WO2003060517A2

WO2003060517A2 - Diffusable adhesive composition for multi-layered dry reagent devices

Info

Publication number: WO2003060517A2
Application number: PCT/IB2003/000055
Authority: WO
Inventors: Lloyd S. Schulman; Michael J. Pugia; Karlheinz Hildenbrand; Spencer H. Lin
Original assignee: Bayer Healthcare Llc
Priority date: 2002-01-15
Filing date: 2003-01-13
Publication date: 2003-07-24
Also published as: AU2003201460A1; WO2003060517A3

Abstract

A multi-layer device for detecting an analyte in a fluid sample includes at least two layers with a fluid diffusible layer disposed between them. At least one of the layers contains a reagent system for detection of the analyte.

Description

DIFFUSABLE ADHESIVE COMPOSITION FOR MULTI-LAYERED DRY REAGENT DEVICES

BACKGROUND OF THE INVENTION

Dry reagent analytical devices typically involve absorbent pads containing dispersed reagent systems which react with analytes (components to be detected) in fluid test samples applied to the device to provide a detectable response. Certain of these devices involve an enzymatic reaction with the analyte in the presence of a peroxidase and a hydroperoxide to cause a detectable color change in a redox dye and are normally based on the use of filter paper as the absorbent pad. Other such devices operate on the basis of immunoreactivity of a labeled antibody located in the reagent device which specifically binds with an analyte in the test sample to provide a detectable response in a specified region of the test device. Nitrocellulose is a preferred base material for this sort of device due to its flow through properties.

These dry reagent devices are inexpensive and convenient to use but suffer from certain limitations. For example, immunoassays require separation of ingredients to operate, which is often achieved by protein binding. Migration of reagents and analytes often presents problems, leading to inaccurate results. In the dry reagent format, such as that described by Greenquist in U.S. 4,806,311, an analyte is bound to a labeled reagent and then passed to a detection zone where the amount of the analyte is measured by the amount of labeled reagent. Unreacted labeled reagent is immobilized by immobilized analyte in the reagent zone. Any labeled reagent-analyte which passes into the detection zone is prevented from back migration by being immobilized in the detection zone.

The assembly and fabrication of multilayered devices has not been completely successful. In EP 0226 465 A2 and U.S. 3,992,158 for example, films have been used to separate layers of reagents. However, these devices require tight control of the pore size and shape and of the thickness of the films. One consequence of such designs is that the reagents cannot be on filter paper, since such papers do not have the well defined pore structures of films or the uniform surfaces needed for uniform thickness. But, filter paper is desirable in multilayered devices since they are well suited for use with many reagents due to their inert nature and high water absorbtivity. Thus, filter paper has been used, along with a nylon mesh covering. Such devices rely on surface contact between the reagent layers and this causes reagents to mix on the surface into one layer. The present invention avoids this result and keeps the reagents in their intended positions.

There are many examples of incompatible chemicals in dry reagent systems. For example, the base in white blood cell reagents causes premature hydrolysis of protease substrate. Iron in occult blood reagents causes premature oxidation of redox dye indicators to their colored form, which is also the result of the presence of iodate in glucose reagents. In the case of copper based tests for creatinine, the copper can oxidize redox indicators such as tetramethylbenzidine to their colored form in the absence of creatinine. Tests for occult blood in urine can be skewed by the presence of ascorbate in the urine test sample which acts as a reducing agent to cause false negative results and urine protein tests can be rendered inaccurate by the presence of buffers in the urine sample being tested. Dry assay devices for determining white blood cells in urine can be influenced by interference due to proteins in the urine sample and whole blood assays, such as blood glucose and blood CKMB, suffer from interference caused by red blood cells. The present invention provides a means for alleviating these problems by joining two layers of a dry reagent device, at least one of which layers contains a reagent for detection of an analyte, with a test fluid permeable layer comprising a blend of an aqueous based polymer dispersion and a water soluble polymer, which blend has been cast and dried to form an adhesive layer.

Previous methods for dealing with these problems have involved separating the reagents into discrete, stacked layers. There are, however, problems associated with the use of the discrete, stacked layer configuration. Thus, the top layer(s) must allow the test sample to pass to the lower layers while continuing to separate certain interfering chemicals and/or biochemicals. For example, metals such as copper or iron should be separated from redox indicators and bases from protease substrates. Oxidants such as iodate and reactants such as ascorbate need to be separated from redox indicators such as tetramethylbenzidine.

These problems are effectively dealt with by derivatizing the permeable, adhesive layer of the present invention with elements which serve to remove interfering substances as they flow through the top layer of the device, through the permeable adhesive layer and into the device's bottom layer. This sort of format requires a permeable, adhesive material to hold the reagent layers together.

