CA2197061A1 - Printed fluid transport devices - Google Patents

Abstract

A backing sheet (101) is provided with a pattern of pathways (131, 132, 133) of e.g. silica or cellulose by a printing process (e.g., screen printing). There may be multiple pathways leading from an eluant application region (117) to a detection zone (116) and thence to a waste reservoir (118). Different pathways may have different fluid traversal times because they differ in length and/or material (e.g., nitrocellulose for slow traversal and fibrous cellulose for rapid traversal by an aqueous liquid). Thus analyte and reagents deposited at depots (112, 113) on different pathways are sequentially delivered to the detection zone. Reagents may be applied by printing. The detection zone may have an electrode assembly (105), also applied by printing, for detecting the effects of analyte.

Description

~ WO96/05510 2197061 r "~ ~ ,~

~R~NTED FLUID TRANSPORT DEVICES
INTRODUCTION
TEcH~ncAL Eb~
The present invention relates to printed fluid transport devices, c~
and methods for their r ' C~ and use as assay platforms. The invention ~uLi~.ulcul.y relates to analytical sensor devices, e.g. for biological samples, such as d~ LI~ ~ ~ ' assay devices.
BACKGROI)ND
The ~ IU~ of a rapid, simple method for carrymg out a range of 1...~.1.. .";. 1 assays would greatly enhance the field of ~ gnrctirc. ~uLil~uk~ in areas such as the health care, el..h, ~ ~ and food industries. For effective"on-site" use, the device should be operated with the minimum amount of manual I5 l.. r.. :l.. /1-1;ll and be suitable for use by non-specialist operators. At present the majority of assays which are performed involve several steps which are often time rrnnl~ning, laborious and require technical trainir~. Sequential additions of assay ul~ (e.g. reactants, substrates, etc.) is an inherent feature of such assays, requiring technical skills and, in many instances, a degree of manual dexterity.The ability to detect and monitor a number of analytes in a given sample using a single test device would facilitate an , ~ IUI~ in the use of such diagnostic tests.
To ameliorate the problems outlined above. devices have previously been descnbed that utili~e liquid flow channels to deliver a timed sequence of reagent additions to sample. GB-B-2 231 150 describes the use of a device CU~ g tvo flow cha~mels leading to a common channel. all formcd of a single sheet of porous material, e.g., filter paper. One of the channels is of greater length so that it delivers the liquid with a relative delay to the common channel. This allows for the sequential timed delivery of reagents, through wpillary liquid flow, to a common site. It is not easy to provide elaborate systems of channels. If a long delay is required, a channel must be made very long amd hence, very; v~ ' ~ This is illl.UU~lUl~lli, and providing two or more chalmels with long delays tends to be; 'l". ~ Ir R. Bunce et al., Anal. Chim. Acta, 249, 263-269 (1991) disclose such devices in which pathways are delimited on a sheet of filter paper by printing L ~ ~ul)hul~i~, regions into the paper using a wax-resist batik technique, so that the non-printed regions provide the wo s6/o~lo 21 g 70 6 ~ P~

pathways. Such methods are of Irmited practical utility in the ,.. ,.. r~ n.~ of assay kits.
US 5,194,133 describes a device that can be used for the analysis of a sample fluid contair~ing a substrate. A single channel is formed by ~ of a suitable material. .'S.. h~ 1y~ the channel is filled with a material capable of acting as a .,hl~ ' separation medium. Biological material can be hl~ol~. I intothe ~.hl~ ' matrix, allowing controlled reactions to occur. Detection is by means of cl~Llu~L~llfi~al sensors located at defined points along the channel.
W. Schrarmn e~al., 'Biosensors '92 P",c~e.,;,.~ published by Elsevier Advanced Technology, Oxford, England) discloses an i.. ,~ y device baving a strip of cl~ g~ material with a detection zone with an ;-- -- --l.;l; . ~i antibody and, upstrearn thereof, a deposit of a reagent (allaly L~,-CI~y~ . conjugate) . A porcelain chip has a printed electrode located in contact with the detection zone. In use, analyte solution passes up the strip and carries the reagent to the detection zone. Analyte and a~ ,~yllll. conjugate compete to bind to the antibody. An enzyme substrate is provided and generates an ~,k,~,LI~a~,Liv~: product at the detection zone. The electrode system provides a signal whose strength varies inversely with the amount of analyte in the analyte solution.

SUMMARY OF THE II~ENT10~
The present rnvention provides for a fluid transport device comprisrng a backingsheet and at least one fluid pathway defined by a pattern of material through which fluid can flow and printed onto the backing sheet. Such pathways can be referred to as guidance pathways that guide fluid flow parallel or 1' l~ -1;- 1~ to the plane of the backing sheet. Desirably, the pathway(s) fluidly ~ with a detection zone, and the device further includes an electrode assembly which provides an electrode means disposed in relation to the detection zone to enable the detection of chemical species. Preferably, the electrode assembly is applied by a printing technique.
The guidance pathway material will typically have capillarity and be made with a bibulous material and/or a cl.l, _ ,' medium.
The present invention further provides for a method of ,.~ .g a fluid transport device comprising providing a backing sheet and printing a material on the ~ wo s6/osslo 2 1 9 7 ~ 6 1 I ~

backmg sheet that provides at ieast one fluid pathway. Preferably, the method comprises providing a backing sheet and, in either order, (i) applymg to it by a printmg technique an electrode assembly; and (ii) applying to it by a printmg technique a material which provides at least one fluid pathway disposed so that a detection zone thereof overlies or underlies at least part of the electrode assembly.

