ANALYTICAL ELEMENT CONTAINING A BARRIER ZONE AND PROCESS EMPLOYING SAME

Abstract: Multizone element, for quantitative analyte determination in whole blood, containing a porous spreading zone separated from a reagent zone by a barrier zone, said barrier zone comprising a non-porous film comprising a polymer of: from 30 to 95 percent by weight of polymerized monomer having the structure from (I) 0.25 to 30 percent by weight of polymerized monomer having the structure (II) from 0.1 to 50 percent by weight of polymerized monomer having the structure (III) wherein: R<1>, R<3>, and R<5> are independently selected from the group consisting of hydrogen and methyl, R<2>is alkyl of from 1 to 16 carbon atoms, R<4> and R<6> are independently selected from the group consisting of alkylene groups having from 1 to 6 carbon atoms, (IV) wherein R<7> is hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, and M is NR<8>R<9>H<+>X or SO3<->X wherein R<8> and R<9> are independently selected from the group consisting of hydrogen and alkyl of 1 to 4 carbon atoms and X is a counterion. A process for employing the element in analyses for analytes in whole blood is also disclosed.

Description: ANALYTICAL ELEMENT CONTAINING A BARRIER ZONE AND PROCESS EMPLOYING SAME The present invention relates to analytical elements for determination of analytes in whole blood samples. Various analytical elements for clinical analysis have been proposed using "dry chemistry", i.e., analytical clinical techniques wherein chemical reagents are incorporated in various substantially "dry-to-the-touch" elements, such as test strips and multizone analytical test elements. U.S. Patent 3,992,158 issued November 16, 1976 discloses improved multizone analytical elements for the analysis of liquids via "dry chemistry". In the element disclosed, there is present a reagent zone and a spreading zone. When the spreading zone of the element was spotted with a sample of aqueous liquid to be tested for a particular analyte, this zone was found to be especially effective in receiv- ing the liquid test sample and distributing such liquid within itself in a manner to provide a sub¬ stantially uniform concentration of analyte for interaction with the reagent components of the element. The reagent zone contains any of a variety of compositions interactive with the desired analyte to provide, for example, a radiometrically detectable species indicative of the presence and/or concentra¬ tion of the analyte. These analytical elements are used in the analysis of a variety of blood components including glucose, uric acid, and protein, e.g. albumin. In these elements, the spreading zone pore size is generally chosen so that the zone filters out undesirable sample components that interfere with an analytical reaction or with detection of any test result produced within the element. It is taught that for analysis of whole blood, porous zones having a pore size of from 1 to about 5 microns are particu¬ larly useful in screening out blood cells. Also, a filter or dialysis zone can be included at an appro¬ priate location in the element. It is stated that in analyzing for blood glucose, a dialysis zone, such as a semi-permeable cellulose membrane, can prevent passage of proteins and other potentially interfering substances to the reagent zone. U.S. Patent 4,258,001 issued March 24, 1981, describes analytical elements that have barrier compositions having a predetermined selective permeability permitting only selected species to come into fluid contact with particular zones of the multizoned element, such barrier compositions being described in U.S. Patent 4,066,403 issued January 3, 1978. These elements can be used to analyze either serum, plasma, or whole blood. The barrier compositions described in U.S. Patent 4,066,403 that are specifically mentioned therein are cellulose acetate butyrate, cellulose propionate valerate, poly(methyImethacrylate) , and cellulose acetate. Published European Patent Application 10425 is directed to a semi-permeable composite membrane comprising a thin semi-permeable film of a polymeric material deposited on one side of a microporous substrate. The polymeric material is prepared by cross-linking a soluble polymer containing at least 30 mole% of a recurring unit of the formula * I ■ CH 2 -C- Y OMPI wherein , for example , among a number of possibili¬ ties , Y can be a carbonyl group 0 5 II (-C-) ; Ri can be a hydrogen atom or a mono- valent organic radical containing 1 to 20 carbon atoms; π R 2 can be a hydrogen atom; R 3 can be an alkylene group containing 2 to 5 carbon atoms; and Rt, can be a hydrogen atom or a methyl group. Cross-linking is carried out through the , e amino group by means of a polyfunctional compound. These membranes can be used for reverse osmosis. It is said that reverse osmosis can be used for removing impurities from liquids such as water or blood. The polymer used in preparing the se i- 2 Q permeable membranes can be a copolymer containing up to 70 mole% of one or more comonomers. These polymers can be blended with up to 50 mole % of another polymer having the ability to form a water-soluble or aqueous emulsion. The polymer is 2e deposited on a microporous substrate by applying a solution of the base polymer to the substrate. After such deposition, the polymer is cross-linked. Japanese Kokai 129790/79 describes polymers similar to those of the above European application. In use, the polymer is fashioned into a thread-like or granular form and placed in a column. The leucocytes remain in the filter and the erythrocytes are hemolyzed and pass through the filter along with the . plasma. »- Many of these materials, as well as others (see Examples 5-8, infra), were tested by us in an effort to provide an improved element of the "dry assay" type for the determination of analytes , particularly glucose, in whole blood. None, however, were satisfactory. In some instances, the membranes were too permeable; in others, they were not perme- able enough. In either case, the result was an inability to differentiate meaningfully among various analyte concentration levels. Thus, a need remains for analytical elements of the type used in so-called "dry assays", that are improved by the inclusion therein of barrier zones particularly suited for separating analytes from the several interferents present in whole blood. This is the problem to be solved by the present invention. The present invention provides a multizone element, for quantitative analyte determination in whole blood, containing a porous spreading zone separated from a reagent zone by a barrier zone, the barrier zone comprising a non-porous film comprising a polymer of: from 30 to 95% by weight of polymerized monomer having the structure 1 R I 2 CH 2 -C-C00R , (I) from 0.25 to 30% by weight of polymerized monomer having the structure 3 R 0 I II CH 2 -C-C-A-R -M, and (II) from 0.1 to 50% by weight of polymerized