CA2144976A1 - Assay devices using subsurface flow - Google Patents

Abstract

The present invention is directed to improving the performance of assays using a disposable assay device which includes a porous material in liquid communication with a capillary track. In particular, the capillary track is used in conjunction with the solid support to direct test sample and assay reagents directly to a defined reaction site on or in the porous material. Signal devel-opment at the reaction site indicates the assay result. The present invention is also directed to the construction of a disposable as-say device which includes a capillary track. In particular, the cappillary track is formed by printing a fluid insoluble material, in the reverse image of the desired capillary track, on a film layer and then capping the printed material. Alternatively, the capillary track is formed by printing a fluid insoluble material, in the reverse image of the desired capillary track, on a porous material which is then sandwiched between two film layers.

Description

~VO 94/09366 2 1 4 4 9 7 6 PCI/US93/08751 ASSAY DEVlCES USING SUBSURFACE FLOW

BACKGROUND OF THE INVENTION

5 1. Field of the Invention In general the present invention relates to assay methods and devices for the detection of an analyte in a test sample. In particular, the invention relates to novel test devices designed to provide for the rapid transfer of fluid to a rea~ c me",brane by means of a capillary track. In addition, the invention relates to novel 10 methods for forming a capillary track.

2 . Related Art Various analytical procedures and devices are cG"""only elllrl~Jod in assays to delt,r",i"e the pl~sence and/or amount of suLsldnces of interest which may be15 present in biological or non-biological fluids. Such substances are cG",monly termed "analytes". The abiiity to use materials which specifically bind to an analyte of interest has created a bl"geoni"g diag"ostic device market based on the use of binding assays.
Binding assays jIICGr~JOraI~ specific binding members, typified by antil,Gdy 2 0 and antigen immunoreactants, wherein one member of the specific binding pair is labeled with a signal-producing compound. For example, in a binding assay the test sample suspected of containing analyte can be mixed with a labeled anti-analyte ar,liL,ody, i.e., labeled reagent and incuh~t~d for a period of time sufficient for the immunGreaction to occur. The reaction mixture is suhsequently analyzed to detect2 5 either that label which is ~Csoi -IHd with an analyte/labeled reagent complex (bound labeled reagent) or that label which is not cor"?lexed with analyte (free labeled reagent). As a result the presence or amount of label in one of these species can be cGr,~lalt:d to the presence or amount of analyte in the test sample.
The solid phase assay format is a col"r"only used binding assay technique.

3 0 There are a number of assay devices and procedures wherein the presence of an analyte is indicated by the binding of the analyte to an labeled reagent and/or an immobilized complementary binding member. The immobilized binding member is bound or becomes bound during the assay to a solid phase such as a dipstick, te~ flow-through pad paper fiber matrix or other suitable solid phase 35 material. The binding reaction between the analyte and/or assay reagent(s) results in a distribution of the labeled reagent between that which is immobilized upon the WO 94/09366 ~ PCI/US93/Ol~L

solid phase and that which remains free. The presence or amount of analyte in the test sample is typically indicated by the extent to which the labeled reagent becomes immobilized upon the solid phase material.
Flow-through pads for immobilizing and detecting an analyte are well-known in the art. For example, Tom et al. (United States Patent No. 4,366,241) disclose a bibulous strip with an immunosorbing zone to which the test sample is directly applied and wherein the assay result is detecP~
The use of reagent-impregnated teststrips in specific binding assays is also well-known. In such procedures, a test sample is applied to one portion of the tesl~ . and is allowed to migrate or wick through the strip. Thus, the analyte to be detected or measured passes through or along the strip, possibly with the aid of an eluting solvent which can be the test sample itself or a sepdldt~ily added solution. The analyte ~l~iy~ates into a capture or cletection zone on the teststrip, wherein acomplementary binding member to the analyte or labeled reagent has been 1 5 immobilized. The extent to which the analyte becomes bound in the det~clion zone can be determined with the aid of the labeled reagent which can also be illcG,~,ordled in the l~t~tli~. or which can be applied separately.
An early teststrip device is described by Deutsch et al. in United States PatentNo. 4,361,537. In general, the device col"priaes a material capabl~ of ll~ns,~o,li"g a solution by capillary action, i.e., a wicking or chromatographic action. Different areas or zones in the teststrip contain the assay reagents needed to produce a det~ le signal as the analyte is l,dnspo,led to or through such zones. The device is suited for both chemical assays and binding assays and uses a dcveloper solution to transport analyte along the strip.
2 5 The disadvantages of the conventional porous or absorbent matrix devices include the slow rate of flow of test sample through the teststrip material. In addition, the test sample and mobile reagents are directed to and through the edge of an absorbent pad or layer containing the l~ac-lion site. Such diffuse sample apFI -~tion results in reduced signal production and a slowed rate of signal 3 0 production.
Assay devices have also been constructed of tubes wherein the capillary tube cor,t~i"s an immobilized assay reagent to define a reaction zone for the capture and detection of an analyte of interest (Hibino et al., 4,690,907). In general, the capillary tube is used to collect a predetermined amount of test sample for use in a 3 5 test device.

~0 94/09366 ~' 2 1 4 4 9 7 6 PCI~/US93/08751 Conventional capillary tracks are formed from glass tubes. Glass tubes, however, are usually restricted to simple geometric designs. Glass tracks are also breakable and a biosafety hazard to workers. Other typical capillary tracks are constructed by sandwiching a die-cut material between two pieces of film, wherein 5 one film is typically more hydrophobic than the other for the purpose of promoting fluid movement. This type of device is limited to simple single track designs bec~use of manufacturing limitations involving the placement of the die-cut middle layer.
In still another conventional design, capillary tracks are formed through a process of injection molding. A major disadvantage of this process is the cost of 10 prototyping. Another disadvantage is in the manufacturing process which is limited to piece-part assembly. Also, the use of multiple materials complicates construction and assembly. When dissimilar ~"ate,ials are incGr~,or~ed as different layers, the separate pieces must be spot treated during assembly. For example, asar,dwich layer of adhesive would be needed to mate the pieces lug~tl,er.
15 Alternatively, the ",aterials could be ultrasonically welded or solvent bonded, but the manufacturing limitations remain.