However, in the prior art, the contact between the layers was either insufficient to allow the reactants to pass from one layer to the adjacent layer when that was desired, or the reactants migrated from one layer to another when that was not desired.

There are various diffusable, adhesive compositions which can be used to secure two layers in integrated, multilayered reagent devices. Nerbeck, in U.S. 3,993,451 uses adhesives to secure reagent containing particles to a substrate layer. The particles may be covered with a porous layer through which a component contained within a sample may pass to reach the reagent containing particles. In the device proposed by Nerbeck, the adhesive is not used as a layer which separates reagent layers from detecting layers. Furthermore, the solid particles form separate detecting units which do not rely on movement of the reaction product with an analyte into an adjacent layer for detection.

Japanese Patent 5-18959 discloses the use of a hydrophobic polymer which does not swell in water as an adhesive to secure reagent layers and Japanese patent 5-26875 discloses the use of a porous layer comprising a fluorine containing polymer as an adhesive to secure reagent layers. The polymers used in these Japanese systems are hydrophobic and consequently, they hinder rapid movement of sample fluids through the layers. For rapid testing, the sample fluid should pass through the layers of the device within less than one second. A water soluble adhesive would permit rapid movement of the sample fluid, but would cause the layers to separate as the adhesive begins to dissolve.

In EP 0 226 465 A2 a multilayer analytical device is described in which several porous sheets are bonded together with an adhesive placed so as to form openings through which liquids could pass. The adhesive itself was not capable of passing liquids so that openings were provided instead. The result being that not all of the available surface is useful and contact between the layers is not uniform.

The Greenquist '311 patent mentioned above also discloses a multilayer device for medical testing. Although the concept is valuable, in practice the multilayer device is not as satisfactory as would be desired. The layers must perform their intended function without interfering with the functioning of the adjacent layers. At the same time, the sample fluid must pass rapidly through the layers so that a result can be determined rapidly. Thus, the layers must act independently while not limiting the movement of the sample fluid. The present inventors have overcome these problems, in the multilayer device to be described below.

In U.S. patent 4,824,640 a transparent layer is disclosed which is useful for containing analytical reagents which consists of a water soluble or water swellable component and an essentially insoluble film forming component. A similar layer is employed in U.S. patent 6,187,268 Bl as an overcoat over a dry reagent layer.

SUMMARY OF THE INVENTION

The present invention involves a device suitable for the detection of an analyte in a fluid sample which comprises at least a first absorbent layer and a second absorbent layer. At least one of the layers contains a reagent system for the detection of the analyte. The layers have dispersed between them an adhesive layer which is permeable to components of the fluid sample and which comprises a blend of an aqueous based polymer dispersion and a water soluble polymer which has been cast and dried to form the adhesive layer.

The fluid diffusable adhesive composition used in the present invention can be used to construct several types of multilayer devices, which at least have a diffusable adhesive composition between two absorbent layers. This type of multilayered reagent can have open or sealed edges. The adhesive layer holds discrete layers together and either of the discrete layers can be attached to a plastic support, such as a strip handle or a cassette, so that the person using the device can avoid direct contact with the sample fluid. Since the adhesive is permeable, it allows reagents and components of the fluid test sample to flow through the adhesive layer.

The multi-layer device can be made so that when a fluid sample is placed on the first absorbent layer, it is spread across the surface of the layer without interacting with the components of the sample. Alternatively, the first absorbent layer may react with interfering components of the sample, permitting the component to be measured (the analyte) to pass through the adhesive layer to the second absorbent layer. Or, the first absorbent layer may react with the analyte, which is measured in place or the reaction product may pass through the adhesive layer to the second absorbent layer, where it is detected. The second absorbent layer may absorb and retain a component of the fluid sample which has passed through the adhesive layer or it may contain a reagent which reacts with the analyte or the reaction product of the analyte received from the first absorbent layer. The adhesive layer can be made so that it prevents the passage of components of the sample by physical separation. Thus, it may serve to concentrate the analyte by passing it while preventing other components from reaching the second absorbent layer. Alternatively, the adhesive layer may contain additives which chemically bind certain of the sample components. In one embodiment, the adhesive layer passes certain components of the sample, leaving the more concentrated analyte on the first absorbent layer.

