CA1069420A - Integral element for analysis of liquids - Google Patents

Abstract

INTEGRAL ELEMENT FOR ANALYSIS OF LIQUIDS
Abstract of the Disclosure The present invention concerns a multilayer element for the analysis of liquids which comprises, in the form of an integral arrangement, (1) a reagent layer including a composition comprising material that is reactive or otherwise interactive in the presence of a substance to be analyzed (analyte) to provide a diffusible, detectable species, and (2) a registration layer that is permeable to the detectable species and within which such species, e.g., a dye, can be detected. The element also includes a spreading layer, preferably separated from the registration layer by the reagent layer, to spread within itself at least a component of liquid applied to the element such that a uniform apparent concentration of a spread component (e.g., analyte) is pro-vided to the reagent layer.

Description

The layers of the element, which are in fluid contact and can be self-supporting or carried on a radiation-tIansmissive support, can also include filter layers to restrain from penetration into a reagent or registration layer blood cells or other filterable components that might interfere with an analysis Or choice. Prererably, any desirable filtering function is performed by one or more appropriately constituted spreading layers or radiation-blocking layers. In preferred operation, a sample of liquid under analysis is applied to the reagent layer o~
the element or, i~ present, to the spreading layer. If the sample contains analyte that the element is intended to detect, chemical reaction or other interaction within the reagent layer provides a detectable species that diffuses, via any intervening layers such as a radiation-blocking layer3 into the fluid contacting registration layer for detectlon there by radiometric techniques such as re~lection spectrophotometry or the like.

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Background of the Invention Field of the Invention Chemical analysis of liquids such as water, foodstuffs like milk, and biological liquids is often desirable or necessary. Various elements to facilitate liquid analyses are known. Such elements have often included a reagent for a substance under analysis, termed analyte herein, which reagent, upon contacting a liquid sample containing the analyte, effects formation of a colored material or another detectable change in response to the presence of the analyte. Such elements include, for example, pH test strips and similar indicators wherein a paper or other highly absorbent carrier is impregnated with a material, chemically reactive or otherwise, that responds to contact with liquid containing hydrogen ion or other analyte and either generates color or changes color. Depending on the selection of responsive material, the change is usually qualitative or, at best, semiquantitative.
In certain fields, it is often required that analytical techniques yield rapid, quantitative results. Much recent development work has attempted to provide elements useful in diagnostic chemical analysis, where testing of biological liquids including body fluids such as blood, serum, urine and the like must produce highly quantitative results, rapidly and conveniently.
Solution chemical techniques have enjoyed broad , :
acceptance in the clinical laboratory environment, particularly in automated analysis. Such teehniques, however~
require analyzer equipment having intricate solution handling and transport capabilities. ~nalytical equipment of the "wet ,~

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chemistry" variety, illustrated for example in U. S.
Patent Nos. 2,797,149; 3,036,893, and 3,526,480 is often expensive and may require skilled personnelg both for operation and to maintain the high level of cleanliness that is needed to avoid sample to sample contamination.
As an alternative to solution chemistry, varlous multi-layer integral elements for non-solution, essentially dry chemical analysis have been proposed. The term "integral", as used herein to describe analytical elements, refers to elements containing two or more desirably dis-crete layers that are superposed, in substantially con-tinuous intimate contact with ad~acent layers in the element, and not separable without damage to the element. Although essentially dry analysis offers substantial storage, handling and other conveniences as compared to wet chemistry, variations of the "dry" approach have enjoyed only limited success and have been used primarily for qualitative and semi-quantitative test purposes.
Description of Related Art A basic variety of integral analytical element is described in U. S. Patent No. 3,092,465. Such multi-layer elements use an absorbent flbrous carrier impregnated with one or more reagents, typically including a color former, over which is coated a semi-permeable membrane.
Upon contact with a test liquid, analyte passes through the membrane and into the fibrous carrier to generate color in an amount related to the concentration of analyte. The membrane prevents passage and absorption of certain interfering components, such as red blood cells, that could impair accurate reading Or the color provided as a test result.

- Analytical elements that rely on absorbent filter papers or.other fibrous media to receive and distribute - a liquid sample hat~e not been popular, compared to wet -. chemical procedures, in appliGations such as clinical .
laboratory testing, presumabiy due to their inability .~ to produce highly accurate, quantitative results. It . is described in the literature that diagnostic elements using -. impregnated bibulous materials, such as fibrous filter papers~ can produce non-uniform test results. In U. S.
. Patent No. 3,050,373, it is mentioned that precipitation can occur in the impregnating solutions, thereby impairing uniform dlstribution of reagent within the bibulous.carrier : or matrix. Also, elements using fibrous, bibulous ~ ; materials are susceptible to the occurrence of a non-uniformity ;.- in test result that is termed "banding." "Banding" is .
; ~ exemplified by a test result occurring to a greater extent in one portion of the region of the element experiencing a test result, such as at the periphery.of the region . penetrated by an applied sample. It is apparently the ~-. result of extensive and extremely non-uniform migration Or rr .~
=. sample components or reagent chemicals within the bibulous ,.~. . material, possibly due to chromatographing, to provide h~gh local concentrations of such chemicals. Gelatin .
. and gelatin-like materials are described :: -in U. S. Patent Nos. 3,061,523 and 3,104g209 as useful ~ ~ ;
. ;.. - constituents of the impregnating solu.tion, due apparently . to their ability to restrain the high rate of such migration .
.. . . .
;~ and consequently to encourage improvements in test result : uniformity. Ho~-ever, gelatin and gelatin-like materials ;- in the fibrous, reagent containing bibulous matrix d~

- .crease the rate of sample upta~e as compared to the more ,~; . . . .
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hiyhly absorbent gelatin-free matrix. Such decreased a~sorp-tion can leave sur~ace liquid on the element and necessitate washing the element to remove the excass prior to making a test determination. As a result, an upper limit on the amount of gelatin to be impregnated into a bibulous matrix is typically specified. Such properties also characterize analytical elements using, ~ithout more, layers solely of gelatin or similar materials, as discussed in U.S. Patent No. 3,526,480.
Integral analytical elements adapted for automated test procedures have al~o ~een described, such as in U.S. Patent Nos. 3,368,872 and 3,526,480. Such descriptions re~er to means for avoiding chromatographic ef~ects Coften called ringing, targeting, doughnuting or ~anding~ in the element by immo~
zing reagent or including a means to decrease the tenaency of an applied sample to exert a ~ashing e~fect on incorporated reagent, as by use of simple porous mem~ers o~er an absorbent, reagent containing material, such as fibrous filter paper~
However, there is no suggestion in such descrtptions of using within an element a means that not only takes up a liquid sample ~ut also provides a uniform apparent concentration of a sample component such as analyte to su~stantially the : entire portion of a reagent layer surface that is contacted by ;.~
an applied sample. 5uch uni~orm apparency of concentration is : :
extremely important in obtainlng test results appropriate '~ -~
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, for interpretatlon by automated readout, ~hether den~itometric, :., colorimetric, fluorimetric, or other~ise~ This is tru0 even :~ '.
in the a~sence of gross non-uniformities such as those intro- ', duced by chromatrographic effects.
~30 ~ ~eans to provi,de ~ome~hat un~orm concentration of analyts to the reagent areas o~ ~n element ~or dr~ analysis . ~ .
has been by a technique that can be termed sample aon~inement~ ..

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Usually, as is described in U.S. Patent No. 3,368,372, a barrier is inc~uded on the element to confine an applied sample in a '' ~' , - 6A ~

predetermined re~ion Or the elèment's surface, with the result that excess liquid is usually present on the element after sample application. This can create inconveniences, as in the handling and cleanup of excess sample remaining on the element and, more seriously, can require extremely precise sample volume delivery when applying sample to the element.
There has been some recognition o~ the need to promote or avoid, as desired, the migration Or material between layers Or integral analytical elements, as is discussed in U.S. Patent os. 2,7613813; 2,672,431; 2,672~432; 2,677,647; 2,923,669;

3,814,670 and 3,843,452. However, this has been in the context Or elements for determining the presence of micro-organisms, and the elements described for such purposes typically include at least one layer comprising a fibrous matrix and require non-discrete layers, the interface of which is a blend of the ad~acent layers.
Until very recently, there was no suggestion in art relatlng to analytical elements of a layer or other means to .
receive sample constituents and to encourage them to distribute within that layer to achieve therein an apparent concentrational ~ .
~ ~ uniformity of analyte, analyte products or other substances to r^. be provided, in such uniform apparent concentration, to an 1~ ~ ` associated layer for analytical reactions or similar activity.