SUMMARY OF THE INVENTION

The present invention is directed to improving the pe.f~,r."ance of assays using a dispcs-b'e assay device which includes a porous material in liquid commu"icalion with a capillary track. In particular, the capillary track is used in conjunction with the solid support to direct test sample and assay reagents directly to a defined rt:a..tion site on or in the porous material. Signal development at the 25 rea~;tion site indicates the assay result.
The present invention is also well suited for enhancing the production of signal at the reaction site. The capillary track directs fluid to a position below the " defined l~:a-;lion site on the porous material such that the fluid does not have to pass through the edge of the porous material, as in conventional teststrip device, to reach 30 the reaction site. Preferably, the test sample is directed to a posilion directly below the reaction site on the porous material. Upon contact with the porous material, the fluid passes radially through the reaction site rather than transversely through the site.
The devices of the present invention are constructed from a capillary track 35 having an inlet and outlet, wherein the inlet receives test sample or test solution, and the outlet is in communication with and directs test sample or test solution to a W0 94/09366 ~ 6~ ;~, PCI/US93/08 porous support containing an immobilized reagent which binds to the analyte an ancillary specific binding member or a labeled reagent to produce a detectable assay result. The outlet port is disposed beneath the immobilized reagent in the porous support. The device may further include a labeled reagent such that the reagent need 5 not be separ~lely cor,~c~ed to the device and such that the assay method is self-performing. The labeied reagent may be contained within the capillary track or within a material or means which is in fluid communication with the capillary track.
In a preferred embodiment a reagent matrix is contained within a drop forming means which is in communication with the capillary track. Assay kits are also 10 contemplated and include the subsurface flow device together with one or morecontainers of reagents necessary to the pe"~r",ance of the assay.
The present invention is also directed to constructing a f~ ,os~hle assay device which includes a capillary track. One method for constructing the capillary track involves applying a printable material to a first film layer thereby forming a 15 core layer and three sides of the capillary track wherein the printable material is d~l)osit~cl as a reverse image of the capillary track on the first film layer. A second film layer is then adhered to the top of the printable material or core layer, thereby forming the fourth side of the capillary track.
An all~r"dli~e method for constructing the capillary track involves applying 20 a fluid repellant printable substance to a length of porous ",aterial therebyimpregnating the porous material wherein a non-impregnated region defines two sides of the capillary track. A first and a second film layer are then adhered to the top and bottom of the porous material thereby forming the top and bottom of the capillary track.
2 5 The present invention can also be adapted for use in the aul.. ",ated didgl,osis of a plurality of sa"" les. Another object of the present invention is to provide a device capable of performing multiple highly sensitive diagnostic tests simultaneously on a single sample in a single device having multiple capillary tracks and reaction sites.
In particular the devices of the present invention can be used in an aulu",aled fashion where the assay reaction can be rapidly performed and ",or,ilc.red with a minimum of sample material.

BRIEF DESCRIPTION OF THE DRAWINGS

3 5 Figure 1 is a side perspective of one embodiment of the present reaction device.

~/O 94/09366 2 1 ~ 4 9 7 6 ~ -~ PCI/US93/08751 Figure 2 is a side perspective of the present reaction device showing two layer construction.
Figure 3 is a side perspective of the present reaction device showing three layer construction. Figure 3a depicts a modified embodiment of the present 5 invention.
Figure 4 is an end view of a reaction device showing a printed layer construction.
Figure 5 is an end view of a reaction device showing a multi-layer construction using a partially impregnated porous material as the middle layer.
Figure 6 is an end view of a reaction device showing a three layer construction.
Figure 7 is a top perspective of an enhanced assay device having directed flow through the reaclion site.
Figure 8 is side perspective of an enhanced assay device having an absorbent 1 5 layer.
Figure 9 depicts predicted chromatographic flow rates in a linear and radial flow format.

DETAILED DESCRIPTION OF THE PRE~t~tu EMBODIMENTS
One exemplary e,.,bodi,..ent of the present invention is shown in Figure 1.
The device (10) includes a main body (12) in which a capillary track (18) extends along at least a portion of its length. The capillary track has a size and dimensions suit~hle for the l.~nspo,l of test sample through the track by capillary action. The 2~ capillary track has an inlet (14) for the introduction of test sample to the device.
The capillary track is in fluid communication with a porous support (20) containing an immobilized specific binding material (22). The capillary track has an outlet(16) in fluid flow communication with the porous support. P~eferably, the poroussupport is positioned such that the outlet of the capillary track lies directly beneath 30 the site of the immobilized specific binding material. For purposes of the present invention, "directly beneath" means that fluid passing from the capillary track to the porous support passes through less than orie-half the largest dimension of the - porous support. More preferably, fluid passing from the capillary track to the porous support passes through less than one-quarter the largest dimension of the35 porous support.

WO 94/09366 2 1 4 ~ ~ 7 6 6 ~ PCI/US93/08~

Before proceeding with the description of the various embodiments of the _.
present invention, a number of terms used herein will be defined.
"Test sample" refers to a material suspected of containing the analyte. The test sample can be used directly as obtained from the source or after p,~l~eal",ent so 5 as to modify its character. The test sample is typically a physiological fluid. The test sample can be pretreated prior to use, such as preparing plasma from blood,diluting viscous fluids, extracting analyte, or the like. Methods of treatment can involve filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents. Resides physiological fluids, other liquid 10 sar, ~les can be used such as water, food products and the like for the pe"u""ance of environmental or food production assays as well as diagnostic assays. In addition, a solid material suspected of containing the analyte can be used as the test sample once it is modified to form a liquid medium or to release the analyte.
"Specific binding member" refers to a member of a specific binding pair, i.e., 15 two different molecules wherein one of the ",ole..u'~s specifically binds to the second ",o'ec lle through chemical or physical means. In addition to antigen and antiLoJy specific binding pair members, other exemplary specific binding pairs include, without limitation, such materials as biotin and avidin, carbohydrates and lectins, cGm,~le ~entary nu~'eot ~'e sequences, complementary peptide sequences, effector and 20 r~cepLu, I. ole c ~les, enzyme c~ ;tu,~- and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an antibody specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g., ribonuc'e--e, S-peptide and ribonuclease S-protein). Furthermore,specific binding pairs can include members that are analogs of the original specific 25 binding member, for example an analyte-analog or a specific binding member made by recGnll,L,ant techniques or molecular engineering. If the specific binding member is an immul,oreactdnt it can be, for example, an antibody, antigen, hapten, or complex thereof, and if an antibody is used, it can be a monoclonal or polyclonal antibody, a recombinant protein or antibody, a chimeric antibody, a mixture(s) or 3 0 fragment(s) thereof, as well as a mixture of an antibody and other specific binding members. The details of the preparation of such antibodies and their suitability for use as specific binding members are well-known to those skilled-in-the-art.
"Analyte" or "analyte of interest" refers to the compound or cGr"posilion to be detected or measured, which has at least one epitope or binding site. The analyte can 35 be any substance for which there exists a naturally occurring analyte-specific binding member or for which an analyte-specific binding member can be prepared.

.NO 94/09366 PCI/US93/08751 2~449'1G 7 Analytes include, but are not limited to toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, sterC.1~, vitamins, drugs (including those administered for therapeutic purposes as well as those administered for illicit purposes), and met~hc!;'es of or antibodies to any of the 5 above substances. The term "analyte" also includes any antigenic substances, haptens, antibodies""acro",olecules and combinations thereof.
"Analyte-analog" refers to a substance which cross-reacts with the analyte-specific binding member, although it may do so to a greater or a lesser extent than does the analyte itself. The analyte-analog can include a modified analyte as well as a 10 fragmented or synthetic portion of the analyte molecule, so long as the analyte-analog has at least one epitopic site in common with the analyte of interest. Anexample of an analyte-analog is a synthetic peptide sequence which duplicates at least one epitope of the whole-n ole~ analyte so that the analyte-analog can bind to an analyte-specific binding member.
"Labeled reagent" refers to a substance COIll,)liS;ll9 a detecl~hlQ label attached to a specific binding member. The attachment may be covalent or non-covalent binding, direct or indirect, but the method of alldch",ent is not critical to the present invention. The label allows the labeled reagent to produce a detectable signal that is directly or inversely related to the amount of analyte in the test sample. The specific 2 0 binding member cor"~,onent of the labeled reagent may be selected to directly bind to the analyte or to indirectly bind the analyte by means of an ancillary specific binding member, which is described in greater detail hereinafter. Alternatively, the specific binding member component may be selected to directly or indirectly bind an immobilized reagent. The labeled reagent can be incorporated into the test device, it 2 5 can be cGr"L,ined with the test sample to form a test solution, it can be added to the device separately from the test sample or it can be predeposited or reversibly imr"~b~ d at the immobilized reagent site. In addition, the binding member may be labeled before or during the pel~-,r",ance of the assay by means of a suitable dll~chl"ent method.
3 0 "Label" refers to any sul,~l~nce which is capatlE of producing a signal that is detect~hle by visual or instrumental means. Various labels suitable for use in the present invention include labels which produce signals through either chemical or physical means. Such labels can include enzymes; enzyme substrates; chromogens;
catalysts; fluorescent compounds; chemiluminescent compounds; radioactive labels;
and direct visual labels including colloidal metallic particles such as gold, coll~
non-metallic par.~-.tes such as selenium, dyed or colored particles such as a dyed WO94/09366 ?~4~G PCI/US93/0~