The water dispersible polymer may be either an anionic or cationic polyurethane dispersion, preferably an anionic polyurethane, in combination with a water soluble polymer, preferably a polyethylene oxide, a polyvinyl pyrrolidone, or a polyvinyl alcohol. In preferred embodiments the permeable adhesive layer can contain exchange resins and ascorbate scavengers to remove buffering and ascorbate interference from the test sample. The cation exchange resins may include those with oxidative anions such as bromate, iodate, periodate, and chromate or those containing polysulfonic acids, polycarboxylic acids, or polyphosphonic acids with transition metal oxidants such as iron, cobalt, or copper. The permeable adhesive layer can also contain protein binding polymers to separate interfering proteins or antibodies from the sample as well as fillers such as TiO₂ or BaSO₄ to adjust the opacity or reflectance behavior of the reagent device. Suitable protein binding polymers include, for example, positively charged polymers such as polyamines and polyamides and negatively charged polymers such as polysulfonic, polycarboxylic, and polyphosphonic acids. These polymers may be incorporated into the permeable layer by mixing into the adhesive formula and coating onto the reagent layers.

DESCRIPTION OF THE INVENTION

MULTI-LAYER DEVICES

In the simplest form, a multi-layer device for detecting an analyte (i.e. a substance to be detected) in a fluid sample includes a first absorbent layer for receiving a fluid sample, a second absorbent layer for receiving and absorbing a portion of the sample from the first absorbent layer, and a diffusable (permeable) adhesive layer disposed between the two absorbent layers, the adhesive layer not only binding the absorbent layers together but being capable of reacting with portions of the fluid sample to prevent their passage or to physically block passage of portions of the fluid sample. Additional absorbent layers and adhesive layers may be added as needed to carry out any particular analysis, as will be evident to those skilled in the art.

The first absorbent layer has several possible functions. It may merely absorb a fluid sample and spread it across the surface of the adhesive layer. Alternatively, it may react with interfering components of the sample, with the analyte passing through the adhesive layer to the second absorbent layer. In another alternative, the first absorbent layer may react with the analyte, which is then measured in place, or the reaction product is passed through the adhesive layer to the second absorbent layer for detection. The second absorbent layer also has several possible functions. It may absorb a portion of the sample passed through the adhesive layer, thereby concentrating the analyte in the first absorbent layer. Alternatively, it may receive a portion of the sample including the analyte and then react with the analyte to provide a product which is measured. In another alternative, the second absorbent layer may receive the reaction product produced in the first absorbent layer and concentrated by passage through the adhesive layer.

The adhesive layer is permeable. Thus, it is capable of making a physical separation of the fluid sample, either passing the analyte and preventing other components from passing through to the second absorbent layer or passing interfering components to concentrate the analyte. In other applications, the adhesive layer may react with certain components of the sample, thus trapping them in the adhesive layer. Or, it may contain additives capable of reacting with certain components and thereby blocking their passage through the adhesive layer.

Those skilled in the art will appreciate that this broad description of the function of the three basic components of the multi-layer device of the invention can apply to many alternative specific applications, some of which are discussed below, although others not mentioned are potentially useful analytical methods, while not departing from the broad description of the invention.

DIFFUSABLE ADHESIVE LAYER

The basic elements of the fluid permeable, adhesive membrane useful in the present invention involve an aqueous based polymer dispersion and a water soluble polymer. The permeability of the membrane can be adjusted by varying the ratio of the polymer dispersion to the water soluble component. Typically, this ratio will range from 50 : 1 to 1 : 1 on a weight basis with a ratio of l0:l to 5:l excess of the film forming polymer dispersion being preferred. An increase in the water dispersible polymer will increase the membrane's permeability, which is desirable when faster flow is desired. Conversely, increasing the concentration of the water soluble polymer will decrease the membrane's permeability in cases where greater contact, and accordingly more mixing of the reagents, is desired. These layers are particularly useful in conjunction with diagnostic dry reagent test devices because they allow penetration of the analyte present in the fluid test sample through the adhesive binding the reagent layers of the device together. Polyurethane dispersions are preferred for use as the dispersible polymer due to their adhesive properties, flexibility and diverse structures. The reaction of a diisocyanate with equivalent quantities of a bifunctional alcohol provides a simple linear polyurethane. These products are unsuitable for use in the manufacture of coatings, paints and elastomers. However, when simple glycols are first reacted with dicarboxylic acids in a polycondensation reaction to form long chain polyester-diols and these products, which generally have an average molecular weight of between 300 and 2000, are subsequently reacted with diisocyanates the result is the formation of high molecular weight polyester urethanes. Polyurethane dispersions have been commercially important since 1972. Polyurethane ionomers are structurally suitable for the preparation of aqueous two phase systems. These polymers, which have hydrophilic ionic sites between predominantly hydrophobic chain segments are self dispersing and, under favorable conditions, form stable dispersions in water without the influence of shear forces and in the absence of dispersants. In order to obtain anionic polyurethanes, such as Bayhydrol DLN, which are preferred for use in the present invention, diols bearing a carboxylic acid or a sulfonate group are introduced and the acid groups are subsequently neutralized, for example, with tertiary amines. Sulfonate groups are usually built via a diaminoalkanesulfonate, since these compounds are soluble in water. The resulting polyurethane resins have built ionic groups which provide mechanical and chemical stability as well as good film forming adhesion properties. Cationic polyurethane dispersions such as Praestol E 150 from Stockhausen