In fact, as was apparently well recognized, the structural ' Sr- i .
and chemical characteristics of bibulous and other fibrous ` materials used in most known analytical elements (such as absorbent cellulosic filter papers, glass fiber papers, wood, -~ etc.) might impair such a result for reasons Or physical - - restraint, non-uniform permeation of sample constituents or : ; undesirable chemical binding. Additionally, the choice of -- fibrous materials can frustrate highly accurate measurement due to severe non-uniformity in properties such as structure and texture. It is known, ror example, that in the preparation L~

of papers, starting ~ibers are often processed to fo~m smaller constituent fibers, called tendrils, that increase the strength of the resultant paper. The term l'fibrous", as used herein to describe materials such as papers and the like, refers to materials prepared using pre~ormed fibers or strands that are present in the finished material. Exemplary fibers used in preparing fibrous materials are described in U.S. Patent No.
3,867,258.
Non-uniformity in the detectable color response or other test result obtained when using integral analytical elements incorporating fibrous materials has been recognized as a problem associated with the use of such elements~ Improved devices using such materials to provide absorbent layers have sought to overcome the gross effect o-f such non uniformity, but they have not succeedPd in avoiding the problem. As an example, U.S. Patent No. 3,723,064 describes an analytical element that includes regions of different effective permeability to an analyte or reaction product of an analyte and produces a plurality of spaced-apart, threshold color indications as an analytical result.
Although the desirability of a smoo~hly continuous response is manifest, an element made as described in the '064 patent can only yield an approximate analytical result, the accuracy of ~hich varies indirectly with increased spacing between thresholds.
If the diference in permeability hetween regions were decreased, to narrow the interval bet~een thresholds in the interest of increased response precision over the intended dynamic range, the complexity of elements made in accordance with the '064 patent would increase dramatically. No suggestion is made as to how one might improve the uniformity and precision of a continuously varying test result and, however optimized, elements of the '064 patent would produce a discontinuous response that would apparently be non-uni~orm v withln each region OI permeability due to non-uniformitles associated with the use of filter papers and other fibrous materials.
U.S. Patent No. 3,791,933 describes a multi-component dev~ce for the assay of enzyme substrates and metabolites., such as in body fluids- However, such device is not an integral element. Rather, it is a clamped array adapted to receive a ~est sample, filter out or otherwise remove large sample constituents (such as proteins) and effect a test reaction to produce a detectable result, such as the generation Or a color.
Although glass fiber paper is disclosed as assi~ting in distri-buting a reaction mixture across a plastic viewing window, ~ch material apparently merely assists the outward diffusion of liquid sample within the glass fiber layer to enlarge the region Or ..
the element exhibiting a test result and thereby render the result more easily visible. There is no sug~estion of any means to :
form within the region of diffusion a concentrational uniformity of analyte, which~ of course, is extremely important for the production of an analytical result that is-of a uniform nature and, as such, precisely detectable.
Improved multilayer integral analytical elements are described in French Patent Application 7,323,599, filed June 28, 1973, now French Patent No. 2,191,734. Such elements can receive a liquid sample and spread the sample within a spreading layer of the element to obtain in the element a uniform apparent concen-tration of analyte, other appropriate sample constituent or analyte product and produce in the presence of analyte an analytical result that, by virtue of its unirormity, can be measured quantitatively by automated devices, using techniques such as spectrophotometry, fluorimetry, etc. Elements disclosed in French Patent No. 2,191,734, include spreading layers and reagent layers that contain a reactive or otherwise interactive . .
. -9 material t-hat, by virtue of its activity, promotes in the element a radiornetrically detectable change, such as a color c hange .
However, color formation or the pro~id~ng of another analytical result often requires a serieS of reactions that can ~e difficult to control and may be sub~ect to chemical or okher interferences. As an example, the fluid under analysis or by-products of the analytical determination can provide within the element constituents that interfere with detection of the test result. It is considered desirable9 therefore, to have an element for dry analysis of liquids in which the materials .
characterizing the test result can be detected without inter-~ ference from such constituents, which may provide unwanted : color, fluorescence or the like.

Accordingly, although the analytical elements of `i French Patent No. 2,191,734 represent a substantial advance in devices for the essentially dry analysis of liquids, -~ elements of different or expanded capabilities would be ~; . desirable.

, Summary of the Invention - The present invention provides novel integral elements . . .
i ` I for analysls of liquids, such as biological liquids. As E- - referred to herein, the terms "integral element" and "integral analytical element" re~er to composite elements including an ~ "integral" array of at least two superposed layers. Elements - of this invention are capable of performing internally a variety of sample handling and/or processing functions.
TheJ do not require expertise in their use and, especially in their preferred embodimen~s, they can produce quanti-., . ~ . .
` ta~ive analytical resul~s withou~ specialized ~ .. -. . : .
"` "'' ' -1~-. . -- ' spottin~ or other p~cedures such as sample confinement, washing or other removal Or excess sample. Further, the results produ~ed b~ elements Or this invention are substantially consistent and rree from deleterious internal variations so that automated means of measuring electromagnetic radiation (radiometric techniques) can be used to detect such results~ if necessary or desirable, with minimal risk of inconsistency.
Stated more particularly, thb present invention provides integral analytical elements composed of multipleg super-posed layers which can provide quickly within the element a detectable change in response to the presence of an analyte in liquid appiied to the element. Elements of this invention can be used ror diagnostic analysis Or biological liquids, such as blood, blood serum or urine, and include, in fluid contact~
(1) a reagent layer that is permeable to at least analyte or . j .
; an analyte precursor and which has therein a composition containing ", ~r , material that is interactive in the presence of analyte to ... . . .
provide a detectable chemical species~ such as a dye, that is diffusible within the element and (23 a registration layer that ~ ~ is permeable to the detectable species and within which the t : detectable species can be detected, such as by radiometric t`.'' `
techniques. Registration layers as described herein are generally prepared without the inclusion of any such chemically reactive : materials or other materlals as would interfere with such detection. The various l~yers of the present elements can . .
~ ~; be carried on a radiation-transmissive support. As use~ i-~. .. . , , ~ - . _ . - -~ herein, the term "radiation-transmissive" describe~ supports ` ` " ` `
`. - and other layers of an ana~ytical element, that pesmit ~- effective passage of electromagnetic radiation used to detect - - an analytical result produced in the el~ment~ Such trans-missiveness includes transmiss~on of electromagnetic radiation ,. . .
- of a wavelen~th or wavelen~ths within the region between ~ about ~00 ~m and 900 ~mt and also o , " . ~
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detectable radiation as is produced by radioactivity. Radiation-tra~smissive layers and ~upports can be transparent, if desired, and this may be beneficial for measurements to be made at low levels of radiation. When the element includes a support, the registration layer is interposed between the support and the reagent layer and usually is ad~acent to the support.
The elements of this invention can include a radlation-bloc~ing layer, which is usually interposed between the reagent layer a~d the registration layer. The radiation-blocking layer is a layer that contains one or more opacifying agents-and inhibits passage in or through such layer of electromagnetic radiation, such as at the wavelength or wa~elengths used for excitation and/or detection of a species within the registration layer. Further, the subject elements can include a sample spreading layer that is in fluid contact wlth other layers of the element, such as the reagent layer and the registratio~ layer.
The sample spreading layer, synonymously referred to herein as a spreading layer or a metering layer, is capable of distributing or metering within the layer substance(s) including an analyte or an analyte precursor in a liquid sample applied to the element to provide, at any given time, a uniform apparent concentration of such substance at the sur~ace of the spreading layer facing, i.e., closer to, the reagent layer. The applied sample need not be confined to obtain such uniform concentration which, although it will be uniform at any point in time can change over a period of time without deleterious effects.