plastic or a stained microorganism, organic polymer latex particles and liposomes or other vesicles containing directly visible substances. A visually detectable label is advantageously used as the label component of the labeled reagent, thereby providing for the direct visual or instrumental readout of the presence or amount of the analyte 5 in the test sample without the need for additional signal producing components at the detection sites.
The selection of a particular label is not critical to the present invention, but the label will be capable of generating a detectable signal either by itself, such as a visually detectable colored organic polymer latex particle, or be instrumentally1 0 delt:cl~ble, such as a fluorescent compound. The label may be detected in conjunction with one or more addilional signal producing components, such as an envme/substrate signal producing system. A variety of different labeled reagentscan be formed by varying either the label component or the specific binding member component of the labeled reagent; it will be appreciated by one skilled-in-the-art 1 5 that the choice involves consideration of the analyte to be detecled and the desired means of dete~;tion.
"Signal producing component" refers to any substance caF~'E of ~ac~;ng with another assay reagent or with the analyte to produce a reaction product or signal that indicates the presence of the analyte and that is det~ by visual or 20 instrumental means. ;Signal production system", as used herein, refers to the group of assay reagents that are needed to produce the desired reaction product or signal.
One or more signal producing components can be reacted with the label to generale a detectable signal. For exd",ple, when the label is an enzyme, al"pl;ficdlion of the del~-,tabls signal is obtained by rea~:ti"g the enzyme with one or more substrates or 25 additional envmes and suL~tldl~:s to produce a det~ E ~eaction product.
"Porous support" refers to any suitable porous, absorbent, bibulous, isollopic or capillary material, which includes the reaction site of the device.Natural, synthetic, or naturally occurring materials that are synthetically modified, can be used as the porous support and include, but are not limited to: papers 30 (fibrous) or membranes (microporous) of cellulose materials such as paper, cellulose, and cellulose derivatives such as cellulose acetate and nitroce!l ~lose;
fiberglass; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon);
porous gels such as silica gel, agarose, dextran, and gelatin; porous fibrous matrixes; starch based materials, such as cross-linked dextran chains; ceramic 35 materials; olefin or thermoplastic materials including films of polyvinyl chloride, polyethylene, polyvinyl acetate, polyamide, polycarbonate, polystyrene, copolymers ~0 94/09366 PCI/US93/08751 214~9~ 9 -of vinyl acetate and vinyl chloride and combinations of polyvinyl chloride-silica; and the like. The porous material should not interfere with the production of a detectable signal.
"Immobilized reagent" refers to a specific binding member that is attached 5 within or upon a portion of the porous support to form a "capture site" or reactive membrane. The method of attachment is not critical to the present invention. Theextent of signal production in the capture site is related to the amount of analyte in the test sample. The immobi~i~ed reagent is selected to bind the analyte, the labeled reagent or a complex thereof. In preferred embodiments, the immobilized reagent 10 binds to the analyte for the completion of a sandv ich cGr, Flx:- Co"",elilive assay formats will also be apparent to those skilled-in-the-art. The immobilized reagent may be chosen to directly bind the analyte or indirectly bind the analyte by means of an ancillary specific binding member which itself is bound to the analyte. In addition, the immobilized reagent may be directly or indirectly immobilized on the 15 solid phase before or during the pe"or",ance of the assay by means of any su'-~le dlldchi"ent method.
Typically, the capture site of the present invention is a delimited or defined portion of the porous support such that the specific binding reaction of the immobilized reagent and analyte is localized or concentrated in a delimited site. Such 2 0 a locP';~ ;on facilitates the detection of label that is immobilized at the capture site in coht,dst to other po,lions of the porous support. The delimited site is typically less than 50% of the porous support, and preferably less than 25% of the porous support. The immobilized reagent can be applied to the solid phase ",aterial by dipping, i"scriLi.,g with a pen, dispensing through a capillary tube or through the 25 use of ,t:agenl jet-printing or any other suitable dispensing techniques. In addition, the capture site can be marked, for exc""ple with a dye, such that the position of the capture site upon the porous support can be visually or instrumentally determined even when there is no label i"""ob 'i~ed at the site.
P~edetermined amounts of signal producing col"ponents and ancillary S O reagents can be i"cGr~ordted within the device, thereby avoiding the need for addilional protocol steps or reagent additions. Thus, it is also within the scope of this invention to provide more than one reagent to be immobilized within the porous support. For example, to slow or prevent the diffusion of the detectable reaction product in an enzyme/substrate signal producing system, the substrate can be 3 5 immobilized by direct attachment to the porous support by methods well-known in WO 94/09366 PCI`/US93/08--the art, or the substrate may be immobilized by being covalently bound to insoluble microparticles which have been deposited in and/or on the porous support.
The immobilized reagent may be provided in a single capture or detection site or in multiple sites on or in the porous support. The immobilized reagent may also 5 be provided in a variety of configurations to produce different detection or measurement formats. Alternatively, the immobilized reagent can be distributed over a large portion of the porous support in a substantially uniform manner to form the capture site.
"Ancillary specific binding member" refers to any member of a specific 10 binding pair which is used in the assay in adcJilion to the specific binding members of the labeled reagent or immobilized reagent. One or more ancillary specific binding members can be used in an assay. For example, an ancillary specific binding member may be used to bind the labeled reagent to the analyte in instances where the analyte itself could not directly bind the labeled reagent. The ancillary specific 1 5 binding member can be illcor~.o,dl~d into the assay device or it can be added to the device as a separdte reagent solution.

Figure 1 depicts a device wherein the capillary track outlet is in direct contact with the porous support and is positioned beneath the ,~a~:tion site of 20 im",obili~ed specific binding material. It is not essential to the present invention that the outlet and porous support be in direct contact. It is sufficient that the components are close enough that fluid in the capillary track will pass from theoutlet to the porous support. Nor is it essential that the outlet be positioned directly under the reaction site. It will be readily appreciated, however, that the closer the 2 5 capillary track outlet is to the reaction site, the less di.,ldnce the test sample and/or assay reagents must travel.
Assay reagents, such as a labeled specific binding member, may be mixed with the test sample, may be sequentially contacted to the test device, or may be included within the capillary track. For example, the labeled reagent may be 3 0 predeposiLed in the capillary track such that contact with the test sample mobilizes the labeled reagent and transports the labeled reagent to the lea~lion site on the porous support. This is to be distinguished from agglutination assay devices wherein the ~ea.iLion of assay reagents and test sample analyte results in the formation of a reaction product which agglutinates and decreases or stops fluid flow within a 35 capillary space.