Chemical Co. may also be used in forming the membrane. One method of preparing cationic polyurethanes is by the reaction of a dibromide with a diamine. If one of these components contains a long chain polyester segment, an ionomer is obtained. Alternatively, polyammonium polyurethanes can be prepared by first preparing a tertiary nitrogen containing polyurethane and then quaternizing the nitrogen atoms in a second step. Starting with polyether based NCO prepolymers, segmented quaternary polyurethanes are obtained.

The most important property of polyurethane ionomers is their ability to form stable dispersions in water spontaneously under certain conditions to provide a binary colloidal system in which a discontinuous polyurethane phase is dispersed in a continuous aqueous phase. The diameter of the dispersed polyurethane particles can be varied between about 10 and 5000 nm. Polyurethane dispersions which are ionic with the ionic radicals being sulphonate, carboxylate or ammonium groups are particularly suitable. Also suitable for use in the present invention are other film forming polymer dispersions such as those formed by polyvinyl or polyacrylic compounds, e.g. polyvinylacetates or polyacrylates, vinyl copolymers, polystyrenesulfonic acids, polyamides and mixtures thereof. By combining the polymer dispersion with a water soluble polymer there is formed a matrix which forms a swellable network like web. The tighter the web, the smaller the pores and the slower the flow of the test fluid through the matrix.

As water soluble polymers the known polymers such as, for example, polyacrylamides, polyacrylic acids, cellulose ethers, polyethyleneimine, polyvinyl alcohol, copolymers of vinyl alcohol and vinyl acetate, gelatine, agarose, alginates and polyvinylpyrrolidone are suitable. This second polymer component is sometimes referred to as the swelling component due to its swellability by absorbing water. Polyethyleneoxides, polyvinylpyrrolidones and polyvinylalcohols are preferred. These polymers can vary widely in molecular weight so long as they are water soluble and miscible with the aqueous polymer dispersion. Polyethylene oxides of a molecular weight from 300,000 to 900,000 g/mol and poly-vinylpyrrolidone having a molecular weight of from 30,000 to 60,000 g/mol are particularly suitable. The molecular weight of the water soluble polymer is not critical so long as they are miscible with the polymer dispersion and allow the incorporation of assay specific reagents such as buffers, indicators, enzymes and antibodies. The finished film should be swellable so as to be permeable to the test fluid. The polymers are dispersed/dissolved in a solvent (preferably aqueous) preparatory to its application to the surface of the dry reagent device by use of a dispenser as in the following examples. In the preferred aqueous casting solutions, aqueous polymer dispersions are mixed with an aqueous solution of the second polymer such as, for example, polyvinyl acetate dispersions with cellulose ethers, polyurethane dispersions with polyvinyl alcohol, polyurethane dispersions with gelatine or polyurethane dispersions with polyvinylpyrrolidone. Normally, a surfactant is added to the formulation to enhance its spreadability and a thickener such as silica gel is added to thicken the formulation to a consistency which facilitates it being spread across the surface of the reagent device. The formulation is then applied to the surface of the dry reagent device, such as by a Myer rod applicator or a wiped film spreader, and dried to remove solvent. Typical dry thicknesses of the permeable membrane range from 1 to 100 mils (0.0254 to 2.54 mm).

USE OF THE DIFFUSABLE ADHESIVE LAYER Protein interference in an assay for white blood cells in urine is alleviated by the protein sticking to the adhesive and not passing through the reagent and buffer interference in tests for urine protein is reduced by either adhering to the adhesive (ion pairing) or being neutralized (proton exchange) with the result being either that the buffer does not come into contact with the reagent or is altered to a non-interfering form which matches the pH of the reagent. The instability of reagents for testing urine creatinine due to the presence of incompatible chemicals when all mixed in one discrete reagent layer is prevented by the diffusible adhesive, since a device can be fabricated to hold two discrete reagent layers, one with copper and the other with a redox indicator. The layers keep copper separated from the redox indicator until it comes into contact with the fluid test sample. The sample presents creatinine to bind with the copper and the copper is liberated from the top layer and mixed with the redox indicator.