monomer having the structure s R 0 I » 6 CH 2 »C-C-0-R -OH (III) C.V.PI where R 1 , R 3 , and R 5 are independently selected from the group consisting of hydrogen and methyl; R 2 is alkyl of from 1 to 16 carbon atoms; R 1 * and R 6 are independently selected from the group consisting of alkylene groups having from 1 to 6 carbon atoms; A is -0- or -N- I? R where R 7 is hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, and M is NR 8 R 9 H + X or S0 3 " X, where R β and R 9 are independently selected from the group consisting of hydrogen and alkyl of 1 to 4 carbon atoms and X is a counterion. By virtue of the present invention, the problem of separating analytaes from interferents has been solved and "dry assay" type elements and processes are now provided from which a meaningful determination of analyte concentration in whole blood samples can be obtained. The problem has been solved by use of the described non-porous film of a particular chemical composition as a barrier zone in the analytical element. Many advantages are provided by the use of the above-described barrier zones, which can be summarized as follows: (1) Whole blood samples, both hemolyzed and non-hemolyzed, can be analyzed. (2) The barrier zone, which is impermeable to large molecular components for the duration of the test, is only partially permeable to the soluble analytes. This partial permeability offers several advantages, i.e, (a) it provides improved kinetics, i.e. greater linearity, and simplifies calibration of the assay, (b) it permits the assay to be insensitive to metered volume, and (c) it permits the use of a variety of detection systems for chosen analytes not useful prior to the present invention. For instance, dyes S . VJ srO having very high extinction coefficients can now be used advantageously. The use of dyes having high extinction coefficients helps to eliminate spectral interferences by enabling the manipulation of the wavelength at which the assay is made. The analytical elements of the present invention comprise at least three zones: a spreading zone, a non-porous barrier zone, and a reagent zone, i.e., a zone in which an analyte or reaction product thereof is acted upon in some way to bring about, either directly or indirectly, a detectable change. A sample applied to the element is first distributed uniformly by the spreading zone so as to present a uniform concentration of material to the adjacent zones, as disclosed, for example, in U.S. Patent 3,992,158. ' The spreading zone is generally the zone upon which the liquid sample to be analyzed, i.e., whole blood, is deposited. A wide variety of spreading and reagent zones are known in the art, the details of which are well described in the patent and other literature. By "non-porous" is meant that the films employed as barriers in the present invention are not of the type described as "microporous membranes" in the Encyclopedia of Polymer Science and Technology, Vol. 8, 1968, page 620. Such microporous membranes are described as having a structure that enables liquids to flow through them according to the normal equations of hydrodynamics and an effective pore size at least several times the mean free path of the molecules, i.e. from several micrometers down to about 100 A (10 nm). Of course, films are con¬ sidered porous, as used herein, if they also have pores larger than this. Although the mechanism of transport through the barrier zone of the elements of the present invention is not known, it is believed that the non-porous films used are of the type "BUREACΓ Oa.PI described on pp. 620 and 621 of the above reference as being "molecular diffusion membranes" or "ultra- filter type membranes". The effective pore size of ultrafilter type membranes is from about 7 to 50 A 5 (0.7 to 5 nm). The "molecular diffusion membranes" have no pores at all in the strict sense of the word. The barrier zones are at least partially permeable to the analyte being determined and are impermeable to interferents for such determination, 10 e.g. hemoglobin. It is understood that the terms "permeable" and "impermeable" as used herein are employed in the practical sense of permeability or impermeability during the time period over which the analyte determination takes place. ■t ζ The polymer that the barrier zone comprises can be prepared by the polymerization of a mixture of monomers comprising from 30 to 95% by weight 1 R 0 I 2 CH 2 -C-C00R , (I) from 0.25 to 30% by weight 3 R 0 5 I H » CH 2 »C-C-A-R -M, and (II) from 0.1 to 50% by weight 5 R 0 0 CH 2 -C-C-0-R -OH (III) where R l , R 3 , and R 5 are independently selected from the group consisting of hydrogen and methyl; R 2 is alkyl of from 1 to 16 carbon atoms; R 1 * and R 6 are independently selected from the group consisting of alkylene groups having from 1 to 6 carbon atoms; A is -0- or -N- 17 R where R 7 is hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, and M is NR 8 R 9 H + X or S0 3 " X, where R β and R 9 are independently selected from the group consisting of hydrogen and alkyl of 1 to 4 carbon atoms and X is a counterion. The composition of these polymers has here been described with reference to the percentage by weight of the monomers that enter the polymerization reaction. It will be understood by those skilled in the art that, in general, the weight percentage of the individual polymerized monomers in the resulting polymer will not be exactly the same as the percen¬ tages given above-. These differences are generally fairly small, however, i.e. on the order of 1 or 2 percentage points. In addition to the above, other units derived from additional monomers can also be present if desired, in order to modify the properties of the polymer for special requirements. Polymers containing units polymerized from monomer (II) are known in the art. See, for example, U.S. Patent 2,923,734; U.S. Patent 3,024,221; U.S. Patent 3,411,911; Japanese Kokai 51-130217; U.S. Patent 3,679,425; Japanese Kokai 54-129790; U.S. Patent 4,130,524; Russian 530887; Japanese Kokai 54-101791; Japanese Kokai 53-16042; Japanese Kokai 52-146443; European Published Application 10425; Japanese Kokai 52-22088; U.S. 3,706,564; U.S. Patent 3,749,577; U.S. Patent 3,813,251; and U.S. Patent 4,055,469. Methods for polymerization are also disclosed. OMPI In the formula 3 R 0 I II 4 CH 2 -C-C-A-R -M, (II) , R 3 is either hydrogen or methyl and A is either -0- or -N- I? R. That is to say, the monomer can be considered to be either an ester or an amide of acrylic or methacrylic acid. Where the monomer is an amide; R 7 is 1) hydrogen, 2) alkyl of 1 to 10 carbon atoms, i.e. methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or an isomer of the foregoing, e.g. isobutyl, neopentyl, or ethylhexyl, or 3) cycloalkyl of 5 to 10 carbon atoms, e.g. cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, methylcyclopentyl, diethylcyclohexyl, or propylcycloheptyl. M designates NR 8 R 9 H