VO 94/09366 PCI~/US93/08751 A second embodiment of the present invention is shown in Figure 2. The device (10) generally comprises a main body (12) constructed from a first surface (6) and a parallel second surface (8) one or both of which are grooved, separated by ~pacers or otherwise constructed such that when the two surfaces are aligned and5 joined a capillary track (18) is formed. The capillary track has a size and dimensions suitable for the transport of test sample and soluble assay reagents through the track by capillary action. The surfaces may be joined by any suitable means including, but not limited to, sonic welding, solvent welding and adhesivebonding. In the adhesive bonding method, the adhesive may be applied by a printing 1 0 means.
Figure 3 represents a further embodiment of the present invention. This e",boJi",ent has, in adherent relationship, a first or bottom wettable, but liquid-occlusive, layer (22), a second or middle liquid occlusive layer (24) parallel to and overlying the first layer (22), and a third or top liquid-occlusive, preferably non-1 5 wettable, layer (26) parallel to and overlying the second layer (24). The thirdlayer (26) may be made from a clear material, such as a clear polycarbonate film, and therefore, the layer may also serve as a window, or viewing area, for observing the capillary track. The second layer (24) is interposed between, and is adhered to, the first layer (22) and third layer (26). For example, the layers are adhered by 2 0 means of an adhesive on each side of the second layer (24) facing the topside of the first layer (22) and the underside of the third layer (26). Typically, the second layer (24) is die cut or prefc,rl"ed to have a slot positioned through its thickness, thereby defining the walls of the capillary track (18) in conjunction with the first (22) and third (26) layers. Thus, when the first, second and third layers are 25 laminated together, a portion of each of the first and third layers serve as the floor and roof, respectively, of the capillary track with part of the walls of the slot of the second layer (24) defining the walls of the capillary track.
The device illustrated in Figure 3 may also include an oplional well-defining means (2) in the third layer (26). The well-defining means is positioned such that 3 0 the it defines an area for receiving the test sample, and it is in fluid flow communication with the capillary track. The bottom of the well may be formed from a corlesponding circular portion of the first layer (22).
In a preferl~d embodiment, the devices of the present invention include a test sample application pad in fluid flow contact with the capillary track. The application 3 ~ pad facilitates the arplic~lion of test sample or reagents to the device and may optionally contain one or more reagents, such as the labeled binding member. The WO 94/09366 2~ PCI~/US93/0 addition of test sample to the application pad serves to elute an assay reagent from the application pad, such that a test sample/reagent mixture emerges from the bottom surface of the application pad. The device may further include a well situated between the application pad and the capillary track inlet such that the test solution exiting the application pad substantially fills the well prior to passing into the capillary track. The application pad may be constructed from a single material or from a plurality of layers. The use of a multi-layered application pad permits the inclusion of multiple assay reagents, even when the reagents are not compatible for extended storage, thereby allowing multiple, separate reagent additions to the test sample. The ~ppl - ticn pad material or a layer thereof may also be sElevl~ d toprovide a filter function.
Figure 3a depicts an alternative embodiment wherein the device includes a drop-forming means (50) which holds an ~pplic~tion pad (55) that contains the labeled reagent. By cor,ta~;ti"g a test sample to the drop-forming means, the labeled reagent is released from the al-~!ic~liol1 pad and forms a drop on the bottom surface of the _pF' c~tion pad. The drop-forming means is situated over the capillary track such that when the drop is rele~-sed from the application pad it is delivered to the capillary track. This optional m~li~ic ~lion provides an added advantage. The addition of fluid or test sample to the reagent-containing application pad serves to deliver a 2 0 bolus of the eluted reagent to the capillary track. Thus, the first fluid mixture entering the capillary track contains a large portion of the eluted reagent, and s~hsequent fluid contains less of the reagent. When the reagent is a labeled reagent, this means that the first fluid delivered to the reaction site on the porous support cor,tvi.,s the largest portion of the labeled reagent. Subsequent fluid cohl-ai"s a lesser 2 5 amount of assay reagent and thereby enhances the clearance of l" ,r~a~;~d reagents from the reaction site. This clearance or washing aspect of the invention helps to stabilize the signal that is produced in the reaction site and decreases interference from the occurrence of background signal in the area surrounding the reaclion site.
In yet another embodiment, as depicted in Figure 4, the sides (24) of the capillary track (18) are formed from a printable material that is insoluble, andpreferably, that aids in adhering the first liquid occlusive layer (22) to a third liquid occlusive layer (26). The material is applied to either the first or third layer, and the sides are then capped by the application of the third or first laminate layer, respectively, thereby defining the capillary track (18).
The first (back laminate) layer may be a flexible plastic or plastic-coated film supplied in either sheet or roll form. The film can also be chosen to have ~0 94/09366 PCI/US93/087~1 21~4976 13 distinct properties such as opacity, biodegradability, etc., or it can be treated to have certain properties, such as hydrophobicity, hydrophillicity or selective biocompatability. The film may be selected from a variety of materials including, but not limited to, polyester, polycarbonate, and other film materials. In addition, 5 the surface or a portion of the surface of the capillary track may be spot treated to create or enhance a desired property. For example, a portion of the capillary track may be spot treated with a hydrophobic material to slow the rate of flow through that portion of the track. Suitable film materials include, but are not limited to, Mylar(E~' film (DuPont, Wilmington, DE) and polyester films (Melinex~D films; ICI Films, 10 Wilmington, DE). Suitable film materials include materials having the following properties or characteristics:

Film Properties Typi~
Thickness: 0.002 inch 0.0002 - 6.0 inches Flexible: 30,500 psi tensile any flexible or rigid material Optical: opaquewhite transparent, opaque, reflective, metallic, or treated Material: polyester any plastic, glass or metal or combination thereof The second (core or sandwiched) layer is conveniently applied via printing 15 techniques, such as screen printing methods which are well-known in the art. The second layer may be applied or cleposil~d, however, by any suitable printing method capab'e of achieving the desired design tolerances for thickness, alignment, or geometric lir"ildlions of the capillary track. It will be appreciated by those skilled-in-the-art that the capillary track dimensions will be selected to accG""~lish the 2 0 desired fluid delivery and timing characteristics which may differ between devices based upon the analyte of interest, the test sample used, inCIlh~tion and l~a~icn requirements, and other assay parameters. The second layer may be printed from an adhesive material, an ink, a dielectric material, or any material that is suitable for printing and for providing the desired thickness or height of the capillary track. The 25 second layer may preferably be formed from an adhesive. More preferably from a pressure sensitive adhesive. Pressure sensitive adhesive materials generally consist of a polymer formulation and are usually vinyl or acrylic based (e.g., UVC