Ascorbate interference with urine occult blood tests can be alleviated by incorporating ascorbate scavengers, such as a metal capable of oxidizing ascorbate bound to a polymer, into the membrane formulation. Polymer bound metal ascorbate scavengers are described in U.S. Patent 5,079,140. Other oxidizing agents such as iodate and persulfate can be immobilized within the porous membrane to serve as ascorbate scavengers. The diffusible adhesive of the present invention can be used advantageously in conjunction with immunoformats to provide sensitive assays for various analytes. For example, a transparent membrane according to the present invention can be prepared with an immobilized anti-binding label antibody contained therein. Typically, this antibody will be immobilized within the membrane by attaching it to a larger entity such as a latex particle which is incorporated into the polymer blend which forms the membrane before it is cast onto the reagent device. Thus, when the binding label on the anti-analyte antibody has the fluorescein structure, such as in the case of fluorescein isothiocyanate (FITC), anti-FITC can be interspersed in the permeable membrane to capture FITC labeled anti- analyte antibody. In addition, anti-analyte antibody labeled with a peroxidase is incorporated into the membrane, so that as test fluid flowing through the membrane analyte contained therein will bind with bound anti-analyte antibody and peroxidase labeled anti-analyte antibody to form a sandwich attached to the membrane thereby preventing the peroxidase from reaching the reagent layer, which contains a peroxide and a redox dye, and providing a colored response. In this embodiment, the response produced by the interaction of the analyte, peroxidase, peroxide and redox dye is inversely proportional to the concentration of the analyte in the fluid test sample.

More generally, non-limiting examples of reagents which may find use in multilayer devices according to the invention include the following: • reagents for reaction with an analyte in the first absorbent layer which receives the fluid sample may include enzymes such as oxidases, reductases, and proteases commonly used in clinical assays; affinity binders such as antibodies, nucleic acids, antigens, and proteins such as are used in both binding assays and reactions in which the analyte is converted to a detachable chemical. • reagents for reaction with an interfering component of the fluid sample may include enzymes to metabolize the interferent, reactants to convert interferent to non- reactive form, and binding agents to trap the interferent.

• reagents for reaction with an analyte in the second absorbent layer may include indicators producing signals in response to the analyte and enzymes or reactants for signal amplification.

• reagents for reaction with an analyte in the second absorbent layer which analyte had been reacted in the first absorbent layer and passed through the adhesive layer include enzymes used in clinical assays and affinity binders used in binding assays and reactions in which a moiety of the analyte is detached.

• additives to the adhesive layer capable of reacting with components of said sample include affinity binders or enzymes for removing interferents or generating signals.

Three examples are provided below, which illustrate alternative embodiments of the invention, although it is not intended to be limited only to these examples. In one example, a layer of filter paper is treated with a reagent solution for the analyte which is to be detected. The treated filter paper is then coated with an adhesive layer of the invention and a second layer of untreated filter paper is added, which can serve to concentrate the reagent which has reacted with the analyte and then migrates through the adhesive layer into the untreated filter paper. In a second example, an adhesive layer includes a material which prevents migration of interfering compounds through the adliesive layer into the reagent layer. A third example includes a top layer with a reagent for the analyte. The product of the reaction of the analyte and reagent passes through the adhesive layer and is detected in the bottom layer.

EXAMPLE I

A diffusible adhesive was prepared as follows: (1) 75 g of a 50 mM monobasic phosphate buffer (Fisher, pH 7.0) and 0.5 g of a Pluronic P75 surfactant (BASF) were added to a 250 mL steel beaker. Then while stirring slowly 0.3 g of octanol followed by 5.0 g of Aerosil 200 silica gel (DeGussa AG) were added to the beaker. The stirring rate was increased to about 2000 rpm for several minutes to achieve complete dispersion of the contents of the beaker. (2) Stirring was continued for about 15 minutes while 40.25 g of a 40 wt % aqueous solution of Bayhydrol D-762 (polyester polyurethane resin, Bayer Corporation) followed by 0.2 g of polyethylene oxide, m.w. 900,000 were added.