_In ~arious preferred embodiments, the spreading layer is isotropically porous; that is, it is porous in every direction ~,~,,,. ,",, $
within the layer. Reference herein to isotropic porosity identifies the fact of porosity in all direction within the spreading layer. It will be understood that the degree of such poros~ty ~ay be variable, if necessary or desirable, for example, re~arding pore size, percen~age of void volume or _12-otherwise. The term isotropic porosity (or isotropically porous) as used herein should not be confused with the terms isoporous or ionotropic, often used with reference to filter membranes to signify those membranes having pores that are continuous between membrane surfaces. Llkewise, lsotropic porosity should not be con~used with the term isotropic, used in contradistinction to the term anisotropic, which signi~ies filter membranes having a thin "skin" along at least one surface of the membrane. See for example, Membrane Science and Technology, James Flinn ed~ Plenum Press, New York (1970). The reagent layer is preferably of substantially uniform permeability to at least one substance spreadable within the spreading layer and to the diffusible detectable species provided in the reagent layer by virtue of the interaction described herein. The registration layer is preferably of substantially uniform permeability to the detectable species.
Uniform permeability of a layer refers to permeability such that, when a homogeneous liquid is provided uniformly to a surface of the layer, identical measurements of the concentration of such fluid within the layer, but made through different regions of a surface of the layer, will usually yield substantially equal results. By virtue of uniform permeability, undesirable concentration gradients can be avoided within, for example, a reagent layer. It is not necessary that all possible measurement techniques produce such results. The desirability of a particular technique and of specific measurement parameters will depend on the physical characteristics of the layer, such as its tendency to transmit, absorb, or scatter radiation. The selection in any instance of an appropriate measurement technique (e.g., colori-metric, densitometric, fluorimetric) and of appropriate measure-ment parameters ~e.g.g aperture size and configuration) will be apparent to those familiar with analytical procedures.

As discussed elsewhere herein, uniform permeability is not considered characteristic of fibrous materials such as filter paper. It ls believed that factors such as variable wicking action within a fibrous material, differences in fiber size or spacing and the like, can effect the formation within such fibrous materials, and also in associated materials in fluid contact therewith, of variations in the apparent concentration Or permeant liquids. This Or course, introduces undesirable bias between test measurements made with.in regions having different apparent concentrations. Uniform permeability Or reagent, registration or other layers within an analytical element is desirable as a means of facilitating the convenient detection of analytical results. The analytical .
; significance of results produced in elements not having .- uniformly permeable layers can be limited. Also, the efficiency of result detection in such elements may be impaired, for ~- example if irregularly occurring concentrational or other ., ~
~- discontinuities, seen by a means of detection, are present ~:~ wlthin an element.
Reference herein to fluid contact between a spreading j layer, a reagent layer and!or other layers of an integral analytical element as described herein identifies the ability of a fluid, whether liquid or gaseous~ to pass in such element ` ~- . between superposed regions of such layers. Stated in another ; ~ manner, fluid contact refers to the ability to transport com-ponents of a fluid between the layers in fluid contact. In the ~`- - case of analysis for nitrogen containing compounds, ammonia or ~, .-.. :, other nitrogen containing gaseous materials may comprise ~luid . ~ passing between such layers. Although layers in fluid contact -~ can be contiguous, they may also be separated by intervening ., ~ .
~.~ lavers. ~owever, layers in the element that physically intervene . . . ,; , ,,~ '-' layers in mutual fluid contact will also be in fluid contact there-~- with and will not prevent the passage of fluid between such layers.

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' ~ 4,'4 ' As used in the specification and claims herein, the term "diffusible" denotes the capability Or a material to move erfectively within an analytical element by diffusion when ` that material is carried in liquid present in the element, :, such as t~e solvent or dispersion medium Or a liquid sample ¦ applied to the element. Similarly, the term "permeable"
; denotes the ability Or a substance or layer to be penetrated i effectively by a material carried, l.e., distributed in as , by dissolution or dispersion, in a liquid.
In operation, an exemplary analytical element of thls inventlon can receive a liquid sample which, if analyte positive, initiates a chemical reaction or other interaction within the reagent layer to provide a ~dlf~usible, preferably .: , - ;' radiometrically detectable species that differs from the reagent `-, layer into the registratio~ layer where it can be detected.

If necessary or desirable, a radiation-blocking layer can be : , provided in the element between the reagent layer and the registration layer. Also, a metering layer can be included ~~ such that an applied sample will pass through the metering ----~ ` layer prior to entering the reagent layer, and analyte or an analyte precursor will be distributed within the metering layer to provide - a uniform apparent concentration of such material at the surface ~ Or the metering layer facing the reagent layer. It is possible s-: - - to obtain such uniform apparent concentration over a wide range ~-~ of sample volumes applied to the element. Due to fluid contact ~i : - between the metering layer and the reagent layer and also to the ;- preferred uniform permeability of the reagent layer to substance ~ i spread within the spreading layer or to products formed by virtue of the action of such substance, uniformly metered constltuents are provided from the spreading layer to the reagent layer and i . . .
can penetrate the reagent layer essentially without the occurrence -- therein, at any instant in ~ime, o~ significant variat1ons in the apparent concentration of such ~u~stance or products ~hereof.
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~ue to the presence of an interàctive (e.g., chemicaliy reactive) material, and â uniform apparent concentration of substance plovided from t~e metering layer to the reagent layer, a uniform quantitative detectable change can be produced in the element.
Such a change, which can be the generation or destruction of coloration or fluorescence, can be detected quantitatively by radiometric techniques and, lr desired, by automatic radiometric sensing devices such as photometric or fluorimetric devices.

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Brief Description of the Drawin~s In the accompanying drawings, each of Fig. 1, Fig. 2, - Fig. 3 and Fig. 4 is an enlarged sectional view of a preferred embodiment illustrating an integral analytical element of this invention.
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Description of Preferred ~mbodiments The integral elements of this invention include a reagent layer in fluid contact with a registration layer that ` ~ is preferably radiation-transmissive. Such elements can have - . .
`~ the layers on a support, pre~erably radiation-transmissive, or ~` if the layers demonstrate appropriate durability and integrity, ~- i a support is not needed.

In one preferred embodiment~ an integral analytical - ~ element of this invention comprises a radiation-transmissive support having thereonj in fluid contact, non-fibrous lnyers inclu~ing - ~ ~; (1) a reagent layer that is permeable to at least analyte or an ~` ~ analyte precursor and which contains 2 composition having material : interactive in the presence Or analyte to provide a dif~usible, -~ detectable species, (2) a radiation-blocking layer that is permeable ., ~ 9, . to the detectabie spec es, and (3~ a radiation-trallsmissive regis-" ; .
- tratior layer that is permeable to the detectable species and ~ within which the detectable species can be detected. Optionally, - ~ the registration layer can in~clude a mcrdant for the detectable ' ~

species. The registration layer is preferably interposed between the support and the radiation-blocking layer, with the radiation-blocking layer interposed between the registration layer and the reagent layer. Also, the reagent layer is preferably of substantially uniform permeability to analyte ~also to an analyte precursor if appropriate) and to the diffusible, detectable species. The registration layer is of such permeability as regards the detectable species. The radiation-blocking layer, although usually not considered disruptive of the apparent concentration of detectable species provided to the radiation-blocking layer from the reagent layer, is desirably of uniform permeability to the detectable species.
Preferred radiation-blocking layers include an opacifying agent such as a pigment, a polymer in appropriate form, such as a blushed polymer, or both.
In accordance with another preferred embodiment of the present invention, there is provided an integral analytical element with a support having thereon, in fluid contact, a reagent layer, a registration layer and, optionally, a radiation-blocking layerg all as described above with respect to the foregoing preferred embodiment. Additionally, however, there is ~ncluded in elements of this preferred embodiment a metering layer, preferably isotropically porous and positioned in the element such that the reagent layer is interposed between the registration layer and the metering layer. In one aspect of the Yarious embodiments of this invention~ the layers are preferably non-fibrous.
Reagent layers in the elements of this invention are desirably uniformly permeable, and optionally porous ~f appropriate, to substance spreadable within the metering or spreading layer and to reaction products thereof or products formed as a result of the interaction of such substance. As used herein the term permeability includes permeability arising from porosity, ability to swell or any other characteristic. Reagent . .