2 1 4 4 9 7 6 ` i PCr/US93/08~
~j i`.4`

8201, UVC 8200, ML 25184; Acheson Colloids, Port Huron, Ml). Suitable pressure sensitive adhesives include, but are not limited to, materials having properties similar to UVC 8201 polyester film:
Adhesive Properties Typical E~ngQ
SolidsContent: 100% solids 20% - 100%
Density: 8.48 Ibs./gal . 5.4 - 13.4 Ibs./gal.
Viscosity: 1700 - 2000 cps. 100 - 750,000 cps.
Color: transparent any Cure: U.V. air dry, heat cure, U.V., solvent evaporation, crosslink Material: Urethane Acrylate any -- acrylic, epoxy, vinyl,conductive, non-conductive, adhesive Coverage: 1600 sq. ft./gal a? 1 mil any -- to meet the desired film thickness Adhesion (90 degree 2 Ibs./in. 0.25 - 100 Ibs./in.
peel test):
Printed Film 1 - 5 mils 2 - 10 mils Thickness:

The printed material may be selected to have a suitable adl,esiv0ness to la,-,i.,dle the top and bottom layers, as well as a suitable hydrophilic cha,dcl~ri~lic to promote the movement of fluid through the capillary track. Alternatively, theprinted material may be selected to have a suitable hydrophobic characteristic to 1 0 inhibit or prevent fluid flow within a portion of the capillary track, thereby cor,t,." ~9 the rate of fluid flow through the device.
The pressure sensitive adhesive is applied in the reverse image of the capillary track, such that the printed area or areas define the thickness of the core layer and the non-printed area or areas form a gap between the first and second 1 5 la",i"ate layers, thereby defining the sides of the capillary track. The pressure sensitive adhesive may be applied using standard screen printing equipment (such as a flatbed screen printer from deHaart Inc, Burlington, MA). Typically, the second layer is applied in a single printing pass to a thickness ranging from 0.0002 inches to 0.010 inches. The layer, however, can be formed to have any desired thickness if 21~97~ 15 '' accomplished in multiple passes. Usually, the layer will not exceed a final thickness of 0.100 inches.
Following application, the pressure sensitive adhesive is cured. For example, the pressure sensitive adhesive may be cured by means of ultraviolet radiation at about 200 watts per inch, or any other power rating that accomplishes crosslinking of the polymer to achieve the desired film properties such as thickness, adhesion and tack. A release liner may be placed over the printed adhesive for handling purposes until device assembly, although assembly can take place immediately after cure. In conventional device construction methods involving a die cut film material with a two-sided adhesive, there are two layers of release liner to be removed prior to assembly, and assembly is more cumbersome. In the present invention, the core layer of printed pressure sensitive adhesive is applied directly to one side of the base or back film, thereby avoiding this handling step.
The third (base) layer may be selected from the same film material as the first lar"i.,dle layer. A different material may be used, huwevcr, to meet desired fluid flow char~ct~ristics or other properties pertinent to the design of the desired capillary track. A plastic or plastic-coated film, chosen for properties of opacity and wetability (e.g., plastic-coated paper board 150HT, Daubert, Dixon, IL; Vistex PC polyester film, Film Speci~lties, Inc., Whitehouse, NJ), is received in a rolhand is applied to the printed core layer via standard web-laminating or pl~cess;,~g techr, ,_es which are well-known in the art. Pressure is applied to bond the back and base layers to the core of pressure sensitive adhesive thereby forming the top and bottom of the capillary track. The base film may be slit, or die punched, or laser cut to produce fixture holes or other features of the desired design such as inlet and outlet ports for the capillary track. The third film layer, like the first, can be treated to have certain properties, such as hydrophobicity, hydrophillicity or selective biocol"palt,bility. Either the whole layer may be treated or at least that portion of the layer which forms the capillary track may be treated. Such treatment ",al~,ials may also be advantageously applied by printing techniques.
3 0 In yet another embodiment, as depicted in Figure 5, the second layer (24) is made of a porous or liquid absorbent material which is selectively implegnal~:d through its thickness with a substance, such as a water-repellent ink, to form an impregnated region (30) and a non-impregnated region (18). The core layer of porous material defines the thickness of the gap between the top and bottom laminate layers, and the impregnated regions of the porous material define the side walls of the capillary track (18). The non-impregnated region remains liquid absorbent, WO 94/09366 P~/US93/0~
.~
2 1 g ~ 1 6 and the impregnated region is made liquid-occlusive, such that the non-imprey"ated region defines a solid capillary track for the passage of fluid via capillary action.
Thus, the non-impregnated region, with inlet and outlet portions, serves as the means for directing the test solution through the device to the overlying porousmaterial .
The porous second or core layer can be constructed using any suitable porous medium which typically has characteristics similar to the porous support materials.
An exemplary porous medium is conventional filter paper (Whatman, UK; or Schleicher & Schuell 410, Keene, NH). The porous medium is generally printed 10 with a pressure sensitive adhesive or ink by means of equipment and printing techniques well-known in the art. The printed pattern defines the capillary track because the pressure sensitive adhesive or ink inhibits fluid-flow through the printed poilions of the porous medium. The use of a pressure sensitive adhesive to print the porous medium has the added advantage of providing the adhesiveness for 15 applyi.,g the base or back laminate layers. The conventional method of deviceasse",bly involved cutting strips or pieces of the porous media and sar,d~iching that media between the back and base layers. With the present method, the roll of printed porous material is simply incorporated into the web process, thereby eliminatingcostly and cor,~pli~t~d pick and place operdlions. Printable inks may be formulated 2 0 to contain an assay reagent which is released from the printed layer as the test sample passes over the printed material. The filtering capacity of the porous medium in the capillary track may advar,l~geou~ly be used in the assay protocol.Moreover, different po,lions of the porous medium in the capillary track may be treated to modify the filter of l,~nspo,l cha~cteri~lics of the medium. In acldition, 2~ different portions of the porous medium in the capillary track may be treated to contain one or more assay reagent zones from which a reagent is released upon conl~-;li"g the llanspo,led fluid.
The use of a pril~ tle medium to define the capillary track also provides for limitless design opportunities in terms of the geometric shape and variable 3 0 thicknesses of the capillary track which characteristics may be used to control the rate at which the assay is performed. In addition, it simplifies device manufacture by oli",i"~li"g the need for additional layer materials and material handling, while enhanl,;.,g batch manufacturing procedures. The application of pressure sensitive adhesives and ink materials can be accomplished with any suitable method, including S5 but not limited to, rotary or flatbed screen printing, flexographic printing,lithographic printing, letterpress printing, rotogravure printing, or ink jet V094/09366 2~ 4~9~ PCI/US93/08751 printing. The devices may be printed in either a batch mode (one sheet at a time on either flexible or rigid film material) or in a roll with web processing methods (on primarily flexible film material). The printing processes can be accomplished with standard equipment. Exemplary processes are described in "The Printing Ink 5 Manual" by R.H. Leach; "Handbook of Thick Film Technology" by P.J. Holmes and R.
G. Loasby; or "Handbook of Thick Film Microcircuits" by Charles A Harper. Set upof the printer is within sl~ndar.l parameters and processes known in the art, or as described in the relevant instruction manual or in the above-mentioned texts.
Typical ink properties are also described and for purposes of the present invention 10 the characleristics of suitable inks are similar to the characteristics of the printable pressure sensitive adhesives.
In yet another embodiment as depicted in Figure 6 the sides of the capillary track are defined by a solid middle or core layer (24) of film material which islaminated between a first layer (22) and a third layer (26) using two layers of a 15 suitable adhesive material (28). The middle layer has a slot within its surface partially or co""~letely through the material, such that in co",' ..,alion with the first and/or third layers a capillary track (18) is formed. The adhesive material may also be die cut or printed to complement the slot in the middle layer such that the adhesive material does not form a part of the capillary track.
In an oplional "~I ric-lion of the device the area of the porous support around the immobilized reagent may be at least partially cG",pr~ssed. Figure 7 depicts an embodiment wherein a nitrocellulose material (20) has been cG",pressed in the area around the i",l"~bilized reagent site (22) to aid in the d;.ection of fluid flow through the porous support and reaction site.
2 5 In another embodiment, the device may include an additional absorbent material in contact with the porous support. Figure 8 illustrates an embodiment representing a device which includes an absorbent layer (30) surrounding the rt:aclion site (22) on the porous support (20). The absorbent material serves toincrease the liquid holding capacity of the device such that large test sample or 3 0 reagent volumes may be used.