(3) The coating solution was stirred under a slight vacuum for several minutes to de-gas the solution, after which it was ready to cast on a reagent layer. An albumin reagent layer was prepared by:

(1) Preparing two solutions for sequential application to a filter paper base. The compositions are given in the following table: Albumin Reagent Composition

Pref. Cone. Allowable

Ingredient Function Used Range

1st application

Water Solvent 1000 mL Tartaric add Cation Sensing Buffer 93.8 g (625 mM) 50-750 mM Quinaldine red Background dye 8.6 mg (12 μM) 5-30 μM 2nd application

Toluene Solvent 1000 mL DIDNTB Buffer 0.61 g (0.6 M) 0.1-3.0 mM Lutonal M40 Polymer enhancer 1.0 g 0.54 g/L

DIDNTB = 5'.5'-Dinitro-3'.3'- Diiodo-3.4,5.6-Tetrabromophenosulfonephthalein

(2) Filter paper (Whatman GF/30cm) was treated with the two solutions in sequence to saturate the paper, after which the treated filter paper was dried for 15 minutes at 90°C to produce the top layer reagent.

The adhesive coating solution was cast on the albumin reagent layer to a wet thickness of about 250 μm, after which the adhesive coated albumin reagent on the filter paper was dried at about 90°C for about 5 minutes.

A complete format was assembled in which a layer of glass filter paper (Whatman GF/30cm) was placed on the opposite side of the adhesive layer from the albumin reagent layer. That is, a test device contained three layers, i.e. an albumin reagent layer, a diffusible adhesive layer, and a layer of glass filter paper. This test device was compared with an albumin reagent layer made as described above, but which was not coated with the diffusible adhesive layer.

In the first test, a sample containing 500 mg/L of albumin was applied to the albumin reagent layer without an adliesive coating and the result was compared with another 500 mg/L sample placed on the glass filter paper of the composite device. In the later case the albumin would have to pass through the filter paper and the adhesive layer to reach the reagent layer where it would be detected. In the comparative sample, the reagent layer would give an immediate response. The amount of albumin present was determined by reflectance measurement using a CLINITEK 200 instrument. When no sample had been added to the albumin reagent layer, the reflectance was 93.6% at a wave length of 610 nm at 1 minute from beginning of the analysis. However, when the sample was added directly to the reagent layer without an adhesive layer the reflectance was found to be 12.8%. The reflectance was found to be 13.0 % when the sample was applied to the glass filter paper and reached the reagent layer by passing through the paper and the adhesive. It can be concluded that the filter paper and the adhesive had substantially no effect on the composition of the sample, which passed through them and reached the reagent layer.

In a second test, a much smaller concentration of albumin was used, 1 mg/L. In this case the albumin reagent without an adhesive coating showed a reflectance of 52.4 %, indicating the smaller concentration of albumin in the sample. However, when a sample was placed directly on the albumin reagent layer in the composite device, the reflectance was measured to be 25.4%, indicating a higher response to the same concentration of albumin. It can be concluded that some of the liquid in the sample passed through the adhesive and into the filter paper layer, thus raising the effective concentration of albumin on the reagent layer.

EXAMPLE II

In this example, a binding reagent layer is added to the diffusible adhesive layer to remove either a competing or interferring component, thus permitting the analyte to reach the detecting reagent layer. A protein blocked diffusible adhesive composition was made in a similar manner to the adhesive composition described in Example I, as follows:

(1) To a 250 ml steel beaker was added 150 g of 0.1 m sodium citrate buffer having a pH of 5.5 and 1.0 g of Pluronic L64 surfactant. With slow stirring 0.6 g of octanol was added followed by 12.0 g of Aerosil 200 and stirring continued for several minutes at about 2000 rpm to complete the dispersion of the ingredients.

(2) With continued stirring 110 g of a 40 % aqueous solution of Bayhydrol DLN was added followed by 0.4 g of PEO 900,000. Mixing continued for about 15 minutes.

(3) For each gram of the coating solution completed in step (2), 100 μL of a casein blocking solution was added. The mixture was vortexed in order to produce a homogenous coating solution. The adhesive coating solution was cast onto a peroxidase reagent layer. The peroxidase reagent layer was prepared by: (a) preparing a 10 mg/mL solution of 3, 3', 5 5'tetraethylbenzidine,

(b) dipping a Whatman 3 mm filter paper into the solution,

(c) drying the impregnated paper for 15 minutes at a temperature of 40°C

(d) dipping the dried paper of step (c) in a solution of 1400 U/mL of stock glucose oxidase, and

(e) drying the impregnated paper of step (d) for 20 minutes at 40 °C.

(4) The adhesive - peroxidase reagent layer combination was pressed onto a binding reagent layer, the binding reagent was prepared by:

(a) making a polymer membrane from the following components 17.3 g Dralon L (polyacrylonitrinle)

69.1 g Ultrason E (polyetherpolysulfone)

25.9 g Aerosil 200 (silica)

7.78 Pluriol P 600 (propylene oxide-based surfactant

(b) dipping the membrane into a solution of 4 mg/mL anti-FITC (anti- fluorescein isothiocyanate) in 0.1 M sodium citrate pH 4.5 buffer, and

(c) Drying the impregnated filter paper for 30 minutes at 40°C.