~LQ~ f3 layers can include a matri~ in which the interactive material is ~istributed, i~e., dissolved or dispersed. The choice of a matrix material is, of course, variable and dependent on the intended use of the element. Desirable matrix materials can include hydrophilic materials including both naturally occurring substances like gelatin, gelatin derivatives, hydrophilic cellulose derivatives, polysaccharides such as dextran~ gum arabic, agarose and the like, and also synthetic substances such as ~ater-soluble polyvinyl compounds like poly(vinyl alcohol) and poly(vinyl pyrrolidone), acrylamide polymers, etc. Organo-philic materials such as cellulose esters and the like can also be useful, and the choice of materials in any instance will reflect the use for which a particular element is intended. To enhance permeability of the reagent layer, if not porous, it is often useful to use a matrix material that is swellable in the solvent or dispersion medium of liquid under analysis. Also, it may be necessary to select material that is compatible with the application of an adjacent layer, such as by coating means, during manufacture of the element. As an example, where the formation of discrete layers is desired and the intended analysis will be of aqueous liquids, it may be appropriate to select an essentially water soluble matrix for the reagent layer and essentially organosoluble or organo dispersible ingredients for an adjacent layer, such as a spreading layer. In such manner, mutual solvent action is minimi~ed and a clearly dellneated layer structure can be formed. In many cases, to facilitate the formation within the spreading layer of such apparent concentrational uniformity as is discussed herein, it may be desirable to have the reagent layer Gf lower permeability than is the spre-ading layer itself. Relative permeability can be determined by well-known techniques.
~ ithin the reagent layer is distributed a material that can interact in the presence of analyte, such as the analyte of choice for which the element is optimiæed. Op~ionally, the interactive material can ~ ~s~ eract with a precursor or reaction pro~uct Or such an analyte, if appropriate in Yiew Or t~e analysis Or choice, such as in elements intended to determine cholesterol, which in serum is present in esterified form, and triglycerides, which are often analyzed on the basis Or the glycerol component Or triglycerides. The term "interactive"
is meant herein to refer to chemical reactivity, catalytic activity as in the formation of an enzyme-substrate complex, or any other form of chemical or physical interackion able to produce or promote within the element, such as in the reagent layer, the formation of a diffusible species that is radiometrically detectable, that is, by suitable measurement of light or other electromagnetic .
radiation. The dlstribution of interactive material can be obta~ned by dissolving or dispersing it in the matrix material. Although uniform distributions are often preferred, they may not be necessary if the interactive material is, for example, an enzyme~
Reagents or other interactive materials soluble in the liquid under analysis may advantageously be immobilized in the reagent layer, particularly when the reagent layer is porous. The : .
"- . particular interactive materials that may be distributed within : a reagent layer will depend on the analysis of choice. In the i- - case of many analyses, enzymes such as oxidase materials like .
glucose oxidase or cholesterol oxidase may desirably be included - as interactive materials within a reagent layer of an element - - intended for the analysis of analyte that ls a substrate for ;- - such enzyme. As an example, an oxidative enzyme can be - incorporated into a reagent layer kogether with peroxidase :~ -~ or ~ peroxidative material and a material or composition that, upon oxldation in the presence of peroxidase ~or another ~. ~ substance having peroxidakive activity) and the hydrogen - ~ perox1de formed upon in~eraction Or an oxidase and its ~ubs~ràte, provides a dye or other ~te~table ~p~ies. ~n the practice of this invention, the detectable species is ~. .
~ diffusible such that it can move into the permeable registrati~n .~, ,-' . .
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layer. Such diffusivity can be imparted to detectable species not inherently diffusible by means known to those skilled in chemical synthesis, usually by the addition of chemical groups that impart the desired solubility. Where aqueous liquids are to be analyzed, solubilizing groups such as hydroxyl groups, carboxyl groups, sulfonic acid groups and the like can be useful for purposes Or solubilization.
Materials or compositions that contain an oxidizable material and can provlde a detectable species include certain dye-providing compositions. In one aspect, dye-providing compositions can include a compound that, when oxidized, can couple with itself or with its reduced form to provide a dye.
Such autocoupling compounds include a variety Or hydroxylated compounds such as orthoaminophenols, 4-alkoxynaphthols, 4-amino-5-pyrazolones, cresols, pyrogallolg guaiacol, orcinol, catechol, phloroglucinol, p,p-dihydroxydiphenyl, gallic acid~ pyrocatechoic acid, salicyclic acid, etc. Compounds of this type are well known and described in the literature, such as in The Theory of the Photo~raphic Process, Mees and James Ed, (1966), espe.cially at Chapter 17. In another aspect, the detectable species can be provided by oxidation of a leuco dye to provide the corresponding dyestuff form. Represen-tative leuco dyes include such compounds as leucomalachite green and leucophenolphthalein. Other leuco dyes, termed oxichromic compounds, are described in U.S. Paten~ No. 3,880,658 and it is rurther described that such compounds can be diffusible with appropriate substituent groups thereon. The non-stabilized oxi-chromic compounds described in U.S. Patent No. 3,880,658 are considered preferable in ~he practice of this invsnti~nc In yet another aspect~ the detectable species can be provlded by dye-providing compositions that include an oxidizable compound capable of undergoing oxidative condensation with couplers such ,.

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as those containing phenolic groups or activated methylene ~roups, together with such a coupler. Representative such oxidizable compounds include such compounds as benzidene and its homologs, p-phenylenediamines, p-aminophenols, 4-amino-antipyrine, etc: A wide range of such couplers, including a number of autocoupling compounds, is described in the literature, such as in Mees and James (supra) and in Kosar, Light-Sensitive Systems, 1965, pages 215-249.
Preferred dye-providing compositions inclùde 4-methoxy-l-naphthol, an autoooupling species, and the combination of

4-aminoantipyrine (HCl) as an oxidizable compound together with .
~: 1,7-dihydroxynaphthalene as a coupler.
? .
- To facilitate the detection Or any change produced - - in an element as described herein, such as change in coloration, optical density or fluorescence, the elements of this invention include a layer to receive any reaction products or other .~ - .
materials produced in a reagent layer~ the relative presence . ~ - or absence of which relates to detection of an analytical ,. .
,~ - result. Such a layer, referred to herein as a registration ~- layer, is permeable to detectable species formed in the element ... ..
. and is in fluid contact with a reagent layer. The registra-: tion layer may be separated from reagent layer(s) by a radiation-~;. -. .
blocking layer, such as a reflecting and/or opaque layer, to ~- facilitate result detection by various radiometric techniques.5 ' ` .
: The registration layer, which is also desirably swellable in liquid under analysis, can include hydrophilic colloids such ~ .~
~: as those useful in reagent layers. The registration layer ~- should also be radiation-transmissive to facilitate result detection. Additionally, where the detectable species produced ,. . , ~
in the element is a dye, the registration layer may contain mordant material for the dye~ such 2.S those described as t~ - useful Lmage dye mordants in color photograph.c films and ~`~ papers. ~xemplary mordants are materials such` as vinylpyridine .