X or S0 3 ~X. Thus, these polymerized monomers are in the form of an amine salt or a sulfonate salt. In the case of the amine salt, it is preferred that the salt be the hydrohalide, especially the hydrochloride. Where the polymerized monomer contains a sulfonate group, it will generally be convenient for it to be in the form of an alkali metal salt, such as Na + or K+, although other salts can be used as long as the monomer retains water solubility. Thus, X can be a positively charged ion such as Na +, K+, or H+ or a nega¬ tively charged ion such as chloride, sulfonate, or the like. As stated above, R 8 and R 9 are indepen¬ dently selected from the group consisting of hydrogen and alkyl of 1 to 4 carbon atoms, i.e. methyl, ethyl, propyl, butyl, or isomers of the foregoing. It is preferred that at least one of R 8 and R 9 be - B U REAT hydrogen and most preferred that they both be hydrogen. Finally, R* in the above formula repre¬ sents an alkylene group having from 1 to 6 carbon atoms, i.e., methylene, ethylene, propylene, butylene, pentylene, hexylene, or isomers of the foregoing. R 1 * can, of course, be substituted, if desired, with any non-interfering substituent. In the above formula, it is preferred that A be oxygen. It is more preferred that A be oxygen and R 1 * be ethylene. It is most preferred that A be oxygen, R* be ethylene and R s be methyl. As noted above, polymerized monomer (II) constitutes from 0.25 to 30 percent by weight of the polymer. When the monomer- is derived from sulfonic acid or sulfonate salt group containing acrylic monomers (such as 2-methacryloyloxyethyl-l-sulfonic acid, sodium salt), it is preferred that this monomer be employed in a range from 1 to 25 percent by weight and most preferred that it be employed in the range of from 2 to 5 percent by weight. When the monomer is derived from an amine salt of an acrylic or methacrylic amide or ester (such as 2-aminoethyl methacrylate hydrochloride) , the preferred range is from 2 to 30 percent by weight and most preferably from 5 to 20 percent. Where monomer (II) is a sulfoester, it can be prepared using any method known to be suitable. For example, in U.S. Patent 2,923,734, it is dis- closed that an α-methylene carboxylic acid and an aliphatic hydroxy sulfonic acid in the free form are interacted by heating, optionally while dispersed in an inert liquid medium capable of forming an azeo- trope with water and while azeotropically distilling water out of the reaction mixture to form the corres¬ ponding carboxylate ester. The reaction is generally carried out at a temperature between 50° and 200°C. I J UREATT oypi In U.S. Patent 3,024,221, there is disclosed a method for preparing the sulfoesters by reacting the appro¬ priate acyl halide with the salt of a hydroxy sul¬ fonic acid, generally at a temperature in the range of 0° to 200°C, although the particular temperature employed depends upon the nature of the specific reactants. Examples of hydroxy sulfonic acids (and their salts) that can be employed to form the sulfo¬ esters include 2-hydroxyethane sulfonic acid, 2-hy- droxy-1-propane sulfonic acid, and 1-hydroxy-2-butane sulfonic acid. α-Methylenecarboxylic acids or acyl halides include, for example, acrylic acid, meth¬ acrylic acid, acryloyl chloride, and methacryloyl bromide. Where monomer (II) is a sulfonate or sulfo- amide, it is preferred that it be selected from the group consisting of 2-methacryloyloxyethyl-1-sulfonic acid, sodium salt; sodium 3-acryloyloxypropane- sulfonate; and sodium 2-acrylamido-2-methylρroρane sulfonate. Where monomer (II) is an amine salt of an acrylic or methacrylic amide or ester, it is prepared by the neutralization of the appropriate primary, secondary, or tertiary amine. It is preferred that a primary amine be used. Such amines, as pointed out above, are known in the art and include, for example, aminoethyl acrylate, aminoethyl methacrylate, amino¬ methyl acrylate, aminomethyl methacrylate, aminobutyl acrylate, N-methylamino ethyl methacrylate, N-ethyl- aminomethyl acrylate, and N,N-diethylaminoethyl methacrylate. Where monomer (II) is an amine salt, it is preferred that it be 2-aminoethyl methacrylate hydrochloride. In the formula 1 R I 2 CH 2 »C-C00R , (I) R 1 is hydrogen or methyl and R 2 is an alkyl group of from 1 to 16 carbon atoms, i.e. methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, penta- decyl, hexadecyl, or isomers of the foregoing. It is preferred that R 2 be butyl, more preferably n-butyl and, most preferably, that R 2 be n-butyl and R l be methyl. Polymerized monomer (I) constitutes from about 30 to 95 percent by weight of the polymer. Preferably, it is present in the polymer in a range of from about 40 to 75 by weight, more preferably, from about 50 to 70 percent by weight. In the formula R 0 I ll 6 CH 2 »C-C-0-R -OH, (III) R 5 is either hydrogen or methyl and R 6 is an alkylene group of from 1 to 6 carbon atoms, i.e. methylene, ethylene, propylene, butylene, pentylene, hexylene, or an iso er of the foregoing. It is preferred that R 6 be ethylene and more preferred that R 6 be ethylene and R 5 methyl. Polymerized monomer (III) constitutes from about 0.1 to 50 percent by weight of the polymer. Preferably, it is present in an amount of from about 10 to 30 percent by weight, more preferably from about 15 to 25 percent by weight. As mentioned above, additional polymerized monomers can be included in the polymers of the barrier zones. For example, it has been found useful, although not always necessary, to cross-link the films of these polymers to a small extent in OYPΪ order to control their structural stability in the presence of water. To accomplish this, units can be included in the polymer having moieties, such as hydroxyl groups, carboxyl groups, and active methyl- ene groups, capable of reacting with cross-linking agents. The amine groups of the amine salts des¬ cribed above inherently have such capability. Thus, where the polymers contain one of these amine salts, cross-linking can be carried out by treating the polymer with a cross-linking agent. In addition to using these amine groups as cross-linking sites, or as an alternative to them where they are not present, as in the above-described polymers containing sulfoester units, additional units in the polymer derived from, for example, mono¬ mers containing active methylene groups in their side chains can be included. Such monomers have been described, for example, in U.S. Patent 3,459,790 and U.S. Patent 4,215,195. The polymer can contain up to 20 percent by weight of such polymerized monomers. Preferably, the polymer contains from about 0.1 to 15 percent by weight of these polymerized monomers, more preferably, from about 5 to 10 percent by weight. Active methylene groups are methylene groups between activating groups, for example, electronega¬ tive groups such as carbonyl. Malonic esters, aceto- acetic esters, cyanoacetic esters, and 1,3-diketones