An analytical element for assay of complex fluids, such as blood, the element comprising two reagents and a barrier composition separating the reagents. Such an element is particularly useful in the analysis of blood for urea nitrogen and cholesterol content.
|
14. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. urease; and 2. a reagent capable of interacting with ammonia to form a detectable product;
the improvement comprising a barrier composition separating substantially all of said urease from said reagent, said composition being substantially uniformly permeable to ammonia vapor and substantially impermeable to liquid bases.
27. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. urease; and 2. a reagent capable of interacting with ammonia to form a detectable product;
the improvement comprising a barrier composition separating said urease and said reagent, said composition being substantially uniformly permeable to ammonia vapor and substantially impermeable to liquid bases, said reagent including an ammonia-bleachable dye.
20. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. urease; and 2. a reagent capable of interacting with ammonia to form a detectable product;
the improvement comprising a barrier composition separating said urease and said reagent, said composition being substantially uniformly permeable to ammonia vapor and substantially impermeable to liquid bases, said reagent including a 1,4-substituted quinolinium salt which deprotonates in the presence of NH3 to produce a dye.
19. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. urease; and 2. a reagent capable of interacting with ammonia to form a detectable product;
the improvement comprising a barrier composition separating said urease and said reagent, said composition being substantially uniformly permeable to ammonia vapor and substantially impermeable to liquid bases, said reagent including a 1,2-substituted quinolinium salt which deprotonates in the presence of NH3 to produce a dye.
51. In an integral element useful in the analysis of a fluid sample for a predetermined analyte, the element including
1. a reagent capable of producing ammonia vapor from the fluid sample; and 2. a reagent capable of interacting with said ammonia to provide a detectable change;
the improvement comprising a barrier composition separating substantially all of said reagent (1) from said reagent (2), said barrier composition being substantially uniformly permeable to said ammonia and substantially impermeable to interferants. 40. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. a layer comprising urease in a sample intercept area; and 2. a reagent capable of interacting with ammonia to form a detectable change;
the improvement comprising a barrier composition separating substantially all of said urease from said reagent and having a sample intercept area that is at least coextensive with the sample intercept area of the layer comprising said urease, said barrier composition within its sample intercept area being uniformly permeable to ammonia vapor and impermeable to liquid base. 1. In an integral element useful in the analysis of a fluid sample for a predetermined analyte, the element including
1. a reagent capable of interacting in the presence of said analyte to produce a decomposition product; and 2. a reagent capable of interacting with said decomposition product or an intermediate to provide a detectable change;
the improvement comprising a barrier composition separating substantially all of said said reagent (1) from said reagent (2), said barrier composition being substantially uniformly permeable to said decomposition product and substantially impermeable to interferants.
48. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. a layer comprising urease in a sample intercept area; and 2. a reagent capable of interacting with ammonia to form a detectable change;
the improvement comprising a barrier composition separating said urease and said reagent and having a sample intercept area that is at least coextensive with the sample intercept area of the layer comprising said urease, said barrier composition within its sample intercept area being uniformly permeable to ammonia vapor and impermeable to liquid bases, said reagent including an ammonia-bleachable dye. 44. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. a layer comprising urease in a sample intercept area; and
2. a reagent capable of interacting with ammonia to form a detectable change; the improvement comprising a barrier composition separating said urease and said reagent and having a sample intercept area that is at least coextensive with the sample intercept area of the layer comprising said urease, said barrier composition within its sample intercept area being uniformly permeable to ammonia vapor and impermeable to liquid bases, said reagent including a 1,2-substituted quinolinium salt which deprotonates in the presence of NH3. 45. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. a layer comprising urease in a sample intercept area; and 2. a reagent capable of interacting with ammonia to form a detectable change;
the improvement comprising a barrier composition separating said urease and said reagent and having a sample intercept area that is at least coextensive with the sample intercept area of the layer comprising said urease, said barrier composition within its sample intercept area being uniformly permeable to ammonia vapor and impermeable to liquid bases, said reagent including a 1,4-substituted quinolinium salt which deprotonates in the presence of NH3. 29. An integral multilayered element useful in the analysis of a fluid sample for a predetermined analyte, the element comprising, in superposed relationship,
1. a first layer which includes an enzyme capable of complexing with analyte to produce a decomposition product; 2. a second layer which includes a reagent capable of interacting with said decomposition product or an intermediate to form a detectable change; 3. a barrier composition separating substantially all of said enzyme from said reagent, said composition being substantially uniformly permeable to said decomposition product and substantially impermeable to interferants; and 4. a non-fibrous isotropically porous spreading layer.