BRIEF DESCRIPTION OF TIIE DRA~1VINGS
Some ~ o 1; . ~ of the invention are further described below, by way of e~ample, with reference to the ~Vlll~ou,~illg drawings.
EIG. Lis a plan view of an assay device which is a first ~.. -'.. >l: -:
FIG. 2 is a section of II-II in FIG. 1; FIG. 3 is a section of m-m in FIG. 1.
FIGS. 4-6 are plan views of the second, third and fourth ~ l.v.l;. ~b EIG. 7 is a plan view of a fifth I ~, FIG. 8 is a section on VIII-VIII
in FIG. 7.
~IGS. 9 and 10 are plan views of the sixth amd seventh e .I,o.l; .
~IG. 11 is a plan view of a test card.
FIG 12 is a three .1 ,. .~ cross section of a portion of a detection zone shown in FIG 11.
EIG. 13 is a control current measured using a test card in the absence of an analyte.
FIG. 14 is a current measured usmg a test card in the presence of an analyte.

DESCRIPTION OF SPECIFIC EMBODIMENTS
The present invention provides for printed fluid tr;msport devices. Such devicesfmd application where fluid flow must be directed or timed. Printed fluid transport devices can be used as assay platforms to permit convenient and rapid Ill~ ul~ llb of analytes, as well as for the ec~ mir~l and efficient mass production of disposable test cards. "Test cards" refers to printed fluid flow devices that can be used for assays of analytes m fluids, ~alL~,uhllly m small and convenient assay kits. The printed devices amd techniques disclosed herem are particularly useful for the lll~lur~ul~ of 21 9 7 ~ 6 ~ r wos6/osslo r~.,~ . /L~

test cards that use an clc~uu~ uical deTection means, such as, but not limited to, printed electrode assemblies discussed herem.
The fluid transport devices comprise a backing sheet and at least one fluid guiding pathway deflned by a pattern of material through which fluid can flow. As illustrated in the ~GS. of various l ,.. l,n l;,.. ~I~ of the invention, the fluid guiding pathways permit fluid to be transported from a sample application site TO at least the detection zone. Typically, as illustrated in various , 1,o.1;,., ,'~ disclosed in the PIGS., such as ~GS. 1-10, fluid transport through printed fluid guiding pathways will be parallel to the plane of the backing sheet, with minimal fluid transport P . 1~....1,...1A .
to the plane of the backing sheet. Usually, fluid transport ~ 1~ -T;- ~ to the plane of the backing sheet (provided by the buffer or sample stream in the fluid guiding pathway) will be less than 10% of the fluid transport parallel to the plane of the backing sheet. Fluid transport ~ ..1A. to the plane of the backing sheet will increase in relation to fluid transport parallel to the plane of the backing sheet in some regions of the fluid guiding pathways, such as thicker layer regions in waste zones that contain a large amount of wicking rnaterial in relation to the reagent, liquid application and detection zones.
Generally, printed fluid guiding pathways provide directional fluid transport through a printed fluid guiding pathway. Directional fluid transport relates to net transport of fluid through the material or materials forming a printed fluid guiding pathway and between at least two points along the printed fluid guiding pathway. For example, buffer enters a liquid application zone and is transported through the matnx or materials forming the printed fluid g uding pathway in a direction away from the initial site of liquid Apr1i~tinn i.e., directional transport. Typically, printed fluid guiding pathways will provide directional fluid transport through a detection zone during detection of an analyte. In most instances, fluid transport is not stopped in the fluid detection zone. In some ~ of the invention, such as those with certain types of flow A~ .,.s..~, as dlscussed herein, fluid transport will be assisted by non-matrix fluid transport regions that have capillary flow parallel to, or in generally the same direction as, the fluid transport through the printed fluid guiding pathways.
Many ~ . l.o.~ of the invention will lack non-matrix fluid transport regions. ~uch non-matrix fluid transport regions mclude those comprised of a capillary track, tube, 2197~61 j,, i !~
6/05~ ~ r_.,. . ,~
5.
channel or slit formed by two planar surfaces (such as glass) ~ u~fl..~ly distanced to permit capillary flow; such non-matrix fluid transport regions are recognized by the air or gaseous gap between two surfaces that permit capillary flow of the fluid.The printing oechniques disclosed herein provide for any desired patoern of fluid guiding pathways. Generally, a defned prinoed patoern of a fluid guiding pathwayrefers to a patoern of prinoed maoerial that will permit directional fluid transport.
rUILL~,I-uulc different "inks" or "pasoes" can be applied to produce fluid guiding pathways that can include fluid guiding pathway portions having different flow properties. Generally, the oerm pasoe relaoes to maoerials at a of lû to 60% (w/v) typically in waoer or an organic solvent. Preferably pasoes are used in the 20 to 40% (w/v) range. Printing c~ usually range from I to 25 poise at 25~C, preferably from 4 to 18 poise at 25~C and more preferably at 6 to 12 poise at 25~C. The oerm "flow properties" may refer to properties affecting liquid flow along a fluid guiding pathway or to properties affecting the CU~ UI~ C of soluoes by carrier liquids as in cll., O ~' /. Maoerials can be used to change soluoe/solvent RF
values. RF value refers to the ratio of the distance moved by a particular soluoe to that movcd by a solvent front. For example, printed fluid guiding pathways can be ~ll~ulur~c~ulcd with a printcd layer or layers comprised of two or more different maoerials ("multi-maoerial") providing different raoes of fluid transport. Multi-material fluid guiding pathways can be used when it is desirable to modify reoention times of reagents in fluid guiding pathways (such as reduction of retention times, as discussed herein for conjugaoes, or to increase retention titnes to allow reactions to occur, e.g., antibody-analyte ;"t..~ '), prevent non-specific binding, improve the assay sensitivity or improve the ~ ludu~,;h;liLy of analyoe assays. Such multi-rnaterial fluid guiding pathways can be c . . ~l ., t~ .1 as a l S uO~ . ~ prinoed layer, where a plurality of maoerials are mixed together and then prinoed. Multi-material fluid guiding pathways can be also made by printing multiple layers of materials with different flow properties on top of one another. Multi-material fluid guiding pathways are examples of some types of flow r ' ' further described herein.
Prinoed fluid guiding pathways can be provided with regions containing reagent substances, by including reagent substances in the "inks" used to produce them or by a subsequent printing soep. A preferred printing oechnique to use is screen printing.