are examples of compounds containing such groups. Such monomers include, for example, 2-acetoacetoxy- ethyl methacrylate or 2-acetoacetoxyethyl acrylate, N-(2-cyanoacetoxyethyl)acrylamide, 4-cyanoacetyl-l- methacryloylpiperazine, N-(2-proρionylacetoxybutyl)- acrylamide, N-(2-valerylacetoxyphenyl)acrylamide, N-4-(acetoacetoxymethyl)- benzylacrylamide, N-4- (acetoacetoxybenzyl)- phenylmethacrylamide, N-(2- acetoacetoxyethyl) crylamide, N-(3-acetoacetamido- propyl)methacrylamide, N-(2-acetoacetamidoethyl)meth- acrylamide, 4-acetoacetyl-l-methacryloylpiperazine, acetoacetamidoethyl methacrylate, 4-acetoacetyl-l- acryloylpiperazine, N-(2-propionylacetoxyethyl)acryl- amide, and N-(2-valerylacetoxypropyl)methacrylamide. The preferred active methylene group-containing mono¬ mer used in the polymers employed in this invention is 2-acetoacetoxyethyl methacrylate. Conventional photographic hardeners can be used as the cross-linking agents to cross-link these polymers. These compounds are well known in the art and an extensive listing of them appears in British Patent 1,478,811 in the paragraph bridging pages 7 and 8 of that patent. Typical hardeners include formaldehyde and free dialdehydes, such as succin- aldehyde and glutaraldehyde, blocked dialdehydes, α-diketones, active esters, sulfonate esters, active halogen compounds, s-triazines and diazines, epoxides, aziridines, active olefins having 2 or more active bonds, blocked active olefins, carbodiimides, isoxazolium salts unsubstituted in the 3-position, esters of 2-alkoxy-N-carboxydihydroquinoline, N-carbamoyl and N-carbamoyloxypyridinium salts, halogen-substituted aldehyde acids, Onium substi¬ tuted acroleins, and the like (see Research Disclo- sure; Item 17643; December 1978, page 26). A pre¬ ferred cross-linking agent for use in the present invention is bis(vinylsulfonylmethyl)ether. The preferred polymers of the barrier zone of the improved elements of the present invention are synthesized from the following monomers: n-butyl methacrylate, 2-methacryloyloxyethyl-l-sulfonic acid, sodium salt, 2-acetoacetoxyethyl methacrylate, 2-hydroxyethyl methacrylate, 2-aminoethyl methacryl¬ ate hydrochloride, and 2-ethylhexyl methacrylate. The preferred polymers include: poly(n-butyl meth- acrylate-co-2-methacryloyloxyethyl-l-sulfonic acid- co-2-acetoacetoxyethyl methacrylate-co-2-hydroxyethyl -gϋ EA Of/PI _ methacrylate) (60/5/10/25; 70/2.5/10/17.5; or 60/10/- 10/20) ; poly(2-ethylhexyl methacrylate-co-methacryl- oyloxyethyl-1-sulfonic acid-co-2-acetoacetoxyethyl methacrylate-co-2-hydroxyethyl methacrylate) (50/2.5/10/37.5); and poly(n-butyl methacrylate-co- 2-aminoethyl methacrylate hydrochloride-co-2-hydroxy- ethyl methacrylate) (50/15/35); where the numbers in parentheses are weight percentages of the monomers in the polymerization mixture. The polymers employed in the practice of the present invention can be prepared at a wide variety of temperatures, since the temperature will depend upon such variable features as the specific monomers used, the duration of heating, pressure employed, and the like. However, the polymerization temperature generally does not exceed about 100°C and, most often, is in the range of about 50° to about 90° C. The polymerization is generally carried out in solution in organic solvents, e.g. the lower alcohols, dimethyl sulfoxide, dimethylformamide, and the like, and then, if desired, the polymeric product is dispersed in water. Latex polymerization can also be employed although the barrier properties of the polymers prepared in this way have been inferior to those obtained using solution polymerization. The pressure employed in the polymerization, if any, is usually only sufficient to maintain the reaction mixture in liquid form, although either superat ospheric or subatmospheric pressures can be used. The concentration of total polymerizable monomer in the polymerization mixture can be varied widely with concentrations up to about 60 percent by weight and, preferably about 20 to about 40 percent by weight, based on the weight of the monomers plus solvent, being satisfactory. Suitable catalysts for the polymerization reaction include, for example, the free radical catalysts, such as hydrogen peroxide, cumene hydro- peroxide, azo type initiators, and the like. In redox polymerization systems, the usual ingredients can be employed. If desired, the polymer can be isolated from the reaction solvent by coagulation, or other separa¬ tion procedures suitable for this purpose, e.g. dia- filtration. Optionally, in forming the barrier zones, these synthetic polymers are blended with various amounts of a hydrophilic colloid, preferably gela¬ tin. The gelatin can constitute up to 70% of the total weight of the polymer and the gelatin. In particular, the inclusion of gelatin in the barrier zones has been found to be especially useful where the synthetic polymer employed is one of the butyl methacrylate/aminoethyl methacrylate hydrochloride/- hydroxyethyl methacrylate copolymers referred to above. These polymers have a low permeability, even to the analytes being determined, and it has been found that the presence of gelatin enhances the diffusion of the analyte. The semi-permeable barrier zone physically impedes the passage of large molecular components that cause chemical interference for the duration of the test. The zone also has only limited perme¬ ability to the analyte. Owing to the minimal quantity of analyte actually passing through the barrier zone and being subsequently detected, the assay is less sensitive to the volume of the sample applied, making it possible to use random quantities of blood samples, such as might be obtained from ear and finger pricks. Also, the small quantity of analyte present permits the use of dyes that have very high extinction coefficients. These high- extinction dyes, heretofore not useful in some assays, permit a greater selection of wavelengths at which the assay can be monitored — ideally, away from the wavelength region of spectral interference. Additionally, the relatively high dye density generated can allow the assay to be spectrally monitored off peak. These options are instrumental in avoiding spectral interferences. It is necessary that the analytical elements of the present invention contain the above-described spreading, barrier, and reagent zones. Of course, for some applications, it may be desirable to have more than one of these zones, i.e. two or more spreading zones and/or two or more barrier zones and/or two or more reagent zones.. This is the case, for example, where a given analyte has to react sequentially with two or more reagents that could, if coated together in one zone, react with each other. Further, the elements optionally include additional zones having specialized functions required for a given analysis or, perhaps, necessary for the convenient manufacture of the element. For example, it is a common practice to