(d) Drying the treated membrane for 30 minutes at 40°C.

(5) The combined layers were dried at room temperature for 1-2 hours to complete preparation of the analytical device. In a test, the sample contained both BSA-FITC (bovine serum albumin - anti- fluorescein isothiocyanate) and HRP-FITC (horseradish peroxidase- anti-fluorescein isothiocyanate), the later competing with the BSA-FITC. As the sample passes through the adhesive layer, there is a separation of BSA-FITC from the HRP-FITC. The HRP- FITC which reaches the peroxidase reagent and a color is developed, indicating its presence and is measured by reflectance on a CLINITEK® 50 analyzer. When only

HRP-FITC was present, the reflectance was found to be 62.6 %, while when BSA-FITC was present the relectance was 45.7 %, indicating that HRP-FITC is capable of passing through the membrane when an excess of FITC is achieved

A comparative test was made in which the binding layer and the peroxidase reagent layer were placed in contact with each other without the intermediate adhesive layer. In that case, there was no difference observed between the two samples. That is, there was no separation of the sample containing both BSA-FITC and HRP-FITC. It can be concluded that it was not possible without the adhesive to keep the competing analyte separated, even though an excess of FITC was present in the binding layers.

EXAMPLE III

This example illustrates the use of a multi-layer device similar to that of Example II for measuring digoxin. The reagent containing a substrate capable of detecting peroxidase and the protein binding layer were prepared as described in Example II. Then, those layers were placed on either side of a diffusible adhesive layer previously described to produce a three-layer device. The combined layers were cut into strips, each strip being covered with a polystyrene strip having square openings which served as sample wells. Test samples were prepared containing 0, 25, 50, and lOOμg/mL of digoxin and 50 ml of a 50 mg/ml solution digoxin-BSA-HRP (digoxin-bovine serum albumin- horseradish peroxidase), and 50 mg of a 100 μg/ml solution of anti-digoxin labeled FITC. 45 μL of each sample mixture was added to a sample well on a strip to bring the sample into contact with the protein binding layer. The sample passed through the top layer and the adhesive layer into the reagent layer where a color response was developed.

Measurements made by a CLINITEK® 50 reflectance spectrometer indicated that the digoxin was reaching the reagent layer proportionally to its concentration in the sample, as shown in the following table.

Table 3

Con. of Digoxin in Test Sample % Reflectance

0 85% 25 65%

50 51%

100 40%

In a comparative test in which no adhesive layer was included, no variation in response was found from the reagent layer, indicating that without the adhesive layer competition did not take place.

Claims

WHAT IS CLAIMED IS: 1. A multi-layer device for detection of an analyte in a fluid sample comprising: (a) at least a first absorbent layer for receiving said fluid sample;

(b) at least a second absorbent layer for absorbing a portion of said sample received from said first absorbent layer; and

(c) an adhesive layer disposed between said first and second layers, said adhesive being diffusable to fluids and comprising a blend of an aqueous polymer dispersion and a water soluble polymer which has been cast and dried to form said adhesive layer.

2. A device of claim 1 wherein said first absorbent layer absorbs and spreads said fluid sample over said device.

3. A device of claim 1 wherein said first absorbent layer comprises a reagent for reaction with an analyte in said sample.

4. A device of claim 1 wherein said first absorbent layer comprises a reagent for reaction with interfering components of said sample.

5. A device of claim 1 wherein said second absorbent layer absorbs and retains a component from said sample.

6. A device of claim 1 wherein said second absorbent layer comprises a reagent for reacting with an analyte in said sample.

7. A device of claim 5 wherein said component from said sample is the product of a reaction of an analyte in said first absorbent layer.

8. A device of claim 1 wherein said adhesive layer is capable of making a physical separation of said fluid sample.

9. A device of claim 1 wherein said adhesive layer is capable of reacting with components of said sample and thereby trapping them in said adhesive layer.

10. A device of claim 1 wherein said adhesive layer contains additives capable of reacting with components of said sample and thereby preventing their passage through said adhesive layer.

11. A device of claim 1 wherein said water dispersible polymer is an anionic polyurethane_dispersion in combination with a water soluble polymer.

12. A device of claim 1 wherein said water dispersible polymer is an anionic polyurethane dispersion in combination with a cationic acrylic dispersion as the water soluble polymer.

13. A device of claim 1 wherein said water dispersible polymer is a cationic polyurethane dispersion in combination with a water soluble polymer.