.. . , , . = ~

2 ~ 9 7 0 6 1 ~
WO9610~SIU ~_I/L.
6.
Such regions are usually referred to as reagent zones. Non~ llr~ d reagents are preferably printed on materials thal release the reagents quickly to the fluid flow to allow for rapid hydration of reagents and less reagent retention. Inert materials are especially suitable for tbis purpose.
Screen printing techniques are also preferably used for printing fluid guiding pathways, ~;u~ ,liviLy strips, electrodes and their associated conduction tracks, and for printing reagents at specific fluid guiding pathway locations. Air-brushing may also be used to print fluid guiding pathways. Ink jet printmg cam be used for printing reagents but is not generally suitable for depositing particulate material greater than 20-S~L.
The present invention makes available an improved technique of controlling the flow of liquid and the subsequent timing of reagent delivery in an assay device.GP~ mPtri~ ly defined and printed fluid guiding pathways cam be used to guide tbe fluid flow and cam be made of disparate ~,1.., ~ , ' materials (such as cellulose, silica gel, silica includmg silica modified to increase the hJdIUIJIIUb;~ Y of the silica, starch, agarose, alginate" ~ or polyacrylic polymer gel and mixtures of such materials) to facilitate ~ ,.ul retention of individual reagents and to assist and enhance the sequential delivery of the assay l , Different physical, as well as chemical, properties can be used to affect transit times, e.g., densely packed small particles produce longer transit times than loosely packed larger particles of the same material. Regularly shaped particles can be deposited to form a closely packed regular structure which facilitates the passage of ~,.u~ material.
Generally, printed fluid guidmg pathways, especially screened printed fluid pathways, comprise thin layers of matrix sufficiently thick so as to provide enough fluid for a detectable signal and to ensure uniformity. Typically, a single printed layer can vary from 5 to 500~ m thickness and preferably, 5 to 100~ or 40 to 100~ in thickness and more preferably 40 to 100~ m thickness. Fluid guiding pathways canbe made of multiple layers or single layers wherem the total layer thickness is usually 50 to 500,u and preferably, 75 to 300~.
"Silica" includes silica derivatives, which can be selected to meet the , r.~ c~ of a particular assay~ The average silica particle for prmted fluid guidmg pathways usually ranges from 2~L to 100~1 and preferably, 5~ to 25~L or 10~ to 50~.

2l97Q6l t~
. ~ WO 96/05510 ' i! .'~ ~ P~

For example, there are cu~ll."~,~lly available silica-bearing groups to which can be ;". ,~1, t;, .I such as silicas with h~llu~ullob;~, groups, ~ ti~ul~uly phenyl, benzyl, two aromatic rings and substituted phenyls, as well as ODS2-Silica (5~) and Spherisorb S5-Epoxide (both from Phase S~pDr~ti~-n~). Phenyl silica generally ~ S refers to a phenol group attached a silane group via a short carbon linkage, preferably, 1 to 4 carbons in length and more preferably, by a single carbon. Phenyl silica is preferred for printing fluid guiding pathways leadmg up to or adjacent to the detection zone, and more preferably, phenyl silica with am average particle size of 5~ is used.
Generally, the role of silicas with h~Lu~llob;c groups, such as phenyl silica, is to act an ' 1j7Dt;~r~ matrix fo} reagents, such as a capture antibody, amd as a matrix that has a minimum of non-specific conjugate binding when blocked.
Derivatized silica is typically used with a cellulose derivative for printing c~ A silica paste is made with a solvent at a .~ of 10 to 60%
(w/v) and preferably, 40 to 60% (wlv). Often the solvent is water. Other solvents can be used such as methanol, ethanol, propanol, butanol, 2-bu~v~.y~llyl acetate (99~O) and 1-(2-elLu~ alu~y) ethyl ester, either individually or in ~ ' ~ Pastes from silica with h~U~ UIJ;~ groups such, as phenyl silica pastes, are preferred. The cellulose derivative mixed with the silica paste is preferably h~dIU~ CIIUIO~ and typically mixed at a ratio of .2:1 to 3:1 ratio (w/w of paste to hy~u.~y~;llyll,~llulOse solution), preferably, .5:1 to 2:1 (w/w) and more preferably, 1:1 (w/w). ~Iydlw~y~lyluellulose solutions are typically aqueous-based, but other solvents as Icnown in the art can be used, so long as they are compatible with the printing process of choice.
Hydlu~ lyl~ellulose solutions are typically 1 to 25% (w/v), preferably 5 to 15% and more preferably, 9 to 11%. Calcium phosphate is the preferred bmder, although calcium sulphate, gelatme and other binders as known m the art can be used.
Typically, binder U~ are 5 to 40% (w/w) with a preferred c~ ... of 8 to 14% and a most preferred cn ~ of 12.5% (wlw). Mi~lu..~vh.~ can be used to erh~mce h~- ~b. ~ of the paste. Such derivatized silicas cam be blocked with blocking materials as described herein and taught m the art. Proteins can be " ' on dervatized silicas, pAlli~ulally phenyl and epoxide silicas, such as proteins required for the assays described herein and known in the art. For example, 219~61 WO 96/OS510 ' Y~ .'C /L3~
. ~,,~ i,,.,g~, capture antibodies, otber capture ligands or a member of a receptor pair can be ....
"Cellulose" includes cellulose derivatives. For example, nitrocellulose may be used to provide a patbway portion that tends to }etard (polar) solutes, while fibrous cellulose may be used to give rapid flow. Variations rn nitro-cellulose flow properties are well-known in the field of ~,lu~ y . Cellulose acetate is ~ ,ulAfl~ useful for exclusion .. "I " A ,.. ~, as discussed herein. Cellulose acetate usually has am acetyl content of 20 to 90%, and preferably 30 to 50%.
Using printing techniques such as screen printing, well-defmed, complex and rc,uludu~ lc pathway patterns can be laid down on an inert backing material. Porous material can be deposited in layers, by successive printing steps, to achieve desired pathway thickness for desired flow rates. A thicker pathway portion tends to exhibit a lower flow rate. Different fluid guiding pathways and/or different portions of fluid guiding pathways can differ m thickness dependent on, for instance, the desired timing of solutes at a merged zone.
For screen printing, screens are as selected in relation to the size amd viscosity of the particulate c"",~ being prmted. Low viscosity generally requires higher counts per inch for uniform printing of layers. For pastes, larger mesh size (i.e., low-coumt per inch) screens are usually better. Generally, screen hole size is 2 to 3 times larger than the average particle size being printed. Screens are often coated with an emulsion to protect them from the solvents used in the printing c.,.,.~
A backing sheet may comprise thin plastics material, e.g., PVC (Polyvinyl Chloride) sheet. Backing sheets can be provided with a more polar or h.ydlu~Jlulic coating to enhance the adhesion of the fluid guidance pathway material. A resin cn~rrri~irn loaded with carbon (e.g., Electrodag 423SS graphite-based polymer thick fllm ink, Acheson Colloid Co., Plymouth, GB) may be used to coat backing sheets not relying on CIC~ LIU~ ,.ll;.,dl detection. A cull~ iu~l emulsion paint may be generally suitable for both ele~ u~.h.,ul;~,al and nu~. eL.~ ucll, lll;,,AI devices, such as Dulux or other emulsion paints known in the art. An emulsion paint is a water-thinnable paint made from an emulsion or dispersion of a resin (generally synthetic) in water. The resin may be polyvinyl chloride, an acrylic resin or the like. Other backing sheets, e.g., of glass or other ceramic material, may not require coatings. Backing sheet - ~ "
.~ WO96/0551~ 21g7061 r~l"~ f~J