use additional zones where needed in order to promote or control adhesion between other zones. Such zones are commonly referred to as "binder" zones or "subbing" zones and are well known in the art. Another type of zone that can be employed in these elements, and that is referred to in the examples below, is known as a "gel pad" zone. Such zones comprise gelatin, a surfactant to improve the coatability of the gelatin as a zone, and a conven¬ tional hardener, such as bis(vinylsulfonylmethyl)- ether. These zones have been found to be useful in the manufacturing process in order to prevent blocking between one zone and another. Another type of zone that is particularly useful in these elements is known as a radiation- blocking zone. Such zones are also known in the art and have been described, for example, in U.S. Patent 4,042,335 and U.S. Patent 4,166,093. The use of a radiation-blocking zone together with the barrier zone in the elements of the present invention permits a wide choice of wave¬ lengths for the assay, ranging from 250 to 900 nm. Still other zones that can be employed in the present elements include registration zones, detectable species migratio -inhibiting zones, filtering zones, and the like, such as those that have been described in U.S. Patent 3,992,158; U.S. Patent 4,042,335;. U.S. Patent 4,066,403 (Reissue 30,267); and U.S. Patent 4,166,093. The above-described zones, when combined, can be self-supporting or can be carried on a support. Useful support materials include a variety of polymers, such as cellulose acetate; polyethylene terephthalate; polycarbonates; and polyvinyl compounds, such as polystyrene; glass or metal; and paper. A support of choice for any particular element will be compatible with the intended mode of result detection. For example, for fluorimetric detection wherein fluorimetric emission within the element is detected as the emission is transmitted from within the element through the support to an external detector, it is desirable to employ as a support material a material that exhibits a low degree of background fluorimetric emission. Thus, preferred supports include supports that are radiation-transmissive with respect to the particular radiation employed to provide detectable changes within the element. IJUREA OMPI Preferably, the various zones referred to above are present in the elements of the present invention as superposed contiguous layers. Materials In the examples below, whole blood samples were obtained from local medical facilities. Alkanol XC* (sodium alkyl naphthalene sulfonate) and Zonyl FSN* (a fluoroche ical surfactant) were obtained from DuPont, Wilmington, DE. Polyvinyl pyrrolidone was obtained from GAF Corporation, Atlanta, GA. Triton X-100* was purchased from Rohm and Haas of Philadelphia, PA. Glucose oxidase was purchased from Sigma, St. Louis, MO, and peroxidase was purchased from Miles Laboratories, Elkhart, IN. All other compounds were obtained from Eastman Kodak Company, Rochester, NY, and, unless otherwise noted, were of reagent grade. Methods Whole blood samples were prepared for patient calibration tests according to the following procedures to obtain a broad dynamic range: Low glucose level samples (below 80 mg/dL) were diluted or depleted according to the following methods: 1) plasma was removed from centrifuged samples and replaced with HSA (human serum albumin) and saline to keep the electrolyte balance or 2) glycolysis was allowed to occur. High glucose level samples (above 80mg/dL) were prepared by the addition of glucose. OiV l General Element Formats: I Spreading Layer II Spreading Layer Binder Layer (optional) Binder Layer (optional) Gel Pad Layer Radiation-Blocking Layer (optional) (optional) Barrier Layer Barrier Layer Reagent Layer Reagent Layer Support (optional) Support (optional) Example 1: No Barrier Zone (Comparative Example) Test Element I was prepared as follows: a polyethylene terephthalate support was overcoated with a reagent zone comprising [2(3,5-dimethbxy-4- hydroxypheny1)-4,5-bis(4-dimethylaminoρhenyl) imida- zole] (a leuco dye), dimedone, 2,4-di-n-pentylphenol, deionized gelatin, poly(methyl acrylate-co-2-acryl- amido-2-methylρroρane sulfonic acid-co-2-acetoace- toxyethyl methacrylate), weight ratio 88.8/4.7/6.5, Zonyl FSN*, peroxidase (POD), glucose oxidase (GOD), dimethyl glutaric acid (DMG) (pH 6.0), and bis(vinyl sulfonylmethyl ether) (BVSME) ; and a spreading zone comprising poly(vinyl toluene- co-p-t-butylstyrene-co-methacrylic acid) beads, weight ratio 61/37/2, 20-40 μ in diameter, poly- (n-butyl acrylate-co-styrene-co-2-acrylamido-2- methylpropane sulfonic acid) weight ratio 76/19/5, and Zonyl FSN # . The element was spotted with 10 μL aliquots of three whole blood samples having glucose levels of 88, 199, and 392 mg/dL, respectively. The reaction was monitored at room temperature using a spectrophoto eter by measuring reflectance densities at 800 n over a 7 minute period. No meaningful distinctions among the glucose levels tested could be obtained with this element. -BU EACT OMPI _ Example 2: No Barrier Zone (Comparative Example) Test Element II was prepared as follows: a polyethylene terephthalate support was overcoated with a reagent zone comprising [2(3,5-dimethoxy-4- 5 hydroxyphenyl)-4,5-bis(4-dimethylaminophenyl)- imidazole], poly(methyl acrylate-co-2-acrylamido-2- methylpropane sulfonic acid-co-2-acetoacetoxyethyl methacrylate) weight ratio 88/5/7, deionized gelatin, 2,4-di-n-ρentylphenol, dimedone, Zonyl FSN*, 3,3- 10 dimethylglutaric acid (pH 6.0), peroxidase, glucose oxidase, and BVSME. A second zone was coated over the reagent zone and comprised 1.1 gm/m 2 (100 mg/ft 2 ) of deionized gelatin, .048 gm/m 2 (4.5 mg/ft 2 ) of Zonyl FSN « , and .019 gm/m 2 (1.75 15 mg/ft 2 ) of BVSME. A spreading zone was coated over the second zone and comprised poly(vinyl toluene-co- p-t-butylstyrene-co-methacrylic acid) beads, weight ratio 61/37/2, 20-40 μ in diameter, poly(n-butyl- acrylate-co-styrene-co-2-acrylamido-2-methyl propane 20 sulfonic acid) sodium salt, weight ratio 70/20/10, Kelzan* (a xanthan gum thickener commercially available from Kelco Co.), and Zonyl FSN*. The element was spotted with whole blood samples having glucose levels of 66, 131, 229, 309, 25408, and 588 mg/dL. The results were poor with crossover being exhibited for the 131 and 229 mg/dL samples and for the 588 mg/dL sample with the 309 and 408 mg/dL samples. By "crossover" we mean that graphs of reflection density vs. time for two or more 0 different concentrations intersect, or "crossover" one another. Readings were made at 700 nm and over a 5 minute period. This shows that a "barrier" layer prepared from gelatin, as described, for example, in U.K, 5 Published Application; 2,069,131 A, is not useful. Example 3: Barrier Zone Test Element III was prepared having the same reagent and spreading zones as- those of Test Element II in Example 2. A barrier zone of