10. In an integral, multilayered element useful in the analysis of a fluid sample for a predetermined analyte, the element including, in superposed relationship:
1. a first layer which includes at least one reagent capable of interacting in the presence of the analyte to produce a decomposition product; and 2. a second layer which includes at least one reagent capable of interacting with said decomposition product or an intermediate to provide a detectable change;
the improvement comprising a barrier composition separating substantially all of said first and second layer reagents from one another, said barrier composition being substantially permeable to said decomposition product and substantially uniformly impermeable to interferants; said barrier composition being contiguous with and between said two layers.
5. In an integral, multilayered element useful in the analysis of a fluid sample for a predetermined analyte, the element including, in superposed relationship:
1. a first layer which includes at least one reagent capable of interacting in the presence of the analyte to produce a decomposition product; and 2. a second layer which includes at least one reagent capable of interacting in the presence of said decomposition product or an intermediate to provide a detactable change;
the improvement comprising a barrier composition separating substantially all of said reagents in said first and second layers from one another, said composition being substantially uniformly permeable to said decomposition product and substantially impermeable to interferants; one of said reagents being distributed within said barrier composition.
33. A process of analyzing a liquid for the presence of a predetermined analyte, comprising the steps of
a. depositing a sample of the liquid on an element comprising a first reagent capable of producing a radiant energy detectable species and having in association with it a binder, a second reagent capable of producing from the analyte a decomposition product which is interactive with said first reagent; and a barrier composition separating substantially all of said second reagent from said first reagent, said composition being substantially uniformly permeable to said decomposition product and substantially impermeable to interferants; whereby the analyte and the second reagent react to produce a decomposition product that selectively permeates through the barrier composition where it reacts with the first reagent; and b. measuring said detectable change.
34. In an integral multilayered element useful in the analysis of a fluid sample for a predetermined analyte, the element including, in superposed relationship:
1. a first layer which includes a sample intercept area, and reagent in said area capable of interacting with the analyte to produce a decomposition product; and 2. a second layer which includes at least one reagent capable of interacting with said decomposition product or an intermediate to form a detectable change;
the improvement comprising a barrier composition separating substantially all of said first and second layer reagent from one another and having a sample intercept area that is at least coextensive with said sample intercept area of said first layer, said composition within its sample intercept area being uniformly permeable to said decomposition product and impermeable to interferants.
36. In an integral, multilayered element useful in the analysis of a fluid sample for a predetermined analyte, the element including, in superposed relationship:
1. a first layer which includes a sample intercept area and a reagent in said area capable of interacting with the analyte to produce a decomposition product; and 2. a second layer which includes a reagent capable of interacting with said decomposition product or an intermediate to form a detectable change;
the improvement comprising a barrier composition separating substantially all of said first and second layer reagents from one another and having a sample intercept area that is at least coextensive with said sample intercept area of said first layer, said barrier composition within said sample intercept area being uniformly impermeable to interferants; said barrier composition incorporating one of said reagents within said coextensive sample intercept area.