materials include polyurethane, polyester, poly, l , pol~lJu~ldtJpolyester blends and polyalkylenes, either singly o} m ~"" h: ~;""~ thereof. Titanium oxides canbe blended with such rnaterials to improve p.. r.... ~ f ~ ,.. t~ , U.s. Patent 5,238,737, the methods of which are herein ~"~u-l,, ' by reference.
For electrodes, a variety of electrode assemblies can be used, imcluding two-and tbree-electrode-based ' ' Two-electrode assemblies are preferred because of the ease of operation and printmg of the electrodes and conduction tracks. Working electrodes are preferably catalyzed carbon based, such as rhodinized carbon electrodes like MCA 4 (MCA, ~:lmhntlfo~-, United Kingdom). Other electrodes based on a c~ . of carbon and transition metals, preferably platinium, can be used to faci,itate low potential oxidation of enzyme product. Such electrodes help reduce b.l.,hyluu--d noise associated with measuring assay products, such as H2O2, at higher voltages (600-700mV versus Ag/AgCI reference electrode) required by other types of electrodes, e.g., pure carbon or pure platium group based electrodes. Generally, it is al~ ~ to i... ~u-~!~ in a two-electrode system an auxiliary/reference of sufficient size so as not to Irmit the current required for the ~ For reference electrodes, Ag/AgCI is typically used in the range of 10 to 90% Ag, although fordisposable test cards, 10% is preferable. Electrodes are preferably printed so as to maximize detection, for instance by locatmg the electrode broadside to the fluidtransport, as well as creating an hl~ld;f;~ lg pattern between the reference andworl~ing electrodes.
Cf~ rting polymers in the form of a layer or film can be used in c~
with the printed electrode ~ccrmh~ s Usually, the conducting polymer is a h~t~.u~u.ll~Li~ conducting polymer, like a puly,uyllvle, a poly( I i~l~ylene vinylene), a ,uuly(rulyhllc;vmylene),apolyfurmorapolyUIiuAuhc~lt. Mediatoro,.. ,l.ù.. ,.l~canalso be used with printed electrode ~ccf mhli~C such as, but not limited to, ferrocenes, ferrocene derivatives, non-ferrocene mediators (e.g., carbon-boron .~ ,u~
(including the carboranes)), Viologens (N,N'-diaL~cyl of or diaryl derivatives of 4,4'-bipyridyl), one~ conductors (mcluding the salts of TCNO), phenazine dyes (includingphenazinem~fhn~ r' andphenazine ;' ~I' )andm~t~ .l"lil~s (including ~,.UI,~vlll.,-C) and transition metal complexes, ~ il,ul~ly those in which the mediator comprises at least one or two organic rings.