the present invention was substituted for the "second zone" of Example 2, however, by replacing the 1.1 gm/m 2 (100 mg/ft 2 ) of deionized gelatin with 0.90 gm/m 2 (84 mg/ft 2 ) of poly(n-butyl methacrylate-co-2-methacryloyloxy- ethyl-1-sulfonic acid-co-2-acetoacetoxyethyl meth- arcylate-co-2-hydroxyethyl methacrylate) sodium salt, weight ratio 60/5/10/25, and 0.17 gm/m 2 (16 mg/ft 2 ) of deionized gelatin. Zonyl FSN » and BVSME were also present in this zone in the same concentration as in Element II. The element was spotted with whole blood samples having glucose levels of 57, 107, 227, 372, 478, and 623 mg/dL. Good distinction between levels was obtained and the levels were distinguished in the proper order, i.e., low to high glucose, indicating that the barrier zone had successfully screened the chemical interferents. Readings were made at 700 nm and over a 5 minute period. Example 4: Barrier Zone Test Element IV was prepared according to general format I, shown above, and comprised the following zones, each coated over the preceding zone. A polyethylene terephthalate support was coated with a reagent zone comprising: [2(3,5-di- methoxy-4-hydroxyphenyl)-4,5-bis(4-dimethylaminophenyl) imidazole], dimedone, 2,4-di-n-pentylphenol, deion¬ ized gelatin, Zonyl FSN«, BVSME, peroxidase, glucose oxidase, dimethyl glutaric acid (pH 6.0), and poly(methyl acrylate-co-2-acrylamido-2-methylpropane sulfonic acid-co-2-acetoacetoxyethyl methacrylate, weight ratio 88.8/4.7/6.5; a barrier zone comprising poly(n-butyl methacrylate-co-2-methacryloxyethyl-l- sulfonic acid-co-2-acetoacetoxyethyl methacrylate- co-2-hydroxyethyl methacrylate) sodium salt weight ratio 60/5/Ϊ0/25, (0.5 g/m 2 ), deionized gelatin (0.04 g/m 2 ), and Zonyl FSN» (0.05 g/m 2 ); a gel pad zone comprising deionized gelatin, Zonyl FSN « and BVSME; a binder zone comprising poly(vinyl pyrrolidone) ; and a spreading zone comprising poly- (vinyl toluene-co-p-t-butyl styrene-co-methacrylic acid) beads, weight ratio 61/37/2, 20-40 μ in diameter, poly(acrylamide), and Triton X-100. The element was spotted with 10 μL aliquots of four whole blood samples having glucose levels of 66, 105, 214, and 408 mg/dL, respectively. The reaction was monitored at room temperature using a spectrophotometer by measuring reflectance densi¬ ties at 700 nm over a 7 minute period. Good distinc¬ tion between levels was obtained and the levels were distinguished in the proper order, i.e., low to high glucose. Example 5: Barrier and Radiation-Blocking Zones Test Element V was prepared according to general format II, described above, and was prepared in the same manner as Test Element IV, except that a radiation-blocking zone was coated over the barrier zone and the gel pad zone was excluded. The radia¬ tion-blocking zone comprised titanium dioxide, gela¬ tin, Zonyl FSN«, and BVSME. The test procedure was identical to that in Example 4. Compared with the results of Example 4, background density was greatly diminished by the radiation-blocking zone, and the discrimination between glucose levels improved, especially at the higher levels where more density is observed. Example 6: Polymer Variations in the Barrier Layer Test Elements VI-X, with variations in the polymer used in the barrier zone, were evaluated for performance in the discrimination of glucose levels "SUREST pyri in whole blood. All elements were spotted with whole blood samples (10 μL aliquots). The reactions were monitored at 720 nm over a 3 minute period at 25°C, using a spectrophotometer. A. Poly(n-butyl methacrylate-co-2-methacryloyloxy- ethyl-1-sulfonic acid-co-2-acetoacetoxyethyl methacrylate-co-2-hydroxyethyl methacrylate) sodium salt, weight ratio 60/5/10/25 (12:1, polymer:gelatin) Test Element VI was prepared according to format I above in the following manner: a poly¬ ethylene terephthalate support was overcoated with a reagent zone; a barrier zone as described in Example 4 comprising the above polymer, except that the barrier zone also contained BVSME (0.01 g/m 2 ); a gel pad zone comprising deionized gelatin, Zonyl FSN # , and BVSME; and a spreading zone comprising the polymer beads of Example 4, Zonyl FSN # , poly- (n-butyl acrylate-co-styrene-co-2-acrylamido-2-methyl propane sulfonic acid), weight ratio 76/19/5; and Kelzan*. Excellent calibration of the element was demonstrated, with glucose levels ranging from ,40- ,410 mg/dL, indicating this polymer along with gelatin is especially suitable in the barrier zone of the present invention. B. Poly(n-butyl methacrylate-co-2-methacryloyloxy- ethyl-1-sulfonic acid-co-2-acetoacetoxyethyl methacrylate-co-2-hydroxyethyl methacrylate) sodium salt, weight ratio 70/2.5/10/17.5 (12:1, polymer:gelatin) Test Element VII was prepared according to format I, described earlier, as follows: a reagent zone, comprising deionized gelatin, leuco dye, 2,4-di-n-ρentylphenol, dimedone, Zonyl FSN«, BVSME, dimethyl glutaric acid (pH 6.0), peroxidase, and glucose oxidase was coated over a polyethylene tere- OMPI phthalate film support. A barrier zone, comprising poly(n-butyl methacrylate-co-2-methacryloyloxyethyl- 1-sulfonic acid-co-2-acetoacetoxyethyl methacrylate- co-2-hydroxyethyl methacrylate) sodium salt, weight ratio 70/2.5/10/17.5 (0.5 g/m 2 ), deionized gelatin (0.04 g/m 2 ), Zonyl FSN « (0.05 g/m 2 ), and BVSME (0.01 g/m 2 ) was coated over the reagent zone. Finally, a spreading zone comprising beads of poly- (vinyl toluene-co-p-t-butylstyrene-co-methacrylic acid) weight ratio 61/37/2, 20-40 μ in diameter, Zonyl FSN β , Kelzan # , and poly(n-butyl acrylate- co-styrene-co-2-acrylamido-2-methyl propane sulfonic acid), weight ratio 70/20/10, was coated over the barrier zone. Good discrimination between glucose levels ranging from ^30-^400 mg/dL in whole blood samples was demonstrated indicating the usefulness of this polymer along with gelatin in the barrier zone of the present invention. C. Poly(2-ethylhexyl methacrylate-co-2-methacryl- oyloxyethy1-1-sulfonic acid-co-2-acetoacetoxy- ethyl methacrylate-co-2-hydroxyethyl methacryl¬ ate) sodium salt, weight ratio 50/2.5/10/37.5 (12:1, polymer:gelatin) Test Element VIII was prepared according to format I, described above, differing from Test Element VI as follows: the barrier zone comprised ρoly(2-ethylhexyl methacrylate-co-2-methacryloyloxy- ethyl-1-sulfonic acid-co-2-acetoacetoxyethyl meth- acrylate-co-2-hydroxyethyl methacrylate) sodium salt, weight ratio 50/2.5/10/37.5, (0.5 g/m 2 ), deionized gelatin (0.04 g/m 2 ), Triton X-100 (0.05 g/m 2 ), and BVSME (0.01 g/m 2 ); and the gel pad zone comprised deionized gelatin, BVSME, and Triton X-100. Samples tested ranged from ^40-^420 mg/dL glucose. The calibration results demonstrated that this polymer along with gelatin is also suitable for use in the barrier zone of the present invention. D. Poly(n-butyl methacrylate-co-2-aminoethyl meth¬ acrylate hydrochloride-co-2-hydroxyethyl meth- acrylate), weight ratio 50/15/35 (1:1, gel:- polymer) Test Element IX was prepared according to format I, described above, as follows: a reagent zone, as described above, was coated over a poly- ethylene terephthalate support. A barrier zone comprising ρoly(n-butyl methacrylate-co-2-aminoethyl methacrylate hydrochloride-co-2-hydroxyethyl meth¬ acrylate), weight ratio 50/15/35, (0.5 g/m 2 ), deionized gelatin (0.5 g/m 2 ), and Zonyl FSN* (0.05 g/m 2 ) surfactant was coated over the reagent zone. A spreading zone, as described above, was coated over the barrier zone. The test element was evaluated using whole blood samples with glucose levels ranging from ^30-^285 mg/dL. Reflectance densities were monitored. The calibration results demonstrated this polymer along with gelatin (in a 1:1 ratio) to be useful in the barrier zone of the present invention. E. Poly(n-butyl methacrylate-co-2-methacryloyloxy- ethyl-1-sulfonic acid-co-2-acetoacetoxyethyl methacrylate-co-2-hydroxyethy1 methacrylate) sodium salt, weight ratio 70/2.5/10/17.5 (100% polymer and no gelatin) Test Element X was prepared to evaluate the barrier zone performance without gelatin in that zone. The element was prepared according to format I as follows: the reagent zone had the same composi¬ tion as that zone in test Element VII, except that the BVSME concentration was approximately doubled; the barrier zone comprised poly(n-butyl methacryl- ate-co-2-methacryloyloxyethyl-l-sulfonic acid-co-2- acetoacetoxyethyl methacrylate-co-2-hydroxyethyl methacrylate) sodium salt, weight ratio 70/2.5/10/- 17.5, (0.5 g/m 2 ), Zonyl FSN« (0.05 g/m 2 ), and BVSME (0.01 g/m 2 ); and the spreading zone comprised beads of ρoly(vinyl tolueneco-p-t-butyl styrene-co- methacrylic acid), weight ratio 61/37/2, 20-40 μ in diameter, Zonyl FSN* , and ρoly(n-butyl acrylate- co-styrene-co-2-acrylamido-2-methyl propane sulfonic acid), weight ratio 70/20/10. The test element was spotted with 10 μL aliquots of three whole blood samples having glucose levels of 86, 191, " and 403 mg/dL, respectively. The reaction was monitored at room temperature using a spectrophotometer by measuring reflectance densities at 800 nm over a 7 minute period. After about 4-5 minutes, the results obtained showed improvement over elements that were similar but had no barrier layer in that distinctions between the glucose levels could be observed. The results, however, were inferior to those obtained where gelatin was present in the barrier zone. Example 7: Patient Calibration of the Dry Element Using the Poly(n-butyl methacrylate- co-2-methacryloyloxyethyl-1-sulfonic acid-co-2-acetoacetoxyethyl methacryl- ate-co-2-hydroxyethyl methacrylate) sodium salt, weight ratio 60/5/10/25 Barrier Zone The dry element for whole blood glucose, Test Element VI having a poly(n-butyl methacrylate- co-2-methacryloyloxyethyl-l-sulfonic acid-co-2-aceto- acetoxyethyl methacrylate-co-2-hydroxyethyl meth¬ acrylate) sodium salt, weight ratio of 60/5/10/25, was calibrated using patient samples ranging in glucose levels from ^54-^354 mg/dL. Test Element VI was prepared as described in Example 6. The assays also were carried out as described in Example 6, except that the patient samples were used as their own references. The calibration results representing patient data obtained over a three-day period demonstrated that the improved elements of the present invention are effective in determining whole blood glucose. Example 8: Barrier Layer Effects on Determination of Glucose on Whole Blood vs Slightly Hemolyzed Plasma vs Grossly Hemolyzed Plasma A. No Barrier Zone Test elements were prepared as described in Example 1 (containing no barrier zone) . The elements were spotted with 10 μL samples having glucose levels ranging from ^80-^430 mg/dL in various matrices as shown in Table I. Table I Matrix Glucose Level Whole Slightly 1 Grossly 2 mg/dL Blood Hemolyzed Plasma Hemolyzed Plasma 80 X X 88 X 199 X 232 X X 392 X 431 X X 1 "Slightly Hemolyzed Plasma" is the equivalent of about 50 mgs/deciliter of hemoglobin. 2 "Grossly Hemolyzed Plasma" is the equivalent of about 7 gms/deciliter of hemoglobin. No meaningful distinction between the glucose levels tested was obtained especially at early read times, regardless of matrix, i.e., whole blood or lysed red cells in plasma. B. With Barrier Zone Test elements were prepared according to the following: A polyethylene terephthalate film support was coated with a reagent zone comprising gelatin, BVSME, leuco dye (as in Example 1), Zonyl FSN», POD, GOD, dimedone, di-n-pentylphenol, and DMG at pH 6.0; a barrier zone comprising poly(n-butyl meth- acrylate-co-2-methacryloyloxyethyl-l-sulfonic acid- co-2-acetoacetoxyethyl methacrylate-co-2-hydroxyethy1 methacrylate) sodium salt, weight ratio 70/2.5/10/- 17.5 (0.5 g/m 2 ) and gelatin (0.04 g/m 2 ); and a spreading zone comprising the polymer beads of Example 1, 20-40 μ in diameter, 2% by weight poly(n-butyl acrylate-co-styrene-co-2-acrylamido-2- methyl propane sulfonic acid), weight ratio 70/20/10, and Zonyl FSN*. Good distinction between glucose levels tested was obtained regardless of matrix. Example 9: Poly(n-butyl methacrylate-co-2-amino- ethylmethacrylate hydrochloride-co-2- hydroxyethylmethacrylate) (wt. ratio 50/15/35): gelatin, 2:3 weight ratio Test elements for the determination of whole blood glucose were prepared as described in Example 6D except that the barrier layer comprised poly(n- butyl methacrylate-co-2-aminoethyl methacrylate hydrochloride-co-2-hydroxyethylmethacrylate) (50/15/35) and gelatin in the ratio of 2:3 (instead of 1:1). Surfactant, Zonyl FSN* (.08 g/m 2 ), and hardener, BVSME (.06 g/m 2 ), were also included in that layer. The results indicated good discrimina¬ tion between glucose values of from 76 to 554 mg/dL. Example 10; Poly(n-butyl methacrylate-co-2-meth- acryloyloxyethyl-1-sulfonic aci -co- 2-acetoacetoxyethyl methacrylate-co- 2-hydroxyethyl methacrylate) sodium salt (wt. ratio 60/5/10/25): gelatin, 7:1 weight ratio. Test elements were prepared as described in Example 6B except that the polymer:gelatin weight ratio was 7:1 (instead of 12:1) and the weight ratio of the monomers polymerized to form the polymer of the barrier zone was 60/5/10/25 (instead of 70/2.5/10/17.5). The results showed good discrimination of glucose levels from 58 to 600 mg/dL using this higher proportion of gelatin. Example 11: Comparative Example: Cellulose Acetate Butyrate (CAB) as Barrier Zone Composi¬ tion in Glucose Element (as disclosed in U.S. Patent 4,258,001) A test element was prepared by overcoating a polyethylene terephthalate support with a reagent zone comprising gelatin, poly(methylacrylate-co-2- acrylamido-2-methylproρane sulfonic acid-co-2-aceto- acetoxyethyl methacrylate) weight ratio 88.8/4.7/6.5, Alkanol XC, DMG, dimedone, 4-isopropoxy-l-naphthol, poly(n-butylmethacrylate-co-styrene-co-2-acrylamido- 2-methyl propane sulfonic acid) weight ratio 50/40/10, GOD, POD, and BVSME. A second