32. An integral element useful in the analysis of a fluid sample for a predetermined analyte, the element comprising
1. an enzyme capable of complexing with said analyte to produce a decomposition product; 2. a dye precursor capable of forming a detectable change and having the formula ##STR20## wherein: n represents a positive integer of 1 to 10; X.crclbar. represents an acid anion; R1 and R2 are the same or different and each represents either hydrogen, a substituted or unsubstituted alkyl group, an alkoxyalkyl group, a carboxyalkyl group, a sulfoalkyl group, a sulfato alkyl group, an acyloxyalkyl group, an alkoxycarbonylalkyl group, an aralkyl group, an alkenyl group, or a substituted or unsubstituted aryl group; and Z1 and Z2 are the same or different and each represents the nonmetallic atoms necessary to complete a heterocyclic nucleus; and 3. a barrier composition separating said enzyme and said dye, said composition being substantially uniformly permeable to said decomposition product and substantially impermeable to interferants. 50. An integral element useful in the analysis of a fluid sample for a predetermined analyte, the element comprising
1. a layer which includes an enzyme in a sample intercept area, capable of reacting with said analyte to produce a decomposition product; 2. a dye precursor having the formula ##STR24## wherein: n represents a positive integer of 1 to 10; X.crclbar. represents an acid anion; R1 and R2 are the same or different and each represents either hydrogen, an alkyl group, an alkoxyalkyl group, a carboxyalkyl group, a sulfoalkyl group, a sulfato alkyl group, an acyloxyalkyl group, an alkenyl group, or an aryl group; and Z1 and Z2 are the same or different and each represents the nonmetallic atoms necessary to complete a heterocyclic nucleus; and 3. a barrier composition separating said enzyme and said dye and having a sample intercept area that is at least coextensive with said enzyme sample intercept area, said composition within its sample intercept area being uniformly permeable to said decomposition product and impermeable to interferants. 26. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. urease; and
2. a reagent capable of interacting with ammonia to form a detectable product; the improvement comprising a barrier composition separating said urease and said reagent, said composition being substantially uniformly permeable to ammonia vapor and substantially impermeable to liquid bases, said reagent including a dye precursor that deprotonates in the presence of NH3 and has the formula ##STR19## wherein: n represents a positive integer of 1 to 10; X.crclbar. represents an acid anion; R1 and R2 are the same or different and each represents either hydrogen, a substituted or unsubstituted alkyl group, an alkoxyalkyl group, a carboxyalkyl group, a sulfoalkyl group, a sulfato alkyl group, an acyloxyalkyl group, an alkoxycarbonylalkyl group, an aralkyl group, an alkenyl group, or a substituted or unsubstituted aryl group; and Z1 and Z2 are the same or different and each represents the nonmetallic atoms necessary to complete a heterocyclic nucleus. 47. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. a layer comprising urease in a sample intercept area; and 2. a reagent capable of interacting with ammonia to form a detectable change;
the improvement comprising a barrier composition separating said urease and said reagent and having a sample intercept area that is at least coextensive with the sample intercept area of the layer comprising said urease, said barrier composition within its sample intercept area being uniformly permeable to ammonia vapor and impermeable to liquid bases, said reagent including a chromogen that deprotonates in the presence of NH3 and has the formula ##STR23## wherein: n represents a positive integer of 1 to 10; X⊖ represents an acid anion; R1 and R2 are the same or different and each represents either hydrogen, an alkyl group, an alkoxyalkyl group, a carboxyalkyl group, a sulfoalkyl group, a sulfato alkyl group, an acyloxyalkyl group, an alkoxycarbonylalkyl group, an aralkyl group, an alkenyl group, or an aryl group; and Z1 and Z2 are the same or different and each represents the nonmetallic atoms necessary to complete a heterocyclic nucleus. 21. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. urease; and 2. a reagent capable of interacting with ammonia to form a detectable product;