14. A device of claims 11 or 13 wherein said water soluble polymer is at least one member of the group consisting of a polyethylene oxide, a polyvinylpyrrolidone and a polyvinylalcohol.

15. A device of claim 2 wherein said first absorbent layer is a filter paper.

16. A device of claim 3 wherein said reagent for reaction with an analyte in said first absorbent layer is a member of the group consisting of oxidases, reductases, and proteases used in clinical assays, and antibodies, nucleic acids, antigens, and proteins used in binding assays.

17. A device of claim 4 wherein said reagent for reaction with an interfering component of said sample is a member of the group consisting of enzymes to metabolize the interferent, reactants to convert the interferent to non-reactive form, and binding agents to trap the interferent.

18. A device of claim 5 wherein said second absorbent layer is a filter paper.

19. A device of claim 6 wherein said reagent for reacting with an analyte in said second absorbent layer is a member of the group consisting of indicators producing signals in response to the analyte and enzymes or reactants for signal amplification.

20. A device of claim 7 wherein said product of the reaction of an analyte in said first absorbent layer is detected by a member of the group consisting of enzymes used in clinical assays and affinity binders used in binding assays and reactions in which a moiety of the analyte is detached.

21. A device of claim 10 wherein additives to said adhesive layer capable of reacting with components of said sample are members of the group consisting of affinity binders or enzymes for removing interferents or generating signals.

22. The device of claim 1 further comprising additional absorbent layers disposed between said first and second absorbent layers, each of said additional absorbent layers being separated from the closest neighbor absorbent layer by an additional adhesive layer, said adhesive layer being diffusable to said fluid and comprising a blend of an aqueous polymer dispersion and a water soluble polymer which has been cast and dried to form said adhesive layer.

23. A method of detecting an analyte in a fluid sample comprising applying said sample to a multi-layer device of claim 1 and measuring the amount of analyte present in said sample.

24. A device for detection of an analyte in a liquid sample comprising a liquid permeable layer capable of adhesion, said layer comprising a blend of an aqueous polymer dispersion and a water soluble polymer which has been dried to form said liquid permeable layer, said liquid permeable layer being disposed between dry reagents or absorbent layers for transferring portions of said liquid sample.

25. A device of Claim 24 further comprising additives which chemically react with components in said liquid sample.

26. A device of Claim 25 wherein said additives include exchange resins to remove buffering components and ascorbate scavengers to remove ascorbate interference.

27. A device of Claim 25 wherein said additives include protein binding polymers to separate interfering proteins or antibodies.

28. A device of Claim 25 wherein said additives include fillers to adjust opacity or reflectance .

29. A device of Claim 24 wherein the liquid permeability of said liquid permeable layer is adjusted by changing the ratio of said aqueous polymer dispersion to said water soluble polymer.

30. A device of Claim 29 wherein said aqueous polymer dispersion is a polyurethane dispersion and said water soluble polymer is at least one member of the group consisting of a polyethylene oxide, a polyvinyl pyrrolidone and a polyvinyl alcohol.

' 31. A device of Claim 30 wherein the ratio of said aqueous polymer dispersion to said water soluble polymer is 50: 1 to 1 : 1 on a weight basis.

32. A method of analyzing a liquid sample wherein said sample is contacted with absorbent layers or dry reagents which react with analytes in said sample to provide a detectable response characterized by including a liquid permeable layer capable of adhesion between said dry reagents or absorbent layers, said liquid permeable layer transferring portions of said liquid sample.

33. A method of Claim 32 wherein said liquid permeable layer is a dried blend of an aqueous polymer dispersion and a water soluble polymer.

34. A method of Claim 32 wherein said liquid permeable layer concentrates said sample by passing liquid in said sample through said layer.

35. A method of Claim 32 wherein said liquid permeable layer comprises additives which chemically react with components in said liquid sample.

36. A method of Claim 35 wherein said additives include exchange resins to remove buffering components and ascorbate scavengers to remove ascorbate interference.

37. A method of Claim 35 wherein said additives include protein binding polymers to separate interfering proteins or antibodies.

38. A method of Claim 35 wherein said additives include fillers to adjust opacity or reflectance.

39. A method of Claim 32 wherein the liquid permeability of said liquid permeable layer is adjusted by changing the ratio of said aqueous polymer dispersion to said water soluble polymer.

40. A method of Claim 39 wherein said aqueous polymer dispersion is a polyurethane dispersion and said water soluble polymer is at least one member of the group consisting of a polyethylene oxide, a polyvinyl pyrrolidone and a polyvinyl alcohol.

41. A method of Claim 40 wherein the ratio of said aqueous polymer dispersion to said water soluble polymer is 50:1 to 1 :1 on a weight basis.