compounds Or the type disclosed in U.S. Patent No. 2,484,430;

polymers containing quaternary ammonium groups such as those disclosed in U.S. Patent Nos. 3,625,694; 3,758,445;
3,709,690; 3,488,706 and 3,557,006. An exemplary mordant is rl~N-dimethyl-N-benzyl-3-maleimidopropylammonium chloride.
As mentioned previously, elements of this invention can include a radiation-blocking layer, preferably interposed between a reagent layer and the registration layer. Radiation-blocking layers are permeable to the detectable species rormed in the element and serve to inhibit passage of electromagnetic radiation, such as at the wavelength or wavelengths used for detection. Using such a layer, color or other potential interferants to result detection can be kept from the regis-tration layer. Such layers include an opacifying agent that, by virtue of its absorbance, reflectance or the like, provides a radiation inhibiting effect when incorporated into the layer.
In o~e aspect) the radiation-blocking layer can include a matrix containing an opacifying agent, such as a pigment like carbon or other inorganic pigment such as a metal salt like t1tanium dioxide~ zinc oxide, barium sulfate, etc. Blushed poly~er~, ~hich are generally reflective in nature, can ~o~prise the opacifying agent and layers ~f such blushed polymers as are useful in spreading layers can be used also as radiation-blocking layers. It will be appreciated that if a microporous, blushed polymer layer is used as a radiation-blocking layer, ` ;
such layer can also serve as a filtering layer.
In one preferred aspect, blushed polymer layers can also incorporate a reflective inorganic pigment, such as the highly reflective pigments mentioned elsewhere herein, to enhance spreading and/or reflectiv}ty. The amount of pigment that can be included in a layer together with blushed polymer is highly variable, and amounts of from about 5 percent by weight to about 1,000 percent by weight of pigment based on the weight of blushed polymer are preferred, with a pigment concentration of from about 100 weight percent to about 600 weight percent pi~ment based on the blushed polymer being most preferred.
In one embodiment, the integral elements of this invention can include an isotropically porous spreading layer in fluid contact with a reagent layer. The spreadin~ layer is a layer, isotropically porous or otherwlse, that can accept a liquid sample, whether applied directly to the spreading layer or provided to it from a layer or layers in fluid contact with the spreading layer, and within which the solvent or dispersion medium of the sample and at least one solute, dispersoid (constituent of the dispersed or internal phase) or reaction product of solute or dispersoid is distributed such that a uniform apparent concentration of such substance, i.e. solute, dispersoid or reaction product thereof (which can be an analyte or an analyte precursor), is provided at the surface of the spreading layer facing the reagent layer(s) Or the element. It will be appreciated that such an apparent concentration can be achieved with concentration gradients present through the thickness of or otherwise in the spreading layer. Such gradients do not present any difficulty to obtaining quantitative test results and can be accommodated using known calibration techniques.
The mechanism of spreading is not rully understood, but i~ is believed that spreading results from and is limited by a combination of forces such as hydrostatic pressure of a liquid sample, capillar~ action within the spreading layer, surface tension of the sample, wicking action of layers in fluid contact with the spreadi~g layer, and the like. As will be appreciated, the extent of spreading is dependent in part on the volume of liquid to be spread. However, it should be emphasized that the uniform apparent concentration obtained with spreading is substantially in~ependent of liquid sample volume and will occur with varying degrees of spreading. As a result, elements of this invention do not require precise sample application techniques. However, a particular liquid sample volume may be desirable for reasons of preferred spread times or the like. Because the elements of this invention are able to produce quantitative results using very small sample volumes that can be entirely taken up within a conveniently sized region of the spreading layer (e.g. one square centimeter), there is no need to remove excess moisture from the element after application of a liquid sample. Further, because spreading occurs in the spread~ng layer and the spread substance is provided to the fluid contacting reagent layer and without apparent substantial lateral hydrostatic pressure3 there is not the "ringing" problem often seen with prior analytical elements.
The spreading layer need only produce a uniform apparent concentration of spread substance per unit area at its surface facing a reagent layer with which the spreading layer is in fluid contact, and it is very convenient to determine whether a particular layer can be suitable for spreading purposes. Such uniformity of apparent concentration can be determined by densitometric or other analytical techniques, as by scanning the appropriate surface or reagent layer or other associated layer to determine the apparent concentration of spread substance or of any reaction product based on the concentration of spread substance. The following test is lntended only as an example and the selection of materials or test parameters does not indicate, expressly or by implication, that other materials or parameters would not be suitable for similar purposes.
In conducting such a test one can apply to a trans-parent photographic film support material, such as subbed poly (ethylene terephthalate), a transparent gelatin layer at a gelatin coverage of about 200 mg/dm2. The gelat~n may vary in hardnessS but for testing purposes a layer of gelatin hardened ~?~

to swell the layer thi~ness by about 300~O when ~ersed for

5 minutes in 22C water ls suitable. When dry, the gelatln layer will have a thickness Or about 30 microns. Over the gelatin layer can be applied~ such as by coating from solution or dispersion, the layer to be evaluated for spreading purposes.
Spreading layers can be designed to have widely varyin~ dry thieknesses, and a thickness of from about 100 to about 200 microns, is convenient ~or test purposes. A~ter drying the layers, a sample of test solution or dispersion can be applied to the surface of the spreading layer under evaluation, preferably in a small quantity so that not all portions of the layer are wetted by the applied sample, but desirably su~ficient to create a wetted region such as one having a circular area of about 8-10 millimeters in ~iameter. The selection Or a test solution or dispersion is a matter of choice and will depend in part on the type o~ sample or analyte to which the layer will be exposed under conditions of actual usage. For low mo'ecular weight materials, aqueous dye solutions can be us~d and a .0005 weight percent solution of Solatine Pin ~
is acceptable. For higher molecular weight materials such as proteins, an aqueous dispersion of bovine albumin dyed with Solatine Pink~ can be used. After applying the liquid sample to the layer under evaluation and allowing the liquid sample to disappear from the surface of and be taken up lnto the layer, the test element can be turned over and the bottom surface of the pr~posed spreading layer can be viewed through the transparent support material and gelatin layer. Ir, prior to substantial evaporation of solvent or disperslon medium, the test element exhibits a well-defined colored ~pot of a substantially uniform color density when scanned by a densitometer having an aperture of about 5 microns by~l00 microns, then spreading ~nd the achievement of a uniform apparent concentration at the bott~m surface of the ~est layer and/or in the gelatin layer has taken place. B~ substantially uniform density is meant a density across the spot, with the exception of its periphery, having maximum and `` ~ 0 minimum values ~ot more ! than about + 10 rro~ the mean density.Due to edge effects, non-characteristic density gradien~s may arise at the spot periphery but need have no errect on the significance of an analytical result. Peripheral area can vary between spots, but it will usually not be more than about 20% of the entire spot and may be less.
As mentioned herein, useful spreading or metering layers can be iso~ropically porous layers. Such layers can be prepared using a variety of components. In one aspect, particul~te material can be used to form such layers, wherein the isotropic porosity is created by interconnected spaces between the particles. Various types of particulate matter, all desirably chemically inert to sample components under analysis, are useful. Pigments, such as titanium dioxide, barium sulfate, zinc oxide, lead oxide~ etc., are desirable.
Other desirable particles are diatomaceous earth and micro-crystalline colloidal materials derived from natural or synthetic polymers. Such microcrystalline materials are described in an article entitled, "Colloidal Maa~omolecular Phenomena, Part II, Novel Microcrystals of Poiymers" by 0. A.

. . .
Battista et al published in the Journal of Applied Polymer Science, Vol. II, pages 481-498 (1967). Microcrystalline cellulose, which is commercially available from FMC Corporation under the name Avicel~, is an example of such a colloidal material which is satisfactory for use in the present invention.
Spherical particles of uniform size or sizes, such as resinous or glass beads, can also be used and may be particularly desirable where uniform pores are advantageous, such as for selective filtration purposes. If a particulate material of choice is not adherent, as in the case Or glass beads or the ,,,, ,, , , , ., , " ., . , , ~,~.. =,.. ... . . . ... ..... ....... . .