2197o61 . . a~
WO 96/05510 ~ r~l"
10.
For corlduction tracks, Ag is typically used in printing , The size of these tracks is minimized to reduce noise created by increase surface area.
Typically, such tracks are shrouded by insulation material, which is often coated or in many cases, printed. Insulation material is usually a matt vinyl emulsion paint. Other insulation and conductive track materials may be used and known in the art, so long as they can be adapted for use in printing ~ described, especially for screen printing.
Culld~ ivily strips can be used with test cards described herein to trigger electrode monitoring of a reaction product. Typically, col,ll..~liv,l.y strips are printed along the length of the test card and made from a graphite-based ink.
The methods of l l~ulurh..iulc disclosed herein are applicable to the mass production of devices. Universal tes~ cards designs are permitted because printing screens with universal patterns can be used for a myriad of different assays by simply changing the reagents, and possibly the electrodes, but without changing the pattern of the fluid guiding pathways. The test card design may also be varied as required, by simply ~ ~ alternative screens for a~lulJlh~i~, parts of the production process.Prmting techniques are universally applicable to tbe deposition (in precise locations) on the fluid guiding pathways of reagents such as: biological material(s) (e.g.,antibody, labelled antibody, antigen, labelled antigen, antibody fragment, enzyme, cell receptor, mtact cell or nucleic acid), clc~ u~.h~.llli~,~lly active compoumd(s) e.g., mediators such as ferrocene, tetrathiafulvalene and Meldola blue) and necessary unlabelled or labelled substrate(s), e.g., glucose.
The detection zone can be made to ~..., .. ""l,t a variety of optical detection methods, mcluding visual, fluu-u u.,i i." cr~ , reflective, ~ and those methods based on absorbance and l" .~ .:'l- .. e. The assay techniques discussed herein and known in the art can be combined with the use of printed fluid guidmg pathways to optically measure many types of analytes, mcludmg those discussed herein, including organisms, cells, proteins and small molecules, such as therapeutic drugs, drugs of substance abuse, steroids, and naturally occurring hormones. For many optical methods, such as visually based assays, printed fluid guiding pathways can be used m detection zones to locate reaction products for easy detection. For example, thecapture moiety of a capture assay can be immohili7~d m the detection zone to allow WO 96/05510 ~ 1 ~ 7 0 61 r~

detection of a 1 , ' '- reaction product, such as a colored product. For assays based on the absorbance or LluuluA~i~r,~i spectra (or amplitnde at a particular hr ~r~ l) of a chemical species, the detection zone can provide for Ll~sy~uc~
windows in the backing sheets that permit light to be transmitted or focused through S the backing sheet amd the fluid being ~ncpArrto~l Such windows optionally lack materials used for the printed fluid pathway. If materials for a printed fluid pathway are printed over the window area, such materials should be transparent. Al~.~ ly, fluid guiding pathways cam be printed to form a series of tracks with windows between each track and the tracks being lly,ulul '~, distanced to permit capillary contact between the trachs and fluid movement across the windows. Light scatter from thetracks can be subtracted out as hch~;luulld noise prior to the monitoring of an optical signal generated by a chemical species. Spectral analysis of signal that passes through the window or windows will be ycuLi~ulAIly useful for identifying an optical signal related to a chemucal species.
Optical assemblies described herein can be combined with assays described herein and known in the art. Optical assays are preferably used with the following assay c r ~1"", ~ 1) am antibody or analyoe bmding moiety linked to a color change cl mrr~ont such as horse radish peroA-idase (y~u--id~) (using e.g. 3, 3', 5, 5' L~,u~ull-Lllyllr~ IB), 2, 2rr azinobis (e-ethyl h. . ~ 6-sulphonic acid) r" salt (ABTS), ortho-yh~ " (OPD), 4-chloro-1-napthol (4-CN), 3, 3~ .r~ u~ (DAB), or 3 amino 9-ethyl carbazol (AEC));
alkaline pl~ (using e.g. para- uy~ .,yl phosphaoe, di-sodium salt (PNP), nitro blue t~ ;.A,..l;...,. chloride (NBT), 5-bromo4-chloro-3'-- ' '~1 ' , ' para-toluidine (BCIP), iodulliLla~ r~ m violet, NADP, diaphorase red (formazan), r~- ~y~ . , red, or fast red, napthol AS-MX phosphate)); ~-g;ll~rAt~rcir~co(usinge.g. ortho-lliLIuyll ,-lyl-B-D g_L~,Luyy ~ ~ . ~;-bromo-4-chloro-3-indolyl galA~Luyylcu~u~r;rle); glucose oxidase (using e.g. glucose, po ~Yi~ c~, ortho-dianisine hyLuulllùlidc (Hl02generated converted by EIRP), and ~ I;llr, glucose,peroxidase, 3, 3, 5, 5-tetramethyl berlzidine (TMP) (H2~2 generated converted byHRP); urease (usmg e.g. urea, hyyu.Llulit~, and phenol (Bertholat reaction generates ammonia) (~ y~ .1)); creatine hrnase (using e.g. creatinin and ATP); cholesteroloxidase (using e.g. cholesterol color change formation of cholesterol 4-en 3-one);

_ _ _ _ . _ . . _ . , . _ . _ .