The main body of the devices of the present invention may be formed of a non-wettable material such as a plastic material or a w~ ble material such as a porous material wherein at least a portion has been rendered non well~ble. The capillary track typically has a total length of from about 0.5 to about 6.0 inches preferably from about 0.5 to about 2.0 inches. The structural a"~nyer"ent of the ~ 21~97~
WO 94/09366 PCI~/US93/08--device is generally designed such that about 50 to about 1000 microliters of test sample may be used to perform an assay. Preferably, the device is designed such that about 100 to 500 microliters of test sample is used. It will be appreciated by those skilled-in-the-art that the design will be optimized as needed to provide for the use 5 of that amount of test sample required to perform the desired assay. The capillary track has a diameter suitable for the transport of such samples from the capillary track inlet to the capillary track outlet.
The test sample is introduced to the reaction zone in the porous support by means of the subsurface capillary track. The subsurface capillary track reduces the 10 time required for a unit volume application of test sample to wet the same surface area of the porous support. Because the test sample and assay reagents are delivered directly to the reaction site, the test sample is not required to first pass through a non-lea~,-lion site portion of the porous support. This advanPgeous outcome is described by the D'Arcy Equation.
The D'Arcy Equation expresses flow rate as follows:
h2=kt where h = distance of chromatographic flow k = flow constant t = time 2 0 Where A1 is equal to the area of a porous material in the shape of an elongated strip (A1 = h1 x w1), and A2 is equal to the area of the porous support in the shape of a square or circle (A2 = h22 ~) of the same thickness, the unit volume of test sample applied to each device format will flow as depicted in Figure 9. The linear uptake of a unit volume is subsl~"lially slower than that of radial uptake.
2~ The present invention also provides for the ability to simultaneously perform multiple assays while utilizing a very small amount of test sample, for instance, a single drop. Such a device, in assembled form, has a plurality of capillary tracks.
The device includes a sample application means which communicates with the inlet of the capillary tracks. The outlet of each track is in communication with a porous3 0 support. The individual porous supports are selectively impregnated with a specific binding member suitable for the detection of an analyte of interest. The number of tracks is not critical to the construction of the multiple track devices of the present invention. Alternatively, a single capillary track can include multiple outlets such that different outlets underlie different portions of a single porous support, wherein 35 the different portions of that porous support contain immobilized binding members for different assays.

V0 94/09366 ~-l44q~o PCI/US93/08751 The assay devices of the present invention include a porous support in liquid communication with a capillary track, which support is typically positioned adjacent to, and usually in direct contact with, the capillary track outlet. However, it has been discovered that the devices can include additional zones or layers between the capillary track and the porous support. Such zones may be used to further control the rate of flow between the capillary track and the porous support, may containancillary assay reagents or may be used to prevent or inhibit the transport of test sample interferents into the porous support.
Optionally, the flow rate per unit area of the capillary track can be gradually decreased along the general li,eclion of flow by gradually increasing the spacebetween the floor and the roof of the track along the direction of liquid flow. For example, flow may be decreased by gradually bowing the roof of the track upward and/or gradually bowing the floor of the track downward.
In an all~:",ali~/e embodiment of the present invention, a labeled reagent is posilioned in the interior of the capillary track to form a reagent zone. When a test sample is introduced to one end of a capillary track, the test sample and labeled reagent combine to form a test solution. The test solution is transported through the track. The rate of liquid flow through the capillary track may be cor,t,~JllEd at least in part, by means of the porous support positioned at the distal end of the track. It 2 0 has been found that a porous material, such as paper, may be utilized as the fluid flow control means to provide advantages in both manufacturing and pe,~ur",ance over the coating of the track interior with water-soluble materials such as polyvinylpyrrolidone (PVP).
All types of specific binding assays can be accom~"odated with a device constructed in acco,Jance with the present invention. Moreover, with the inclusion of all necess~ry assay reagents within the device itself, the assays may be madeessentially self-performing once the test sample has been added to the device. For example, a soluble reagent (such as a labeled reagent) is dried within the capillary track during manufacture and is solublized upon contact with the test sample. In3 0 other i"sldnces, a labeled reagent can be contained by a soluble or porous matrix which is posilioned within the capillary track itself. Suitable matrices which can release diffusive materials are well known in the art and include, but are not limited to, paper, sponge and glass fiber materials. In yet another embodiment, a reagent can be dispersed in a solution which is placed in the track.
3 5 The very small size of the reaction devices of the present invention advantageously allows for the rapid and convenient handling of a plurality of devices.

W094/09366 ~144g7~ :~ PCI/US93/08 A device can then be loaded into an aulu",alt:d appardtus which indexes and scans the individual reaction sites for the assay results and records this information forfuture access. The small dimensions of the device also provide for efficient use of sample and reagents. The present invention also provides for diagnostic kits 5 employing the present devices in combination with containers of assay reagentswhich are not incorporated within the device itself, for example, a test sample buffer solution or exl,d~;lion solution.

EXAM PLES

The following examples are provided to further illustrate embodiments of the invention and should not be construed as a limitation on the scope of the invention.

A ~ispcs-'-'e device, as depicted in Figure 3, was constructed from a w~; '~le base layer (22) (7 mil, hydromer-treated polyester; Film Specialties, Inc., Whitehouse, NJ), a die-cut adhesive core layer (24) (3 mil, double sided adhesive coated polyester film; Adhesives P~esearch, Glen Rock, PA), a laser-machined, non-20 wettable, adhesive, la,.,i..dte layer (26) (3 mil, single sided adhesive coatedpolyester film, Adhesive Research) and a microporous nitrocellulose pad (20) (5 micron pore size; Schleicher & Schuell, Keene, NH). The assembly of the v,~: ~lebase, die cut core layer and laser-machined laminate form a subsurface capillarytrack with an inlet (2) at one end and a small outlet (16) at the other. The 2 5 nitrocellulose membrane is laminated over the outlet to allow sample to dispense from the capillary track (18) into the membrane center thus avoiding linear chlol,,alug,dphy from the membrane edge.