zone, to be tested for barrier properties, comprising cellulose acetate butyrate (0.54 gm/m 2 ) was coated over the reagent zone. Finally, a spreading zone was coated over the second zone comprising poly(vinyl toluene- co-p-t-butylstyrene-co-methacrylic acid) beads, weight ratio 61/37/2, 40-45 μ in diameter, poly- (n-butyl acrylate-co-styrene-co-2-acrylamido-2- methylpropane sulfonic acid) weight ratio 76/19/5, and Zonyl FSN β . CMP! The elements were tested as above using 77, 177, and 370 mg/dL glucose, respectively, and reflectance densities were monitored at 720 nm for >5 min. The results indicated that no discrimina- tion between glucose levels was obtained using CAB as the barrier zone. Example 12: Comparative Example: Ethyl Cellulose as Barrier Zone (As in U.K. Patent 922,665) Composition in Glucose Element Element format Beads of Example 1 (20-40μ) Spreading Poly(n-isopropyl acrylamide) Zone Triton X-100* Poly(vinyl pyrrolidone) Barrier Ethyl Cellulose Zone 0.54 g/m 2 / 1.1 g/m 2 / 2.2 g/m 2 / 4.3 g/m 2 Element D/ Element E/ Element F/ Element G Reagent Leuco dye Zone Dimedone* Di-n-pentylphenol Gelatin Zonyl FSN» POD GOD DMG BVSME Poly(methylacrylate-co-2-acryl-amido-2- methylpropane sulfonicacid-co-2-aceto- acetoxyethyl methacrylate) weight ratio 88.8/4.7/6.5 Poly(ethylene terephthalate) support Elements D through G were tested and evalu¬ ated as above using glucose concentrations of 50, 110, 210, 315, and 400 mg/dL, respectively. The elements were monitored at 700 nm for 5 min. Elements D, E, and F demonstrated very poor discrimi¬ nation with crossover between some of the analyte concentrations. Element G, which comprises the highest level of barrier material used (4.3 g/m 2 ), showed a tendency towards discrimination between glucose levels, but had lost considerable density and therefore sensitivity, making this amount of material unsuitable. Example 13: Comparative Example The following polymers were also prepared and tested in the manner of the previous examples and found to be unsuitable as barrier layers for use in the present invention. A. Poly(n-butyl acrylate-co-2-acrylamido-2-methyl- propane sulfonic acid, sodium salt-co-2-aceto- acetoxyethyl methacrylate) (Wt. Ratio 90:6:4) B. Poly(methylacrylate-co-2-acrylamide-2-methylpro- pane sulfonic acid, sodium salt-co-2-acetoacet- oxyethyl methacrylate) (Wt. Ratio 90:4:6) C. Poly(N-isopropyl acrylamide) D. Poly(n-isopropyl acrylamide-co-acrylamide) (Wt. Ratio 90:10) E. Poly(n-isopropyl acrylamide-co-acrylamide-co-2- acetoacetoxyethyl methacrylate) (Wt. Ratios 80:10:10, 75:15:10, 70:20:10) F. Poly(acrylamide-co-x-(2-chloroethyl sulfonyl methyl)styrene) wherein x » vinyl toluene, 2-chloroethyl sulfone (Wt. Ratio 95:5) G. Poly[ethylene-co-4,4'-isopropylidenebis(phenyl- eneoxyethylene) (Mole % 50:50) 5-(4-sodiosul- fophenoxy)-1,3-benzenedicarboxylate-co-tere- phthalate (Mole % 50:50)] Poly[ethylene-co-4, 4* -isopropylidenebis (phenyl- eneoxyethylene) (Mole % 50:50) isophthalate- co-(4-sodiosulfophenoxy) -1 ,3-benzenedicarboxylate (Mole % 50:50)]

Claims: Clai s : 1. A multizone element, for quantitative analyte determination in whole blood, containing a porous spreading zone separated from a reagent zone by a barrier zone, said barrier zone comprising a non-porous film comprising a polymer of from 30 to 95 percent by weight polymerized monomer having the structure 1 R 0 I II 2 CH 2 ■C-C-OR from 0.25 to 30 percent by weight polymerized monomer having the structure 3 R 0 I II H CH 2 «C-C-A-R -M, and from 0.1 to 50 percent by weight polymerized monomer having the structure 5 R 0 I II 6 CH 2 «C-C-0-R -OH wherein: R l , R 3 , and R s are independently selected from the group consisting of hydrogen and methyl, R 2 is alkyl of from 1 to 16 carbon atoms, R 1 * and R 6 are independently selected from the group consisting of alkylene groups having from 1 to 6 carbon atoms, A is -0- or -N- I? R wherein R 7 is hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, and -βU At/ O..:PI M is NR β R 9 H + X or S0 3 " x wherein R 8 and R 9 are independently selected from the group consisting of hydrogen and alkyl of 1 to 4 carbon atoms and X is a counterion. 2. The element of claim 1 wherein the barrier zone further comprises up to 70% by weight of gelatin, based upon the combined weight of the polymer and the gelatin. 3. The element of claim 1 wherein the polymer further comprises up to about 20 percent by weight of a polymerized monomer containing an active methylene group in a side chain. 4. The element of claim 1 wherein the analyte being determined is glucose. 5. The element of claims 1, 2, 3, or 4 wherein A is -0- and R 8 and R 9 are both hydrogen. 6. The element of claims 1, 2, 3, or 4 wherein the polymerized monomer of the structure 3 R 0 I II CH 2 »C-C-A-R -M, is selected from the group consisting of 2-methacryl- oyloxyethyl-1-sulfonic acid, sodium salt; sodium 3-acryloyloxypropane sulfonate; sodium 2-acrylamido- 2-methylpropane sulfonate; and 2-aminoethyl meth¬ acrylate hydrochloride. 7. The element of claims 1, 2, 3, or 4 wherein the polymer is selected from the group consisting of poly(n-butyl methacrylate-co-2- eth- acryloyloxyethyl-1-sulfonic acid-co-2-acetoacetoxy- ethyl methacrylate-co-2-hydroxyethyl methacrylate) ; poly(2-ethylhexyl methacrylate-co-methacryloyloxy- ethyl-1-sulfonic acid-co-2-acetoacetoxyethyl meth- acrylate-co-2-hydroxyethyl methacrylate) ; and poly- (n-butyl methacrylate-co-2-aminoethyl methacrylate hydrochloride-co-2-hydroxyethy1 methacrylate) . 8. A process, for quantitative analyte determination in whole blood, comprising bringing a sample of the blood into contact with a multizone element containing a porous spreading zone separated from a reagent zone by a barrier zone, said barrier zone comprising a non-porous film comprising a polymer of from 30 to 95 percent by weight polymerized monomer having the structure i R 0 I II 2 CH 2 -C-C-0R , from 0.25 to 30 percent by weight polymer¬ ized monomer having the structure 3 R 0 I II n CH 2 -C-C-A-R -M, and from 0.1 to 50 percent by weight polymerized monomer having the structure 5 R 0 I II 6 CH 2 »C-C-0-R -OH wherein: R x , R 3 , and R 5 are independently selected from the group consisting of hydrogen and methyl, R 2 is alkyl of from 1 to 16 carbon atoms, R* and R 6 are independently selected from the group consisting of alkylene groups having from 1 to 6 carbon atoms, A is -0- or -N- G.V.PI wherein R 7 is hydrogen, alkyl of 1 to 10 carbon atoms, or cycloalkyl of 5 to 10 carbon atoms, and M is NR 8 R 9 H + X or S0 3 " X wherein R 8 and R 9 are independently selected from the group consisting of hydrogen and alkyl of 1 to 4 carbon atoms and X is a counterion. "BURΞA " _C.V.PI_ ' .i-0 £ e sr . ATi<3$

Inventors: GROSS ROBERT CHARLES, GROSS SUSAN COOK, GROSS, ROBERT, CHARLES, GROSS, SUSAN, COOK
Applicants: EASTMAN KODAK CO, EASTMAN KODAK COMPANY
Application year: 1983
Main classification: G01N33
IPC codes: C08J5, G01N33