~1~70~ ~' wo s6~0~t10 ~t~ ' r~

Iactate LUUUU~7~C (using e.g. Iactate); lactate dch~lLu~,~a~ (using e.g. Iactate and NAD); uricase (using e.g. uric acid~; malate d~h~lLuL,_~LLa~ (usmg e.g. malate, r--- ~' , and NAD as cofactor); 2)1...,.,.\~ U'f .~ luciferase (using e.g.
Iuciferin and ATP); peroxidase (using e.g. Iuminol (cyclic diacyl hydrazide)); alkaline ~ ' , ' , cholesterol oxidase; glucose oxidase"~-g~ f, and intertase, either individllally or a suitable c~ --- thereof. Optical ~ r ' generating H,07 can also be used for the electrical assemblies discussed herein.
Assays related to the health care field can be performed usmg the methods, cl"..l..l-::....,~ and devices described herem. Such assays include assays for: 1) pathogens such as HIV, hepatitis A, B and C virus, tub~l~ulOs;a, chlamydia, gonorrhea, IIIAI l,f .. :;~ protein marker for A zheimer's disease, Neisseria g~nrlThr,PA Vibrio choler~, syphilis (Treponema pallidum), Herpes viruses, humanpapilloma vitus, ~ (My.ul. ~.. ;.. I h. ~ ), and group A strep; 2) surface antigens of pathogens; 3) antibodies of pathogens (ie. serological assays); 4) therapeutic drugs such as theophyilline, digoxin, caffeine, LL~v,uLylLII~ amikacin, g ' netilmicin, ~Ubl~lLlly~ V~ ,Ulll~._ill, I ~ . . I I ' 1~
phenytoin, primidone, valproic acid, digoxin, diav,uyl~il~, lidocaine, N-ety l,UI .,, A; I ~ , ~UI U~ 1, quinidine, AU uiLI i~ Ly I~ UI Ll iy Iy liu~, i . t~y~lOa~,ul'ulc, P~ ~ ' I' I~ 1, and .,.. I~ L~
5) abused substances such as 1,~1,' , l-If ~ C~ ;A~ cocaine mPt~hrlir~, mPt~ Al,mP, opiates, mPtl~7~ir,nP, ~ yLli~liillC~ and ~ :1.--,.1,~.. -'1.;..~, 6) therapeutic drug monitoring for drugs such as theophylline, lidocaine, diau~uyllllid~, N-~I~C~Y~ I- .; A ~ ~I.,.A;,.-- .:Af, quinidine, flecainide, amikacm, gPntAmirin~ kanamycm, nritilmirin, ~LIc~JLullly~,'ul, tobramycin, ~UI~Ulllyl ' , , phenytom, ~ILIll)lJAlI,;ldl, primidone, valproic acid, ~ ;,,,;Af, LLuLl~ , digoxin, digitoxn, and LyLlL~ulill~ and 7) other analytes such as hCG, LH"B-inhibin, thyroxine and bilirubm.
Assays related to the food, veterinary and cllvilulllll.,llL~l fields can be performed using the methods, c~ and devices described herem. Such assays include assays for: 1) pesticides and c~ such as dioxins, .I;l,.. ,.. r,.,,.,~, PCB's, triazine, aldrin, alachlor, atrazine, bacillus i' ' v ' toxin, BAY SIR 8514, S-bioallethin, chlorosulftiron, cyanzine, 2,4-D, DDT, " '' ~'r methyl, dieldrin, 2197~61 13.
~lirulJ~ulu~ nfln~nlfnn iprodione, kepone, malete hydrazide, metalaxyl, ..~F, -l- .,l., parathlon, paraoxûn, paraquat, ~ L~ uluull~,lol~ 2,4,5-T, terhutryn, .., and warfarin; 2) livestock diseases such as Taxoplasma gondri, Brucella abortus, .~, ' ~ dentatus, If yc r ' ~ bovis, Bovine ' ' Maedi visna virus, swine fever virus, Leptospira; ~ 'J~ and Cul~ VilU ~, 3) anabolic agents such as 17~-estradiol, estrogen, i ul~, 17cY..u~Llly' UllC, IJlU~,_D~..UllC, trenhnlnn~, di~lly' ~- Ul, hexoestrol, and zeronat; 4) toxins and pathogens such as ~'lnctr~ mn botulinum neurotoxin A, B, E, F, G, .~l~y h~loco., ~~ aureus, c ...~luLu~.ill~
A, B, C, D, E, Aflatoxins B1, B2, B4 diol, M1, Q1, Ochratoxin, T-2 toxin, 3'-OH-T-2 toxin, T-2 i ' , HT-2 toxin, group A i,;.l.. dh.. _ ~, roridin A, di~.v~ iu~l~.lvl, deu~lllv' l, 3-Acetyl dcvA~uiv.,L,.lol, d~v~.y~lu~uul, Lul, rubratoxin B, PR toxin, .~ ?, Listeria -_y~i~g~, Escherichia coli, ~brae epp., Yersinia enterocolitica, and lvb~tt~ jejuni.
Assays related to the defense fields can be performed using the methods, ~~and devices described herein. Such assays include Anthrax spores (Bacillus anthmacis), Ebola virus, .~ ,' Jloco~u~ aurens enterofxin B (& others), Yellow fever virus, cloned protem toxims (eg. snake, scorpion), Lassa fever virus, and Ricin, Yersuia pestis.
Cholesterol is preferably measured using cholesterol oxidase/H202 assay system usmg HlO2 sensing electrodes, preferably containing palladium or metal chelated substances, such as, but not limited to, cobalt phthalocyanine' Gihmartin et al., Anat~st, 119:2331-2336 (1994), the methods of which are herein ill~UllJ~ ' ' by reference; and Dong et al., Ana. Chim. Acta, 279:235-240 (1993), the methods of which are heremiU~Ul,U~ ' ' by reference. Such electrode methods can also be combmed with otherassays described herein.
In general, a device embodymg the invention has a detection zone where the applied aralyte produces, directly or indirectly, a detectable pl,. .. " " . e.g., relating to the production of color, ~u~Jlc ~;ull of production of color or the generation (or the ~U~J~JlC~:~;Ull of generation) of a species that is detectable el~L u.l~ lly. Often, the detection zone is located du..~LIc~ull of a merged zone, where two or more fluidguiding pathways merge to form one fluid guiding pathway. Ihe detection zone may WO96/05510 ~ 06i ~ c /~3~

have means for tending to retain, or reduce the loss of, species responsible for the detectable ~ Thus, where a detection zone extends over only a part of the width of a pathway, it may be delimited at one or both sides by barriers to lateral diffusion.
A trapping zone can also be provided in the region of the detection zone. This could be a charged layer which would serve the purpose of c.,... .~ oppositely cbarged species at the detection zone, e.g., Nafion is a suitable negatively charged membrane material. Alternatively, a material chosen on basis of size exclusion could be used, e.g., cellulose acetate.
For example, a device based on the generation of hydrogen peroxide and its ele~llu~h~llu~ll detection could have a trapping layer in the region of the detection electrodes which is adapted to retain hydrogen peroxide.
A device based on the generation of a reduced mediator (e.g., L~"ul~,y may have a trapping layer that retains the mediator by charge attraction.
Exclusion membrane canprotect against fouling of the electrode surface and cam be made from polyulclI~uc as well.
Incomplete removal of unbound antibody conjugate from the electrode site can affect the ~lrullll~ul.,c of the electrode assembly. Unbound antibody conjugate adhering to the carbon surface of an electrode or matrix :lUllUUUdill~ an electrode assembly can lead to erroneous signals. This can occur if the "wicking power" ofmatrix, such as silica, p~ulicul.llly phenyl silica, is inc.lffi~ n- to remove unbound conjugate. F.~l,. . ;" .. ~ involving the deposition of proteim conjugate directly onto the surface of the bare, carbon-based electrodes, followed by extensive washing with water (using a wash bottle), revealed binding between prolein conjugate and the electrode surface.
Decrease in the p.. r.... - ., e of the electrode system due to unbound conjugate cam be overcome by 1) the use of blocking materials to cover potential binding sites on and around the electrode(s) and 2) the use of a size selective membrane over theelectrode that enables a reaction product, such as an enzyme product, for instance, HlO2, to pass through to the electrode surface while preventing the binding of unboumd conjugate to the electrode.