the improvement comprising a barrier composition separating said urease and said reagent, said composition being substantially uniformly permeable to ammonia vapor and substantially impermeable to liquid bases, said reagent including a leuco dye that deprotonates in the presence of NH3 and has the formula ##STR17## wherein: a. k represents 0 or 1; b. m represents 0 or 1; c. n represents or 2; d. each L represents a methine group; e. A represents an electron donating moiety, selected from the group oxygen (--O--), sulfur (--S--), and ##STR18## f. E is an electron-withdrawing group selected from the class consisting of nitro, cyano, ethoxycarbonyl, and halogenated methyl; g. p represents 0 or 1; h. R3 represents either an alkyl group having from 1 to 18 carbon atoms and preferably a lower alkyl group having from 1 to 4 carbon atoms, a sulfoalkyl group, a carboxyalkyl group, a sulfatoalkyl group, an alkoxyalkyl group, an acyloxyalkyl group, an alkoxycarbonylalkyl group, a dialkylaminoalkylene group, a cycloaminoalkylene group, an alkenyl group, or an aryl group; i. X.crclbar. is an acid anion; j. Z3 represents the atoms necessary to complete an aryl ring; and k. Z4 represents the nonmetallic atoms necessary to complete a heterocyclic nucleus. 46. In an integral element useful in the analysis of a fluid sample for urea, the element including
1. a layer comprising urease in a sample intercept area; and 2. a reagent capable of interacting with ammonia to form a detectable change;
the improvement comprising a barrier composition separating said urease and said reagent and having a sample intercept area that is at least coextensive with the sample intercept area of the layer comprising said urease, said barrier composition within its sample intercept area being uniformly permeable to ammonia vapor and impermeable to liquid bases, said reagent including a leuco dye that deprotonates in the presence of NH3 and has the formula ##STR21## wherein: a. k represents 0 or 1; b. m represents 0 or 1; c. n represents 1 or 2; d. each L represents a methine group; e. A represents an electron donating moiety, selected from the group oxygen (--O--), sulfur (--S--), and ##STR22## f. E is an electron-withdrawing group selected from the class consisting of nitro, cyano, ethoxycarbonyl, and halogenated methyl; g. p represents 0 or 1; h. R3 represents either an alkyl group having from 1 to 18 carbon atoms and preferably a lower alkyl group having from 1 to 4 carbon atoms, a sulfoalkyl group, a carboxyalkyl group, a sulfatoalkyl group, an alkoxyalkyl group, an acyloxyalkyl group, an alkoxycarbonyl alkyl group, a dialkylaminoalkylene group, a cycloaminoalkylene group, an alkenyl group, or an aryl group; i. X.crclbar. is an acid anion; j. Z3 represents the atoms necessary to complete an aryl ring; and k. Z4 represents the nonmetallic atoms necessary to complete a heterocyclic nucleus. 2. An element as defined in
3. An element as defined in
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23. An element as defined in
24. An element as defined in
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38. An element as defined in
39. An element as defined in
41. An element as defined in
42. An element as defined in
43. An element as defined in
52. An element as defined in
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now U.S. Pat. No. 3,983,005 entitled "Multilayer Analytical Elements For Use in the Assay of Cholesterol," refiled as a continuation-in-part application of Apr. 7, 1975, Ser. No. 565,897. In one embodiment of this application, the enzyme layer includes cholesterol oxidase, and a hydrolysis medium comprising a combination of protease and lipase which combination saponifies any cholesterol esters present in the sample to "free" cholesterol. Such a hydrolysis system is described in detail in commonly assigned U.S. Application Ser. No. 454,659, entitled "Method for the Enzymatic Hydolysis of Cholesterol," filed Mar. 25, 1974 in the names of Goodhue and Risley. According to this embodiment, the barrier composition is a hydrophilic polymeric material such as agarose; and the preferred indicator layer is a gelatin dispersion of peroxidase, an oxidizable alkyl amine and a naphthol capable of coupling with the oxidized amine. As one might expect the incorporation of a protease into a layer contiguous with an indicator layer whose matrix is composed primarily of gelatin poses certain stability problems since the protease attacks the proteinaceous gelatin as soon as wetting occurs. In this element the function of the barrier composition is to permit migration of a detectable product, namely hydrogen peroxide produced by the cholesterol oxidase-catalyzed reaction of cholesterol with oxygen, while prohibiting passage of the protease. It has been found that, in the embodiment shown in FIG. 5, only the cholesterol and not the esters are capable of penetrating the agarose to reach the enzymes in portion 18d. Accordingly, the uppermost indicator level should extend into the lowermost portion 54 of the spreading layer, at least to the extent that the protease and lipase enzymes are located there. The oxidase enzyme then is located in portion 18d.