I ~ g ~ J

like, it can be treated to obtain particles that can adhere to each other at points of contact and thereby facilitate formation of an isotropically porous layer. As an example of suitable treatment, non-adherent particles can be coated with a thin adherent layer, such as a solution of hydrophilic colloid like gelatin or polyvinyl alcohol 3 and brought into mutual contact in a layer. When the colloid coating dries, the layer integrity is maintained and open spaces remain between its component particles.
As an alternative or in addition to such particulate materials, the spreading layer can be prepared using isotropically porous polymers. It is possible to prepare such polymers using techniques useful in forming blushed polymers. Blushed polymer layers can be formed on a substrate by dissolving a polymer in a mixture Or two liquids, one of which is a lower boiling, good solvent for the polymer and the other o~ which is of a higher boiling poing and is a non solvent or at least a poor solvent for the polymer. Such a polymer solution is then coated on the substrate, and dried under controlled condition. The lower boiling solvent evaporates more readily and the coating can become enriched in the liquid which is a poor solvent or non-solvent. As evaporation proceeds, under proper conditions, the polymer forms as an isotropically porous layer. Many different polymers can be used, singly or in combination3 for preparing isotropically porous blushed polymer spreading layers for use in this invention, typical examples being polycarbonates, polyamides, polyurethanes and cellulose esters such as cellulose acetate.
In preparing integral analytical elements of this invention, 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 problems o~ multiple stripping and lamination steps is to coat an initial layer on a stripping surface or a support, as desired, and thereafter ~o 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 o~ten possible to coat ad~acent layers simultaneously~ uslng hopper coating techniques well-known in the preparation of light-sensitive photographic films and papers. If it is essential or desirable that adjacent layers be discrete, and maintenance of layer separation by adJustment of coating formulation specific gravity is not satisfactory, as possibly in the case of porous spreading layers, the approprîate selection of components for each layer, including solvent or dispersion medium, can minimize or eliminate interlayer component migration and solvent effects, thereby promoting the formation of well-defined, discrete layers.
Any interlayer adhesion problems can be overcome without harmful effect by means of surface treatments including extremely thin application of subbing materials such as are used in photographic films.
For reagent layers, a coating solution or dispersion including the matrix and incorporated interactive materials can be prepared, coated as discussed herein and dried to form a dimensionally stable layer. The thickness o~ any reagent layer and its degree of permeability are widely variable and depend on actual usage. Dry thicknesses of ~rom about 1~
microns to about 100 microns have been convenient, although more widely ~arying thicknesses may be preferable in certain circumstances. For example~ if comparatiYely large amounts of inte-active material, e.g. polymeric materials like enzymes, are required, it may be desirable to use slightly thicker ;
reagent layers.

Radiation-blocking layers and registration layers can be prepared using methods and thicknesses as used when =28-preparing reagent layers, but ~ith constituents appropriate for the particular layer. In the case of registration layers, in addition to their permeability and radiation-transmis-siveness, they are desirably substantially free from any characteristic that might appear as or contribute to mottle or other noise in the detection of an analytical result produced in an integral element of the invention. For example, any variations in color or in texture within the registration layer, as could occur if fibrous materials, e.g. some papers, are used as a permeable medlum, may be disadvantageous due to non-uniform re~lectance or transmittance of detecting energy.
Further, although fibrous materials like filter and other papers are generally permeable overall, some such materials typically can exhibit widely ranging degrees of permeability and may not exhibit uniform permeability, for example, based on structural variations such as fiber dimensions and spacing. ~9 a result, such materials are not preferred in registration layers and other layers of elements of the present invention.
Spreading layers can also be prepared by coating from solution or dispersion. As stated previously, spreading and associated layers of an element are in a superposed relationship such that a spreading layer is in fluid contact with a reagent layer. The range of materials useful for inclusion in any spreading layer is widely variable as discussed herein and will usually include predominantly materials that are resistant to, i.e. substantially insoluble in and non-swellable upon contact with, the liquid under analysis.
Swelling of about 10-40% of the layer's dry thickness may be normal. The thickness of the spreading layer is variable and will depend in part on the intended sample volume, which for convenience and cleanl~ness the spreading layer should be able to absorb, and on the layer's void volume, which also affects the amount of sample that can be absorbed into the layer.

Spreading layers of from about 50 microns to about 300 microns i9~

have been particularly useful. However, wider variations in thickness are acceptable and may be desirable for particular elem~nts.
When preparing an isotropically porous spreading layer, it is useful to have void volume comprise at least about 25% of the total layer volume, and void volumes of from 50-95% may be desirable. Variations in void volume of porous spreading layers can be used advantageously to modi~y element characteristics such as total permeability of the spreading layer or the time needed for sample spreading to occur. As can be appreciated, void volume within the layer can be controlled, for example, by selecting particulate materials of appropriate size, or by varying the solvents or drying conditions when isotropically porous blushed polymers are used in the spreading layer. The void volume of any such layer can be calculated with reasonable accuracy by a variety of techniques such as the statistical method described in Chalkley, Journal of the National Cancer Institute, 4, 47 (1943) and by direct weighing and determining the ratio of actual weight of the layer to the weight of solid material equal in volume to that of the layer, comparably composed of constltuents fro~ the layer. It will be appreciated that the pore size in any case should be su~ficient to permit spreading of initial sample components or other substances desirably provided to a reagent layer.
As mentioned previously herein, the integral analytical elements can be self-supporting or coated on a support.
Useful support materials include a variety of polymeric materials such as cellulose acetate, poly(ethylene terephthalate), polycarbonates and polyvinyl compounds such as polystyrenes, etc.
A support of choice ~or any particular element will be compatible with the intended mode of result detection~ Preferred supports include radiation-transmissive support materials that transmit electromagnetic radiation of a wavelength or wavelengths within the region between about 200 nm and about 900 nm as g~t~

well as radiation ~ue to radioactivity. For fluorimetricdetectlon of analytical results through the support, it ls desirable for the support to transmit over a somewhat wider band that is necessary for non-fluorescence measurements or~ alternatively~ to transmit at the absorption and emission spectra of the fluorescent materials used for detection. It may also be desirable to have a support that transmits one or more narrow wavelength bands and is opaque to adJacent wavelength bands. This could be accomplished, for example, by impregnating or coating the support with one or more colorants having suitable absorption characteristics. When an element includes a support, the reagent layer, the radiation-blocking layer (if present) and the registration layer will usually be interposed in the element between the support and the spreading layer (ir present), which often is the outermost layer in an element.
The components of any particular layer o~ an integral analytical element Or this invention, and the layer configuration of choice, will depend on the use ~or which an element is intended. As stated previously, spreading layer pore size can be chosen so that the layer can filter out undesirable sample components that would, ~or example, interfere with an analytical reaction or with the detection of any test result produced within the element. For analysis of whole blood, porous layers having a pore size of ~rom l to about 5 microns are particularly useful in screening out blood cells, which typically have a size of from about 7 to about 30 microns. If desirable, an element can include a plurality of spreading layers, each of which may be difrerent in its ability to spread and filter. Also; ir a restraint on transport of substances within the element additional to that prouided by spreading layers is needed, a ~ilter or dialysis layer can be included at an appropriate locaticn in the element. As an example, in analyzing for blood glucose, a dialysis layer such as a semipermeable cellulose membrane ' ', o can preverl~ passage of proteins or other potentially interfering substances to the reagent layer.
In the layers of the elernent, it can be advantageous to incorporate one or more surfactant materials such as anionic and nonicnic surfactant materials. They can, for example, enhance coatability of layer formulations and enhance the extent and rate of spreading in spreading layers that are not easily wetted by liquid samples in the absence of an aid such as a surfactant. Interactive materials can also be present in the spreading layer if desirable for a particular analysis. As an example, proteins or other higher molecular weight materials can, for convenience, be divided into more easily spreadable~ lower molecular weight components that may also be more suitable for an analytical reaction, such as by having in the spreading layer an appropriate interactive material such as an enzyme, e.g., a protease or esterase. In layers of the element it can also be desirable to include materials that can render non-active in the analysis of choice, by chemical reaction or otherwi~e, ~aterials potentially deleterious to such analysis. hs an example, ascorbate .": .
' ~ ~

. , -~ ~ ' ; -31a-;

.' ~ - , .

