed in said carrier in additlon to said reagents a polymer in an amount suffl-cient to impart stability to said peroxidase, said amount comprising from about 20 percent to about 50 percent of the total weight of said carrier material plu8 said polymer, said polymer being selected from the group consistlng of:
Poly(methyl acrylate-co-3-acryloyloxypropanesulfonic acid, sodium salt-co-2-acetoacetoxyethyl methacrylate) (weight ratio 88.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 88.75:4.75:6.5);
Poly(methyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 85:10:5);
Poly(n-butyl acrylate-co-3-acryloyloxypropanesulfonic acid, sodium salt-co-2-acetoacetoxyethyl methacrylate) (weight ratio 91.25:4.75:4.0); and Poly(n-butyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-2-acetoacetoxyethyl methacrylate) (weight ratio 85:10:5).
28. An element as described in claim 27 wherein said carrier comprises a hydrophilic colloid selected from the group consisting of gelatin, a gelatin derivative, a cellulose derivative, a polysaccharide, an acrylamide, poly(vinyl alcohol), and poly(vinyl pyrrolidone).
29. An element as described in claim 27 wherein said polymer comprises from about 20 percent to about 50 percent of the total weight of said carrier plus said polymer.
30. An element as described in claim 27 wherein the carrier is a layer comprising deionized gelatin coated on a polyethylene terephthalate film support and said carrier is overcoated with a layer comprising deionized gelatin, bis(vinyl-sulfonylmethyl) ether, borate buffer (pH9), a subbing layer comprising poly(n-isopropyl acrylamide) and a blushed-polymer, spreading-reflecting layer comprising cellulose acetate and titanium dioxide.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US83120477A | 1977-09-06 | 1977-09-06 | |
| US831,204 | 1977-09-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1133813A true CA1133813A (en) | 1982-10-19 |
Family
ID=25258530
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA309,175A Expired CA1133813A (en) | 1977-09-06 | 1978-08-11 | Analytical elements with improved reagent stability |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JPS5450393A (en) |
| CA (1) | CA1133813A (en) |
| DE (1) | DE2838877A1 (en) |
| FR (1) | FR2402209A1 (en) |
| GB (1) | GB2004062B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4388271A (en) | 1980-01-10 | 1983-06-14 | Rohm Gmbh | Rapid diagnostic agents |
| DE3025372A1 (en) * | 1980-07-04 | 1982-01-28 | Behringwerke Ag, 3550 Marburg | AGENT FOR DETECTING PEROXIDATICALLY EFFECTIVE SUBSTANCES AND USE OF POLYVINYLMETHYLACYLAMIDE IN SUCH |
| US4336330A (en) * | 1980-12-15 | 1982-06-22 | Miles Laboratories, Inc. | Device for detecting glucose concentration |
| US4318985A (en) * | 1981-01-29 | 1982-03-09 | Miles Laboratories, Inc. | Method and device for detecting glucose concentration |
| JPS59143959A (en) * | 1983-02-07 | 1984-08-17 | Fuji Photo Film Co Ltd | Dry analytical element |
| JPS6010171A (en) * | 1983-06-30 | 1985-01-19 | Fuji Photo Film Co Ltd | Multilayer analysis element |
| JPH0653074B2 (en) * | 1984-02-24 | 1994-07-20 | 大日本印刷株式会社 | Body fluid test body |
| DE69627439T2 (en) | 1995-09-13 | 2003-12-24 | Kyoto Daiichi Kagaku Kk | Analytical element and method for its production |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3554987A (en) * | 1965-12-20 | 1971-01-12 | Eastman Kodak Co | Novel compounds and photographic materials containing said compounds |
| US3459790A (en) * | 1965-12-20 | 1969-08-05 | Eastman Kodak Co | Polymerizable acrylic acid esters containing active methylene groups |
| FR1588009A (en) * | 1968-10-22 | 1970-04-03 | ||
| US3992158A (en) * | 1973-08-16 | 1976-11-16 | Eastman Kodak Company | Integral analytical element |
| US4038485A (en) * | 1976-03-18 | 1977-07-26 | Miles Laboratories, Inc. | Test composition, device, and method |
-
1978
- 1978-08-11 CA CA309,175A patent/CA1133813A/en not_active Expired
- 1978-09-06 FR FR7825586A patent/FR2402209A1/en active Granted
- 1978-09-06 JP JP10871378A patent/JPS5450393A/en active Granted
- 1978-09-06 GB GB7835848A patent/GB2004062B/en not_active Expired
- 1978-09-06 DE DE19782838877 patent/DE2838877A1/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| FR2402209A1 (en) | 1979-03-30 |
| JPS5450393A (en) | 1979-04-20 |
| DE2838877A1 (en) | 1979-03-29 |
| GB2004062A (en) | 1979-03-21 |
| DE2838877C2 (en) | 1983-10-27 |
| GB2004062B (en) | 1982-03-31 |
| FR2402209B1 (en) | 1983-04-15 |
| JPS5646798B2 (en) | 1981-11-05 |
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Legal Events
| Date | Code | Title | Description |
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| MKEX | Expiry |