The oxidation of the alkyl amine occurs as the result of free oxygen formed by the peroxidase induced decomposition of H2 O2 which passes through the barrier layer from the enzyme layer. The reactions which occur in this assay are as follows: ##STR16##
Reaction (6) indicates the release of free cholesterol from cholesterol and cholesterol-esters complexed with serum lipo-proteins. Reaction (7) shows the cholesterol oxidase reaction. Reaction (8) demonstrates one of the many possible dye-peroxidase systems which may be used to detect H2 O2 production. Here a system involving oxidation of 4-aminoantipyrine and subsequent coupling with the naphthol to form a compound with an absorption maximum at 490 nm is shown.
The barrier composition for this cholesterol assay element preferably is coated at a coverage ranging from about 0.1 to 1 g/m2. A highly preferred embodiment of the present invention utilizes a layer of agarose at a coverage of from about 0.25 to about 0.70 g/m2. Other examples of useful barrier compositions for a cholesterol detection element of this type include poly(acrylamide) resins such as poly(isopropylacrylamide).
In preparing integral analytical elements of this invention heretofore described, the layers can be preformed separately and laminated to form the overall element. Layers prepared in such a manner are typically coated from solution or dispersion on a surface from which the dried layer can be physically stripped. However, a convenient method which can avoid the necessity for multiple stripping and lamination steps is to coat an initial layer on a stripping surface or a support, as desired, and thereafter to coat successive layers directly on those coated previously. Such coating can be accomplished by hand, using a blade coating device or by machine, using techniques such as dip or bead coating. If machine coating techniques are used, it is often possible to coat adjacent layers simultaneously, using hopper coating techniques well known in the preparation of light-sensitive photographic films and papers. Interlayer adhesion problems can be overcome without harmful effect by means of surface treatments including extremely thin application(s) of subbing materials such as are used in photographic films.
If the layers described herein are formed by coating from solutions or dispersions as described in the aforementioned Pryzbylowicz et al application, it is often necessary to include coating aids which impart uniform coating properties to the various layers.
Whatever coating aids are used for this purpose, it is important that they do not inhibit the enzyme of the enzyme layer or any of the indicators present in the indicator layer. Particularly useful coating aids for this purpose include nonionic surfactants such as the octyl phenoxy polyethoxy ethanols commercially available from Rohm and Haas Co. under the Triton tradename (X-100, 102, 165, 305 and 405 being particularly useful), (p-nonylphenoxy) glycerol commercially available from Olin Mathieson Corp. under the tradename Surfactant 10G, and polyethylene glycols such as the Carbowax materials available from Union Carbide.
It may be appropriate to prepare an element, in accordance with the invention, having layers that are initially non-contiguous, and which further can be spaced apart, such as by the use of interleaves as described, for example, in U.S. Pat. No. 3,511,608 or by the use of a resilient absorbent material or deformable supports as described in U.S. Pat. No. 3,917,453 and U.S. Pat. No. 3,933,594. As will be appreciated, if the element has initially non-contiguous layers, it may be necessary to apply compressive force or otherwise provide means to bring layers of the element into fluid contact at the time of its use to provide an analytical result.
In the use of the above analytical tape or element, the blood cells can first be separated from the serum, by such means as centrifuging, and the serum applied to the element. A particularly significant advantage of the analytical element described herein is its ability to be used to analyze either serum or whole blood. Of course, where energy transmission techniques are used, the spreading layer and any other layers must be uniformly permeable to the detecting radiation, and unless some mechanism is provided for removing the undesired residues of whole blood (e.g. wiping off or stripping off the spreading layer before measurement), it is preferable to use blood serum obtained in any fashion to make the analysis.
A typical automated analysis system utilizing the multilayer analytical element of this invention would provide means for disposing the element below a dispenser where a drop of the sample to be analyzed, such as whole blood or serum, would be applied to the surface of the element, then directing the element through one or more processing zones. The analytical measurement would typically be made by passing the element through a zone in which suitable apparatus for reflection, transmission or fluorescence spectrophotometry is provided.