J Human Chorionic Gonadol.. p-., (hC:G) Assay A disposable device was constructed substantially in acco,ddnce with the desc,i~lion of Example 1. Anti-beta hCG antibody was applied to the center of the nitrocellulose pad in a "+" pattern with the two bars intersecting over the outlet in 3 5 the laser-machined laminate. One of the bars included hCG to serve as a positive control for hCG-negative samples. Anti-alpha hCG antibody, and protein st~hili7ers~

~ O 94/09366 214 ~ 9 ~ ~ 21 PC~r/US93/08751 were absorbed on selenium particles (180 nm) to provide the labeled reagent for this sandwich assay.
Dry selenium conjugate pads were prepared by dipping glass fiber strips (Lydall, Inc., Rochester, NH) into a selenium conjugate solution and then passing the 5 material through a drying tunnel. Circles (approximately 0.023 inches in diameter) were punched from the material and held in a molded drop forming meansas shown in Figure 3a.
An hCG test sample (250 to 400 microliters) containing buffer or urine was applied to the assembly. A bolus of selenium conjugate was delivered to the track if 10 the drop forming means was held above the track to allow a hanging drop to form.
The drop fell onto the capillary inlet, filled the capillary track and was transported to the nitrocellulose pad and through the capillary outlet directly beneath the center of the immobilized reagent "+". As the solution was radially l,ar,spoiled in 360degrees through and from the immobilized reagent site, a visible signal was formed 15 at the reaction site in the form of a "+" if hCG was present in the sample, and in the form of a ~_n jf no hCG was present.

Strep A Assay A riisp~s-~le device was constructed substantially in acco,ddnce with the 20 des~;,i,ution of Example 1. Anti-Strep A antibody was applied to the center of the nitrocellulose pad in a "+" pattern with the two bars intersecting over the uptake hole in the laser-machined lar"i.,ate. One of the bars included a protein containing the immunodeterminate recognized by the anti-Strep A antibody and served as a positive control for Strep A-negative samples. Anti-Strep A polyclonal antibodies, 25 with protein stabilizers, were absorbed on selenium particles (180 nm) which served as the labeled reagent for this sandwich assay.
Dry selenium conjugate pads and drop forming means were constructed as described above. Upon ~pp':-~tion of the test sample, a signal developed at the reaction site in the form of a "+" if Strep A immunodeterminate was present in the 30 sample, and in the form of a ~_n jf no Strep A immunodeterminate was present.
Results Upon applying a test sample to the devices of the present invention, the average time for the sample to contact the nitrocellulose pad was five to ten seconds.
3 5 Depending on the test sample hCG concentration, signal could be seen as a ~+r in 30-40 seconds (250 mlU/ml) or between one and three minutes for test sample WO 94/09366 2 1 ~ ~ 9 7 6 PCr/US93/08~

concentrations of 5-25 mlU/ml. Similar results were found for high and low levels of Strep A immunodeterminate in a test sample. It will be appreciated by those skilled-in-the-art that these reaction times may be further modified through reagent optimization. A novel and unexpected aspect of this technology is that the 5 labeled reagent passing from the conjugate pad is concentrated in the first drop dispensed from the drop forming means into the capillary track. Typically, over 50% of the conjugate is concentrated in the first formed drop. Preferably, over 70% of the conjugate is concentrated in the first formed drop. In the most preferred form, over 90% of the conjugate is concentrated in the first formed drop. The 10 immobilized reagent is then subjected to a bolus delivery of labeled reagent with the test sample, and as subsequent test sample passes through the test device ~"rea.;led labeled reagent is cleared from the reaction zone. The resulting signal was thusenhanced as the backy,uund field of the immobilized reaction site changed from pink to white and the immobilized signal remained red.

A di_posable device was constructed from a wettable base (7 mil hydromer-treated polyester, Hydromer, Inc.), a die-cut adhesive core layer (3 mil, double2 0 sided adhesive coated polyester film (Adhesives Research) and a printed filter-paper top layer (S&S grade 410). A hole was punched in the printed part of the filter paper layer to serve as a sample entry means. The assembly of the wettable base,die-cut core layer and printed filter-paper top layer formed a subsurface capillary track with a sample uptake hole in the paper top layer where ink was not deposited.
25 The printed pattern in the track allowed sample to fill the track to a predel~r".i.,ed point. The printed pattern in the top layer allowed solution to travel within the track and react with reagents that were deposited in the wettable, non-printed areas of the top paper layer.

Glucose Test A glucose test device was constructed by using a di;,posal)le device as described in Example 3 and assay reagents for an enzymatic glucose determination. The color 3 5 forming reagent was 4-chloronapthol which was spotted onto the filter paper as 25 mg/ml acetone solution and allowed to dry. Solutions of glucose oxidase and ~'0 94/09366 . ~ PCI/US93/08751 ~14~9~ 23 horseradish peloxidase (Sigma, Inc.; St. Louis, MO) in a phosphate buffer were then spotted on the filter paper in the wettable regions containing the dried 4-ch'clrunapll,ol. Concentrations of glucose oxidase were chosen to allow the color rur",ation reaction to proceed at different rates between each wettable region. The 5 test was begun by adding a buffered solution containing glucose to the entry hole.

Results Upon sample application, the fluid flowed via capillary action through all of the wettable regions and color lur",ation began. Rates were monitored using a light 10 trans",ission device. Each wettable area was monitored independently.

A ~i;pos~l.le device and ~agenl~, as described in Example 3 and 4, were used 1 5 to make a glucose test device. The wettable base layer had a pattern of hyd~uphcb s ink printed down the capillary track. Upon application of a buffered solution containing glucose, the track filled down to the hydluphobic ink section of the track.
The device was attached to a rotating means (e.g., Dremel tool) through a hole in the center of the device. Upon applying centrifugal force to the device, the sample was 2 0 forced past the hydrophobic track section and into a holding area of the track. Upon release of the centrifugal force, the solution travelled up the track into the reagent area where the glucose-deter",ining ~actions began.
Blood sepdldtion was accor.,~' s5~ed in the device by apply 9 a whole blood sample to the sample uptake hole as described above for the buffered-glucose 2 5 solution. Upon application of centrifugal force the blood cells separated out in the bottom of the track past the hydrophobic track area. With the release of the centrifugal force, plasma decanted away from the CGIll,ud~ d cells and travelled up into the reagent portion of the test where the glucose-determining lea~,-lions began.

A ~icpos~hle device is constructed by using a film material as the first or base layer. The film may also be used as the third or top layer. The top layer is cut by a laser to form two holes which provide the capillary inlet and outlet ports once the device is constructed. The bottom surface of the third layer is screen printed with an aqueous adhesive leaving an unprinted area between the two holes which W O 94/09366 2 1 4 ~ 9 7 ~ PC~r/US93/08 -defines the sides of the capillary track. The bottom surface of the third layer provides the top of the capillary track. The adhesive material is cured, and the base layer is placed over the printed material. A suitable amount of pressure is applied to adhere the the base layer, thereby forming the bottom of the capillary track.
A porous support is positioned on the upper surface of the top layer substantially over the capillary track outlet such that the outlet is in fluid flow communication with the porous support. An assay reagent may be immobilized on the porous support before or after the support is attached to the film.

A disposable device is constructed substantially in accorddnce with the technique described in Example 6, with the exception that an adhesive is applied to both the first and third layers.

A di_pc~'e device is constructed suL,stantially in accGI.lance with the technique described in Example 6, with the exception that a two-sided adhesive 2 0 material having suitable release liners and a slot to define the sides of the capillary track is applied to either the first or third layer to form a middle layer. For example, a release liner is removed from one side of the adhesive l"at~rial and aligned over the base layer, and pressure is applied. The second release liner is then removed, the third layer is aligned over the adhesive, and pressure is applied to 25 cG~"plete the construction of the capillary track.