oxidase may be incorporated in an element to remove ascorbate ion ~hich may interfere with analysis for glucose.
In still another aspect, an analysis of choice may require a multi-stage reaction tha~ can best be accomplished in an element having a plurality of reagent layers, each Or which may be adapted to enhance or effect particular reaction stages. As an example3 in the determination of the enzyme known as serum glutamic-oxalacetic transaminase, sequential reactions can be used. Thls enzyme catalyzes the conversion at a pH Or about 7.4 of -ketoglutarate and aspartate ions to the corresponding oxalacetate and glutamate. The oxalacetate can be measured via coupling with the diazonium salt of the dye known as "Fast Ponceau L". To facilitate the first equilibrium that should be established before coupling, it is desirable to separate the reagents and to incorporate each of them into a separate layer to provide a suitable time interval for the first equilibrium to be established without hindering the establishment of this ~irst equilibrium by the premature starting of the second reaction. Thus the ~lutamic acid can be incorpo~ated in a first reagent layer which is coated over a second reagent layer that contains the salt of the dye "Fast Ponceau L".
Integral analytical elements of the present invention can be adapted for use in carrying out a wide variety of chemical analyses, not only in the field of clinical chemistry but in chemical research and in chemical process control laboratories.
They are well suited for use in clinical testing of body fluids~
such as blood, blood serum and urine~ since in this work a large number of repetitive tests are frequently conducted and test results are often needed a very short time after the sample is taken. In the field of blood analysis, for example, the multi-layér element can be adapted for use in carrying out quantitative analyses for many of the blood ~omponents which are routinely measured. ~hus, for example, the element may be readily adapted for use in the analysis of such blood components as albumin, ~g~

bilirubin, urea nitrogen, serum glutamic-oxalacetic transami~
nase, chloride, glucose, uric acid, and alkaline phosphatase as well as many other components, by appropriate choice of test reagents or other interactive materials. In analyzing blood with the analytical element of this invention, the blood cells may first be separated from the serum, by such means as centrifuging, and the serum applied to the element.
However, it is not necessary to make such separation, especially if reflective spectrophotometric analysis techniques are used to ~uantify or otherwise analyze the reaction product formed in the alement as whole blood can be applied diractly to the element and the blood cells filtered out through the action of a filtering layer. The presence of these cells on the element will not interfere with spectropho- -tometric analysis if it is carried out by reflection techniques, with light being transmitted through the support and registration layer and reflected from the radiation-blocking layer or other reflec~ing layer such that aetecting radiation does not intercept the cells. A particularly significant advantag~ of -the integral analytical elements described herein is their ability to be used to analyze either serum or whole bloo~
As can be appreciated, a variety of different elements, depending on the analysis of choice, can be prepared in accordance with the present invention. Elements can be con-figured in a variety of forms, including elongated tapes of any desired width, sheets or smaller chips~ Particular elements can be adapted for one or more tests of a single type or a varlety of tests of different types. In such latter evant, it can be desirable to coat a co~mon support with one or more strips or channels, each optionally o a different composition to form a composite element suited ~or conducting a variety of desired tests.
~ - 33 -~' '", .

Exemplary elements o~ this invention include those illustrated in the accompanying drawings. In Fig. 1 i5 represented an analytical element composed of a radiation-transmissive support 10 1 0 ' ~

_ 33 A ~

,. - - - : - - : : ,. , . . . - , ..

on which is carried a registration layer l2, a radiation-blocking layer 14 ~hich can filter as well as provide a white background for analytical result detection such as by reflection spectro-p~,otom~try, and a reagent layer 16. Detection can be done through the support, which is suitably transmissive at the detecting ~avelength. The registration layer 12 can be a hydrophilic colloid such as gelatin. Reagent layer 16 can be cornposed of a solution or dispersion Or one or more test reagents in a binder such as gelatin, while layer 14 can be a blushed polymer having isotropic porosity and/or such pore size as may be needed for any filtering ~unction it is intended to per~orm. The layers are in fluid contact~ In an alternative embodiment o~ the invention shown in Fig. 2~ the analytical element is composed o~ a radiation-transmissive support 20 bearing a registration layer 22 in fluid contact with a reàgent layer 24 and a spreading layer 27 which can also serve the function of ~iltering and also may provide a -~
suitably reflective background for reflection spectrophotometric detection through support 20. Alternatively, layer 26 may be such that it does not reflect and detection can be accomplished in the transmission mode. Layer 26 can be, ~or example, an isotropically porous blushed ~olymer layer which has been coated or laminated over layer 24. Fig. 3 illustrates a further ... .
embodiment of the invention in which the analytical element is composed of support 30, registration layer 32, a radiation-blocking layer 34 which can be formed from a dispersion o~ a pigment like titanium dioxide in a hydrophilic colloid such as gelatin~ a reagent layer 36, and a spreading layer 38 such as an isotropically porous blushed polymer layer which can serve the functions of spreading and filtering. The various layers are in fluid contact.
A still further embodiment Or the invention is shown in Flg. 4 in which the analytical element is composed of a support 40, a registration layer 42, a radiat1on-blocking/fil~ering layer 44, reagent layer (A) 4~, a reagent layer (B) 48, and a spreading~
filtering layer 5G. Layer 44 can be composed, for example, of .

titanium dloxide in blushed cellulose acetate and layer 50 can ~e composed of diatomaceous earth in blushed cellulose acetate or of glass beads ~utually adhered with a hydrophilic colloid like gelatin.
The present elements are placed in use by applylng to the element a sample of liquid under analysis. Typically 9 an element will be formed such that an applied sample will contact a spreading layer prior to a non-spreading reagent layer and will first contact such spreading layer at its sur~ace farther from such reagent layer. Because analytical accuracy of the present elements is not substantially diminished by variations in the volume of applied samples, especially when a spreading layer is present in the element, sample application by hand or maching is acceptable. For reasons of convenience in detecting an analytical result, however, reasonable consistency in sample volume may be desirable. As discussed previously, the spreading layer is also extremely desirable in minimizing the occurrence Or ringing when soluble interactive materials are used in a reagent layer.
In a typical analytical procedure using the present elements, which could be manual or automated, the element is taken from a supply roll, chip packet or other source and positioned to receive a free drop, contact spot or other form of liquid . ~. .
sample, such as from an appropriate dispenser. After sample application, and desirabl~ after the liquid sample has been taken ~ up by a spreading layer, if present, the element is exposed to - any condltioning, such as heating, humidification or the like, , ~ .
- that may be desirable to quicken or otherwise facilitate : obtaining any test result. If an automated procedure is used~

-~ it can also be desirable to have any spreading layer accomplish . .: .
~ its function within several seconds, but allowing sufficient time - ~ to provide metering, which is contrasted with the -almost instantaneous, unregulated diffusion as can be obtained with absorbent fibrous papers. This can be accomplished conveniently by V

appropriate selection o~ various paramete~s, such as layer thickness, void volume 1n porous layers, etc.
~ fter the analytical result is ob-talned as a detect-able change, it is measured, usually by passing ~he element through a zone in ~hich suitable apparatus for reflection, trans-mission or fluorescence spectrophotometry is provided. Such apparatus ~ould serve to direct a beam of energy, such as light, through the support and the registration layer. The light would then be reflected, such as from a radiation-blocking layer in the element~ back to a detecting means or would pass through the element to a detector, in the case of transmission detection. In a preferred mode, the analytical result is detected in a region of the element totally within the region in which such result is produced. Use of reflection spectrophotometry can be advantag-eous in some situations as it can effectively avoid interference from residues, suc~ as blood cells, which may have been left on or in the layers of the element. Conventional techniques of fluorescence spectrophotometry can al~o be employed if the detect-able species is a fluorescent material. Detection would be 2~ accomplished using energy that excites the fluorescent species and a detector that senses its fluorescent emission. Furthermore, wh~n blood serum is tested or means are provided for eliminating unwanted whole blood residue~, transmission techniques can be . ~
used to detect and quantify the indicating reaction products by directing a flow of radiant energy, for example, U.V. visible or I.R. radiation at one surface of the element and measuring the output of that energy from the opposing surface of the element.
Generally, electromagnetic radiation in the range of from about 20Q to about 90onm has been found useful for such measurements, although any radiation to which the eLement is permeable and which is capable of quantifying the product produced in the reagent ; layer can be used. Various cali~ration techniques can be used to provide ' ? ~ j !

a control ~or the analysis. As one example, a sample Or analyte standard solution can be applied adjacent to the area where the drop Or sample is placed in order to permit the use Or differential measurements in the analysis.
- The ~ollowing example of integral analytial elements are provided to further illustrate the present invention.
.; .