By way of example only, the following are non-exhaustive specific illustrations of the above-described embodiments. Examples 1-6 are directed to elements for BUN analysis, while Example 7 concerns an element for cholesterol analysis as disclosed in the aforesaid continuation-in-part application of Ser. No. 454,621.
A series of 3 test elements for BUN having the format shown in FIG. 1, utilizing different chromogens in the indicator layer, was prepared in the following manner.
An ammonia-permeable, relatively water-impermeable indicator layer comprising the different chromogens numbered 1 through 3 below in Table 1, and cellulose acetate butyrate at 8.55 g/m2 as coated on a poly(ethylene terephthalate) film support from a mixture of organic solvents consisting essentially of dichloroethane and acetone. Each of these coatings was overcoated with an ammonia-permeable, relatively water-impermeable layer of cellulose propionate valerate having 30% by weight valeryl content, at 0.84 g/m2, coated from t-butyl alcohol. The elements were then overcoated with a buffered gelatin (pH 7.5) layer containing gelatin at 5.4 g/m2, urease at 4300 U/m2, dipotassium phosphate buffer at 0.05 g/m2, bisvinylsulfonylmethyl ether as a hardener at 0.06 g/m2, oleic ether of polyethylene glycol as a surfactant at 0.32 g/m2, and ethylene diamine tetraacetic acid tetrasodium salt at 0.40 g/m2.
To complete the element a reflecting-spreading layer comprising cellulose acetate at 6.6 g/m2, titanium dioxide at 46.0 g/m2, and Triton X-405 (an octyl phenoxy polyethoxy ethanol available from Rohm and Haas Co.) at 2.69 g/m2 was applied from a mixture of acetone and dichloroethane (1:1) dual melted with xylene (6.4:1).
The completed analytical elements were tested in the following manner.
Aqueous solutions of reagent grade urea (<10-5 M NH3 contained in a 1 M solution) were prepared in the range 1 to 150 mg urea nitrogen/dl. The normal range in serum is 10 to 20 mg BUN/dl. These solutions were spotted on the test elements (10 μl spots). A spectrophotometer was used to measure the reflection densities generated at appropriate wavelengths. The measurements indicated in Table I were taken after 10 minutes incubation at 50°C
| TABLE I |
| __________________________________________________________________________ |
| Reflection Density |
| Amt. of |
| Wavelength |
| urea concentration in mg/dl |
| Chromo- |
| of 5 10 20 50 100 150 |
| Ex. |
| Chromogen |
| gen Measurement |
| mg/dl |
| mg/dl |
| mg/dl |
| mg/dl |
| mg/dl |
| mg/dl |
| __________________________________________________________________________ |
| 1 1-methyl-2 |
| 1.08 585 nm 0.62 |
| 1.06 |
| -- -- -- -- |
| (2,4-dinitro- |
| gm/m2 |
| phenyl)methyl |
| quinolinium |
| perchlorate |
| 2 6-(2,4- di- |
| 1.08 610 nm 0.45 |
| 0.81 |
| 1.22 |
| 2.06 |
| -- -- |
| nitrophenyl)- |
| 6H-pyrido[2,1- |
| gm/m2 |
| s]isoindolium |
| perchlorate |
| 3 phenolsulfone- |
| 0.8 565 nm 0.13 |
| 0.20 |
| 0.44 |
| 0.71 |
| 0.90 |
| 0.96 |
| phthalein |
| gm/m2 |
| sodium salt |
| __________________________________________________________________________ |
Table I demonstrates the utility of analytical elements of the present invention. It is noted that, because of the high extinction coefficients (near 104) of the chromogens of Examples 1 and 2, it is preferred that measurement be made using kinetic analysis, utilization of a shorter time of reaction, or reading the dye off-peak, for determination of concentrations of BUN in excess of 50 mg/dl.