It will be appreciated by those skilled-in-the-art that the concept~ of the present invention are applicable to various types of assay configurations, analytes, 3 0 labels and device nldl~ The embodiments described herein are intended as examples, rather than as limitations, of assay devices using subsurface flow. Thus, the desc-ri~lion of the invention is not intended to limit the invention to the particular embodiments ~isclosed, but it is intended to encompass all equivalents and subject matter within the scope of the invention as described above and as set forth 35 in the following claims.

Claims (25)

What is claimed is::

1. A device for performing a specific binding assay to determine the presence oramount of an analyte in a test sample, comprising:
a capillary track having an inlet and outlet, wherein said inlet receives test sample or test solution, and said outlet is in communication with and directs test sample or test solution to;
a porous support containing an immobilized reagent which binds a member selected from the group consisting of the analyte, an ancillary specific bindingmember and a labeled reagent, wherein said outlet port is disposed beneath said immobilized reagent in said porous support.

2. The device according to Claim 1, wherein at least a portion of said porous support is compressed in the area surrounding said reagent, wherein the compressed area directs the flow of test sample or test solution through said porous support.

3. The device according to Claim 1, wherein said labeled reagent is contained within said capillary track.

4. The device according to Claim 1, wherein said labeled reagent is contained bya reagent matrix within a drop forming means which is in communication with saidcapillary track.

5. The device according to Claim 1, wherein said labeled reagent is contained bya porous reagent matrix within said capillary track.

6. The device according to Claim 1, further comprising a test sample well communicating with said capillary track inlet.

7. The device according to Claim 1, wherein said capillary track outlet directs the test sample to a bottom surface of said porous support directly beneath saidimmobilized reagent.

8. The device according to Claim 1, wherein said capillary track comprises at least a first and a second capillary track wherein said first capillary track contains a labeled reagent, and wherein said second capillary track directs test sample to said porous support at a faster rate than does said first capillary track, whereby at least a portion of the analyte is transported to the immobilized reagent prior to saidlabeled reagent reaching said porous support.

9. The device according to Claim 1, wherein said capillary track is constructed from a first layer and a second layer of liquid impermeable materials having opposed interior bottom and top surfaces, respectively, which define said capillary track.

10. A method for detecting the presence or amount of analyte in a test sample, comprising the steps of:
a) contacting the test sample to a capillary track having an inlet and outlet, wherein said inlet receives test sample or test solution, and said outlet is in communication with and directs test sample or test solution to, a porous support containing an immobilized reagent which directly or indirectly binds the analyte, wherein said outlet port is disposed beneath said immobilized reagent in said porous support, and wherein a labeled reagent directly or indirectly binds to the analyte to form a labeled complex on said porous support in relation to the presence or amount of analyte in a test sample; and b ) detecting said labeled reagent in said porous support to determine the presence or amount of analyte in a test sample.

11. A method for detecting the presence or amount of analyte in a test sample, comprising the steps of:
a) contacting the test sample to a capillary track having an inlet and outlet, wherein said inlet receives test sample or test solution, and said outlet is in communication with and directs test sample or test solution to, a porous support containing an immobilized reagent which directly or indirectly binds the analyte, wherein said outlet port is disposed beneath said immobilized reagent in said porous support, and wherein a labeled reagent competes with the analyte to bind said immobilized reagent thereby forming a labeled complex on said porous support in relation to the presence or amount of analyte in a test sample; and b) detecting said labeled reagent in said porous support to determine the presence or amount of analyte in a test sample.

12. A method for detecting the presence or amount of analyte in a test sample, comprising the steps of:
a) contacting the test sample to a capillary track having an inlet and outlet, wherein said inlet receives test sample or test solution, and said outlet is in communication with and directs test sample or test solution to, a porous support containing an immobilized reagent which competes with the analyte in directly or indirectly binding to a labeled reagent, wherein said outlet port is disposed beneath said immobilized reagent in said porous support, thereby forming a labeled complex on said porous support in relation to the presence or amount of analyte in a test sample; and b) detecting said labeled reagent in said porous support to determine the presence or amount of analyte in a test sample.

13. A kit for performing a specific binding assay to determine the presence or amount of an analyte in a test sample, comprising:
a) a device involving a capillary track having an inlet and outlet, wherein saidinlet receives test sample or test solution, and said outlet is in communication with and directs test sample or test solution to;
a porous support containing an immobilized binding reagent, wherein said outlet port is disposed beneath said immobilized reagent in said porous support; and b) instructions for the performance of the assay.

14. The kit according to Claim 13, wherein said device further comprises a test sample application pad containing a labeled reagent, wherein said pad is in fluid flow contact with said capillary track inlet.

15. A device for determining the presence or amount of an analyte in a test sample, comprising:
a capillary track having an inlet and outlet, wherein said inlet receives test sample or test solution, and said outlet is in communication with and directs test sample or test solution to a porous support;
wherein said capillary track is constructed from a first and a second film layer, defining a top and a bottom of said capillary track, and a printed core layer applied to said first or second layer in a reverse image of said track and having a thickness which defines side walls of said capillary track; and said porous support containing an immobilized reagent which directly or indirectly binds a labeled reagent in relation to the presence or amount of analyte in the test sample, wherein said outlet port is disposed beneath said immobilized reagent in said porous support.

16. The device according to Claim 15, wherein said printed layer is an adhesive material.

17. A method for constructing a capillary track, comprising the steps of:
a) applying a printable material to a first film layer thereby forming a core layer having a thickness which defines the side walls of the capillary track, wherein said printable material is deposited as a reverse image of the capillary track on said first film layer; and b ) adhering a second film layer to said printable material, thereby forming thefourth side of the capillary track.

18. The method according to Claim 17, wherein said printable material is selected from the group consisting of an adhesive, an ink and a dielectric material.

19. The method according to Claim 17, wherein said printable material is a pressure sensitive adhesive.

20. The method according to Claim 17, further comprising the step of treating the capillary track to modify the hydrophobicity, hydrophillicity or selective biocompatability of said film layers.

21. A method for constructing a capillary track, comprising the steps of:
a) applying a fluid repellant printable substance to a porous material thereby impregnating said porous material wherein a non-impregnated region defines two sides of the capillary track and the thickness of said porous material defines the height of the capillary track; and b) adhering a first and a second laminate layer to said porous material thereby forming a top and a bottom of the capillary track.

22. The method according to Claim 21, wherein said fluid repellant printable substance is an ink.

23. The method according to Claim 21, wherein said fluid repellant printable substance is an adhesive.

24. The method according to Claim 21, further comprising the step of treating porous material to modify the hydrophobicity, hydrophillicity or selective biocompatability of the capillary track.

25. A device for determining the presence or amount of an analyte in a test sample, comprising:
a capillary track having an inlet and outlet, wherein said inlet receives test sample or test solution, and said outlet is in communication with and directs test sample or test solution to a porous support;
wherein said capillary track is constructed from a porous material selectively impregnated with a fluid repellant printable substance wherein a non-impregnated region defines two sides of said capillary track, and wherein said porous material forms a core layer between a first and a second laminate layers which form a top and a bottom of said capillary track; and said porous support containing an immobilized reagent which directly or indirectly binds a labeled reagent in relation to the presence or amount of analyte in the test sample, wherein said outlet port is disposed beneath said porous support.