~ WO96105510 2i9~06i ~ 3 15.
.. .
For blocking materials BSA, casein, goat serum and skirnmed miUc, as well as other blocking materials known in the art, can be used, as long as those materials do not interfere with electrode Ill~ ul~ . Preferably, blocking materials are used that mimic or are derived from a source similar to either the sample being tested or the antibodies used in the assay or a o,~ thereof. L~t lr~lcu~c is easily tested by applying varying amoumts of the candidate blocking material to the electrode assembly and ~ the electrode assembly's sensitivity after successive washes. If blocking materials diminish the electrical signal, the c~ .. U,.I.. ~ can be lowered even to ~ero, and a protective or exclusion membrane can then be used to reduce the affect of non-specific binding. Typically, the electrode structure is printed before the primary capture antibody is i~r~hili7r~d on to the silica which forms a layer directly over the electrode's carbon surface. Following imn~ohili7~tir~n, the silica, such as phenyl silica, is bloclced with either one or several of the blocking materials, which are usually in the 1% to 10% c....~ ~ ~n,~l;r~ range (w/v).
For a ~i~ s~ ,livc exclusion ~ , various materials can be used such as cellulose acetate, porous poly~lu~yl~ , porous nylon, porous polyc.u~ , porous pol~l.c~le, silicon-containing elastomers and similar porous material, either singly or in CU...1J- Cellulose acetate is preferred for exclusion ' The resulting pore srze of the exclusion membrane printed using most materials, such as cellulose materials, ~ ,uLuly cellulose acetate, is dependent on the volatility of the solvent. Less volatile solvents, in general, lead to small pore sizes. Cy. lr,h~ .,nr is ~r~ ' Iy well-suited as a solvent for cellulose acetate. To avoid slow c ~ )UI~L~iUII
of e , ~ low-volatility solvents, such as cy~ and the c~ " ly produced " ..,1", ~ with very small ill-defined pore sizes, higher-volatility solvents, such as acetone, can be mixed with the lower-volatility solvents to achieve the desired pore si_es of . . .1 ", , Mixing of high- amd low-volatility solvents also improves the ~ J" of solutions compared to the use of high-volatility solvents alone.
Generally, cellulose acetate u~ r ~ ;.."~ from 3% to 10% (wlv) can be used with varying mixtures of cy~ 1"1._.,..."" and acetone, such as 9:1, 2:1, 1:1 and .5:1 (v/v), ~ ,l.y. The ~;~, ' ' to acetone ratio (v/v) is preferably .5:1 to 2:1 and more preferably, 1:1. The cellulose acetate ~..1.,~..l".l;.,~ is preferably 3.5 to 8%
(w/v) and more preferably, 4 to 5 % . Other organic solvents can be used, especially ., _ _ _ _ ., . ,, . , _ , _ _ =,, 2~g~061 wo 96/05510 P~~ t 16.
whel~ the solvent of lower volatility has a bo~iling point from 100 to 175~C and the solventofhighervolatilityhasabo,ii~g.pointfrom45to65~C,andthelowervolatility and higher volatility solvent are mixed in at least a .25:1 ratio, lca~Li~,ly.
Preferably, 4% (w/v) cellulose acetate in a mixture of high- and low-volatility solvent is used and preferably, a 1:1 (v/v) mixmte of acetone and ~J~ ,.... .is used to form an exclusion membrane. The acetyl content of the cellulose acetate is preferably at least 407O. Such solutions are suitable for screen-printing, giving a lclJludh~ilJlc exclusion membrane over the electrode area. This technique can also be applied to exclude other assay reagents or sample ~ from the electrodes, such as cell fragments or high molecular weight proteins.
Retention of undesired reagents in guidance pathway matrices can be reduced using flow ~ rlr . ~ Flow ~. . L ,, r ~ are generally of two types: 1) material added to the printed guidance pathway printing solutions to increase fluid flow in the guidance pathway mattices or matrix, especially in the region of a drled reagent or 2) a non-matrix deposit of a dried reagent in capillary contact with the matrix or matrices of a printed guidance pathway. Such flow ~. . . l. .~ ,. ~ are p~llLi.,ul.~lly useful additions to the guidance pathway when retention of reagents in the guidance pathways, such as conjugates, generates a signal large enough to interfere with hhe detection of the signal in the presence of an amalyte. For example, faster flow of conjugate through or around a matrix, IJ~Li~ul~ly a phenyl silica matrix, can enhance the l. r.. ~ of the electrical ul~aul~ in the presence and absence of an analyte, i.e., lower b~ S are produced, the signal-to-noise ratio can be enhanced and the lwlu~;h;lily of the assay can be improved. In the case of some mau~ices such as silica, i ' '~, phenyl silica, dry depositmg a reagent onto the matrix cam lead to mcomplete hydration of reagents, such as a conjugate, by the moving buffer or sample fluid front, leading to a signific,mt proportion of conjugate bemg retained and slowly released from a reservoir or reagent zone.
To improve the flow and release of a reagent to the hydrated matrix of a printedguidance pathway, reagents such as conjugates, are separately printed or deposited using non-printing techniques on the surface of a cover-sheet which is in capillary contact with the printed guidance pathway. Typically, capillary contact will arise from the placement of a cover-sheet m immediate apposition to the guidance pathway.