An analytical element was prepared as follows: An indicator layer comprising the chromogen 1-ethyl-4(2,6-dinitrophenyl) methyl quinolinium ethyl sulfonate at 3.78 g/m2 and cellulose acetate at 6.43 g/m2 was coated on a polyethylene terephthalate film support from a mixture comprised of methanol and acetone (1:2). An ammonia permeable barrier layer of cellulose acetate butyrate having a 27% butyryl content and 21% acetyl content was coated over the indicator layer at 0.8 g/m2 from dichloroethane. A subbing layer of copoly(methylmethacrylate, methacryloyloxytrimethyl ammonium methyl sulfate; 2-hydroxypropyl acrylate; 2-acetoacetoxyethyl methacrylate) in the ratio of 40:20:20:20 at 0.14 g/m2 was then applied from acetone.
The element was then overcoated with a gelatin layer buffered at a pH of 8.0 comprising deionized gelatin at 21.6 g/m2, oleyl ether of polyethylene glycol at 0.65 g/m2, ethylene diamine tetraacetic acid tetrasodium salt at 0.4 g/m2, N,N-bis(2-hydroxyethyl)glycine at 5.4 g/m2, disodium orthophosphate at 0.28 g/m2, dithiothreitol at 0.9 mg/m2, urease at 27,000 U/m2, and bisvinylsulfonylmethyl ether at 0.13 g/m2. A subbing layer of poly-(N-isopropylacrylamide) at 0.32 g/m2 was applied from acetone. A spreading-reflecting layer was overcoated as described in Example 1.
The completed analytical element was evaluated in the following manner. Aqueous solutions containing 7 percent albumin and reagent grade urea in concentrations from 0 to 150 mg urea nitrogen/dl were prepared. These solutions were then applied to the test element in 10 μl aliquots. A spectrophotometer having an interference filter covering a band of 30 nm centered at 463 nm, a Wratten 2B filter and a 0.4 neutral density filter, with an incubation temperature of 40°C, was used to measure the reflection densities after 5 minutes. The following results were obtained.
| ______________________________________ |
| mg Urea Nitrogen/dl |
| Reflection Density |
| in 7% Albumin Solution |
| (5 minutes at 40°C) |
| ______________________________________ |
| 0 0.04 |
| 10 0.26 |
| 25 0.57 |
| 50 0.97 |
| 100 1.69 |
| 150 2.33 |
| ______________________________________ |
An ammonia-permeable, relatively water-impermeable layer containing a diazo coupling system as the indicator was prepared in the following manner.
A sample of 7 mil poly(ethylene terephthalate) film support subbed with gelatin was coated with a layer comprising (1) cellulose acetate butyrate having a 27 weight percent butyryl content and 21% acetyl content at 3.19 g/m2, (2) 2-nitro-4-piperidinobenzene diazonium hexafluorophosphate at 0.635 g/m2, (3) 3-(2-methoxyphenylcarbamoyl)-2-naphthol at 0.84 g/m2, and (4) the oleic ether of polyethylene glycol 1540 at 0.097 g/m2.
The coated element, a light cream color, was then tested by spotting with ammonium hydroxide solutions of concentrations equivalent to 10 and 40 mg percent urea, resulting in the generation of a light green and a darker green color respectively, illustrating the suitability of the diazonium salts as the dye precursor.
An ammonia-permeable, relatively water-impermeable layer containing an ammonia bleachable dye as the indicator was prepared in the following manner.
A sample of 7 mil poly(ethylene terephthalate) subbed as in Example 4 was coated with a layer comprising (1) cellulose acetate butyrate having a 27 weight percent butyryl content and 21% acetyl content at 3.22 g/m2, and (2) 2[β-(2-hydroxy-1-naphthyl)-α-methylvinyl]-1-benzopyrylium perchlorate at 0.322 g/m2.
The coated element, which was dark blue in color, was then tested by spotting with ammonium hydroxide solutions of concentrations equivalent to 10 and 20 mg percent urea. The dark blue color of the element was partially bleached in the area which was spotted with the low concentration of ammonium hydroxide and completely bleached in the area spotted with the higher concentration of ammonium hydroxide. Since the urea equivalency thus tested covers the range of normal BUN, Example 6 illustrates the adequacy of bleachable dyes as the indicator.