; Example 1 On a thick t180 microns) support of poly(ethylene terephthalate), having a gelatin sub, are successively applied:
~` 1) a registration (receiving) layer containing, per square meter, 2.15 g. of gelatin, 2.15 g of a mordant ` (copolymer of styrene and N,N-dimethyl-N-3-maleimido-propylammonium chloride);
-~; 2) a porous, reflective radiation-blocking layer containing, per square meter, 151 g of gelatin and 11.4 g of titanium s" dioxide, , .
- 3) a reagent (analytical) layer containing, per square r ' .. meter~ 17.5 g of gelatin, 1.5 g of 1-naphthol-2-~; sulfonic acid potassium salt, 0.73 g o~ disodium `....
phosphate buffer, 0.45 g of monopotassium phosphate . buffer, 0.38 g of 4-aminoantipyrine (HCl) 1.6 g of .
- glycerine as a plasticizer, 0.09 g of peroxydase. . ., ;
~- (14014 ~/m2) and 0.374 g of glucose oxidase (40440 U/m2);

~-~;- 4) a spreading layer oontaining, per square meter, 97 g 1':~ ', -`
of`cellulose acetate and 65.5 g of titanium dioxide.
The thusly prepared element is used for the analysis Or glucose solutions, the concentrations of which are varied ~rom 0 to - ~ .
- - 800 mg per deciliter. On ~amples of the ele~nt Bre depo~it~d r~ drops, each of which represents lQ ~1 of an aqueous glucose .; solution. After one hour, ~he dens~ty ~f the colorations is measur d by reflection using a ~cBeth Densitometer (Model ,, -.;~ .
TD-504). ~~~ ~ ~
~: .
~ 3 7 Whon ~he glucose solution is ~pplied to th~ surface of the element, it spreads within layer (4) and is metered to layer (3) wherein the glucose reacts with the oxy~en and the water in the presence of the glucose oxidase to provide gluconic acid and hydrogen peroxide; these compounds, in the presence Or peroxidase, react with the 4-aminoantipyrine ; which is then oxidized, the ox~dation product Or the 4-aminoantipyrine then reacts by coupling with the l-naphthol-2-sulfonic acid potassium salt to ~orm a dye which diffuses - out of the reagent layer (3), through the radiation blocking layer (2) and into the registration layer (1) where it is detected using the Macbeth TD-504 densitometer. The results are summarized in the table following Example 2.
~, , ~xample 2 - An analytical element, having a structure identical to that of Example 1 is prepared, except that the registration ~ . , .
~ layer (1) contains gelatin as the only component, at a coating . weight o~ 4.30 g per square meter. The product is used accor-~ . ~
i; ding to the procedures descri~ed in Example 1, and the results r obtained are summar~zed in the table hereunder.
-~ , .: , ~ . - - TABLE
, ....................................... .
Glucose content Density measured -- . of sample by white-light Element_ (mg/deciliter) reflection - ~ Example 1 0 0.28 `- - 100 0.36 ~-; 150 0-37 ~- 200 4 -- - 3oo 0.47 ` - 400 0.52 - -. 600 -53 ~- ~ 800 -53 ____________________ _____~______ ., -.
Example 2 0 0.23 : 100 0.36 - 150 0.42 ~:- 200 n. ~7 . 400 0.55 ' 600 o.56 800 0.60 The invention has been described in detail with particular reference to certain preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

Claims (14)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follow:

1. An integral element for analysis of liquids, the element comprising a radiation-transmissive support having thereon, in fluid contact, an isotropically porous spreading layer capable of spreading within itself substance comprising an analyte or an analyte precursor, a reagent layer permeable to substance spreadable within the spreading layer and comprising a composition comprising material interactive in the presence of analyte to provide a diffusible, detectable species, and a radiation-transmissive registration layer, permeable to the detectable species and within which said species can be detected, wherein the registration layer is interposed between the support and the reagent layer and the reagent layer is interposed between the registration layer and the spreading layer.

2. An integral analytical element as described in claim 1 further comprising a radiation-blocking layer, permeable to the detectable species, interposed between the registration layer and the reagent layer and in fluid contact with other layers in the element.

3. An integral analytical element as described in claim 2 wherein the radiation-blocking layer comprises an opacifying agent.

4. An integral element for analysis of liquids, the element comprising a radiation-transmissive support having thereon, in fluid contact, a water-resistant, isotropically porous spreading layer capable of spreading within itself substance comprising an analyte or an analyte precursor, a water-swellable reagent layer permeable to substance spreadable within the spreading layer and comprising a composition comprising material interactive in the presence of analyte to provide a diffusible, detectable species, a radiation-blocking layer, permeable to the detectable species and comprising an opacifying agent, and a water-swellable, radiation-transmissive registration layer, permeable to the detectable species and within which said species can be detected, wherein the registration layer is the closest to the support of said layers, the radiation-blocking layer is interposed between the reagent layer and the registration layer, and the reagent layer is interposed between the radiation-blocking layer and the spreading layer.

5. An integral analytical element as described in claim 4 wherein the spreading layer comprises a blushed polymer.

6. An integral analytical element as described in claim 4 wherein the opacifying agent in the radiation-blocking layer comprises a water-resistant, blushed polymer.

7. An integral analytical element as described in claim 4 wherein the opacifying agent in the radiation-blocking layer comprises a pigment.

8. An integral analytical element as described in claim 4 wherein the layers are non-fibrous.

9. An integral analytical element as described in claim 4 wherein the spreading layer comprises a blushed polymer and a surfactant, the reagent layer comprises a hydrophilic colloid having the interactive material distributed therein, the radiation-blocking layer comprises a hydrophilic colloid having a pigment distributed therein and the registration layer comprises a hydrophilic colloid.

10. An integral analytical element as described in claim 9 wherein the registration layer further comprises a mordant for the detectable species.

11. An integral analytical element as described in claim 9 wherein the spreading layer additionally comprises an inorganic pigment.

12. An integral element for analysis of liquids, the element comprising a radiation-transmissive support having thereon, in fluid contact, a water-resistant, isotropically porous spreading layer capable of spreading within itself substance comprising an analyte or an analyte precursor and comprising a blushed polymer selected from cellulose acetate and a polyamide, an inorganic pigment distributed in the polymer and a sur-factant, a water-swellable reagent layer, permeable to substance spreadable within the spreading layer and comprising a hydrophilic colloid selected from gelatin, poly(vinyl alcohol), poly(vinyl pyrrolidone), an acrylamide, agarose and a polysaccharide, said colloid having distributed therein material interactive in the presence of analyte to provide a diffusible, detectable species, a radiation-blocking layer, permeable to the detectable species and comprising a hydrophilic colloid selected from gelatin, poly(vinyl alcohol), poly(vinyl pyrrolidone), an acrylamide, agarose and a polysaccnaride, said colloid having distributed therein a pigment slected from carbon, titanium dioxide and barium sulfate and a water-swellable, radiation-transmissive registration layer, permeable to the detectable species and within which said species can be detected, comprising a hydrophilic colloid selected from gelatin, poly(vinyl alcohol), poly(vinyl pyrrolidone), an acrylamide, agarose and a polysaccharide, and a mordant for the detectable species.

13. An integral analytical element as described in claim 12 wherein the composition in the reagent layer comprises glucose oxidase, peroxidase and an indicator composition com-prising a compound oxidizable in the presence of hydrogen peroxide and peroxidase to effect formation of a dye.

14. An integral analytical element as described in claim 12 wherein the indicator composition comprises antipyrene chlorohydrate and 1-naphthol-2-sulfonic acid sodium salt and the mordant in the registration layer comprises a copolymer comprising recurring units of styrene and N,N-dimethyl-N-benzyl-3-maleimidopropylammonium chloride.