An analytical element containing all the necessary materials for the quantitative analysis of total cholesterol in blood serum was prepared in the following manner. A sample of gelatin subbed 7 mil poly(ethylene terephthalate) film support was coated with an indicator layer comprising gelatin (21.5 g/m2), peroxidase (7,000 U/m2), 4-methoxy-1-naphthol (750 mg/m2), 4-aminoantipyrine hydrochloride (635 mg/m2) and 4-amino-5,6-dihydroxy-2-methylpyrimidine (10.8 mg/m2) at a pH of 7∅ A barrier layer comprising agarose (108 mg/m2) was then applied followed by an interlayer comprising poly(n-isopropylacrylamide) (323 mg/m2) and a spreading and enzyme layer comprising cellulose acetate (9.7 g/m2), titanium dioxide (64.5 g/m2), Lipase M (1.08 g/m2), α-chymotrypsin (2.15 g/m2), Triton X-100, an octylphenoxy polyethoxy ethanol available from Rohm and Haas Co., (2.96 g/m2), and cholesterol oxidase (450 U/m2).
To evaluate the coated element a series of blood serum samples containing 122, 244 and 366 mg percent cholesterol were applied to the coated element (10 μl drops). After 12 minutes at 37°C a spectrophotometer with a 660 nm interference filter was used to measure reflection density. A stepwise increase was detected for each increase in cholesterol, as follows:
| ______________________________________ |
| Test Serum |
| (mg % Cholesterol) |
| DR 660 nm (12 min at 37°C) |
| ______________________________________ |
| 122 0.12 |
| 244 0.21 |
| 366 0.31 |
| ______________________________________ |
In these examples, an element similar to the element of Example 1 was prepared, except that the indicator layer was prepared by coating 2.26 g/m2 of 4(2,6-dinitro-4-chlorobenzyl)1-propyl quinolinium ethane sulfonate in cellulose acetate having 40% acetylation, so that the indicator layer itself incorporated a carrier composition, in addition to a separate barrier layer formed by cellulose acetate butyrate (27% butyryl content and 21% acetyl content) disposed between the indicator layer and the enzyme layer. The enzyme layer was buffered to a pH of about 8∅
The analytical elements were evaluated in the following manner:
Aqueous solutions containing albumin and reagent grade urea in 4 concentrations ranging from about 15 to about 115 mg urea nitrogen/dl were prepared. These solutions were then applied to the test element in 10 μl aliquots. Reflection densities at 670 nm were measured after 5 minutes. Results are shown in Table II.
| Table II |
| ______________________________________ |
| Amounts of BUN (mg/dl) |
| Example 15.2 30.0 74.3 114.5 |
| ______________________________________ |
| 8 0.4251 0.6567 1.3280 -- |
| 9 0.4156 0.6475 1.3433 1.8942 |
| 10 0.4117 0.6403 1.3090 1.8859 |
| 11 0.4151 0.6627 1.3152 1.9255 |
| 12 0.4234 0.6536 1.3566 1.9097 |
| 13 0.4113 0.6546 1.3171 1.8790 |
| 14 0.4099 0.6559 1.3106 1.8739 |
| 15 0.4121 0.6428 1.3230 1.9024 |
| 16 0.4157 0.6586 -- 1.8650 |
| 17 0.4094 0.6549 1.3335 1.8800 |
| mean 0.4149 0.6528 1.3268 1.8906 |
| SD 0.0054 0.0071 0.0163 0.0192 |
| % COV 1.30 1.08 1.23 1.01 |
| ______________________________________ |
While the invention has been described in detail with particular reference to preferred embodiments thereof, it will be understood that variations and modifications can be effected within the spirit and scope of the invention.
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| Executed on | Assignor | Assignee | Conveyance | Frame | Reel | Doc |
| May 18 1978 | Eastman Kodak Company | (assignment on the face of the patent) | / | |||
| Jan 18 1995 | Eastman Kodak Company | CLINICAL DIAGNOSTIC SYSTEMS INC | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 007453 | /0348 |
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