wo 96/05510 ~ - ' = r~l .. 5 . ,~3 17.
- Cover-sheets c;m be coated or printed with a matrix, such as a synthetic sponge to act as a quick-release reservoir of the dried reagent. Reagent can be mixed with thematrix or dried directly on the matrix or on the surface of the cover-sheet material.
~or all the cover-sheet; ' discussed herein, it is preferred to locate the - 5 cover-sheet so as to optimize flow tbrough the guidance pathway from the application site tbrough the detection zone. Materials that improve hydration of reagents include materials such as, but not limited to, gelatin, silk fibroins, chitosan, collagen and pol~lyl~id, or ' thereof and preferably, gelatin, chitosan or siLk fibroins, or ~ thereof. Printed fluid guidmg pathway materials can also be mixed with at least one surfactant to improve matrix wetting, especially when multiple matrix materials are used. Such surfactants include r" ~r of 5 7 carbon atoms, Tween 20, hexane sulfonate (preferably), Surfynol (~.I~,l~Lhyld~,yll~liOlelllù1Ly- ' with 30 moles of ethylene oxide), Triton (octyl phenoxy polyethoxy ethanol), Silwet (pol~dLk~d~ idc modified dimethylsiloxanes) and short chain rl~-- ~r ', such as ~r ' or ~1~, ~r ' or longer chain , such as those containing from 8-10 carbon atoms.
Capillary contact with the printed guidrmce pathway can be also established by placing reagents on strips or islamds that lack matrices. Reagents can be directly applied, on backing sheets, ~ ll~ly PVC or coated PVC backing sheets, as strips or islands. Such islands or strips are preferably placed m the printed fluid guidmg pathway. Alternatively, a capillary conductive hole can be designed in the backing strip, with an optionally placed, inert wicking material (e.g., a sponge) inside the hole, to provide for a reagent. Such strips, holes or islands act as a quick-release reagent reservoir that is triggered by the flow of fluid through the ~UIIUUIIdill~ or adjacent guidance pathway to permit hydration of the reagent. Preferably, such reagent islands are triangle shaped, with the angle of least degrees forming the point of the triangle tbat first contacts fluid flow. The term cover-sheet when used without reference to covering or bemg located over a guidance pathway, includes such strips and islands as discussed herein. A cover-sheet may be applied to ensure fluid flow across the dried reagent.
To improve the flow~or release of a reagent to the hydrated matrix of a printed guidance pathway, reagents, such as conjugates and guidance pathways, are provided ., , . _ ,, _ , WO ~/OSS10 2 1 9 7 0 6 1 P~.1,~,3.. /.:3 that are a mixrure of fast and siow ~ Liu~ matrices. Fast l".~.~l....l;..g matrices can be added to the guidance pathway ir~s to make a printed guidance pathway to accelerate the flow of slow-moving reagents that decrease the electrical signal to noise ratio.
The ftrst ~ shown in ~IGS. 1-3, has a PVC backing sheet 1 sbaped to provide a generally lc~,L~ul~;ul~ main portion 3, narrowing to a liquid application stem or zone 5. One face of the sheet 1 has a pattern of .,l..~ medium (e.g., silica or cellulose) which has been applied by screen-prmting a slurry of particles of the medium, followed by c ~ ul~liul- of the solvent, leavmg a porous deposit. This pattern provides an immersion area or zone 13 which covers the stem 5 and a single fluid guiding pathway 14 that extends from the immersion area 13. Its far end is of increased area to provide a fluid resenoir or zone. In this example, it is branched, having two reservoir aTms 16. The width and length of the pathway will be selected to give the maximum advantage for carrying out the assay. If necessary, the pathway can be overlaid with additional slurry of the porous material to increase the thickness and hence, the retention of reagents. Indicated on the pathway is a spotting point 11 where a known volume of sample is to be deposited. Also shown on the pathway is a dry reservoir site or zone 12 for the deposition of other ~ --r ' of the assay(e.g., reagents or substrates). Several such sites could be included per pathway in a typical device, e.g applied by screen printing.
In use, the stem 5 is immersed in a buffer solution, held m a separate or mtegral container. The buffer will flow alûng &e pathway by means of capillary force and ~' ,, ,' force. In doing so, the other ~ of the assay will be taken up and carried from the dry reservoir by the flowing liquid. Eventually, the liquid front will reach the detection area where the reaction can be monitored.
One example of the use of such a device would be for an er,zyme-linked y. The assay could be based on the use of a "capture ~"
involving labelled antigen. Enzyme-labelled amtigen would be complexed with a suitable antibody, ;.. ,T.. I;,. J at a dry reservoir site 12. A suitable substrate for the enzyme reaction would be deposited over the detection area or zone 15. A product of the enzyme reaction is ~ . ly detected e.g., by a color change, el.,~,lu~
signal or an emission of light.

W096/OS510 ' ' ~ ~ r._l~.. s'~ /~
~ 2~ a6l In a typical use of this device, a Icnown volume of liquid sample (e.g., urine) would be applied to the spotting point 11. The stem end of the device would then be immersed m the buffer solution. As the fluid is guided up the pathway, the sample solution will pass over the dry reservoir site. Antigen present in the sample solution S will disp!ace the bound labelled antigen from the im~nhili7Pd antibodies. The released labelled antigen will be carried m the buffer and guided by the pathway to the detection area where a reactlon will occur with the substrate. For example, when using a cn1-..; ...~,. substrate in the presence of this substrate, an insoluble color will be produced, and the intensity of this color will be drt~ rminrd by the . of labelled antigen present. A sample containing a high c of the desired analyte will produce a more intense color. Waste reservoirs are sited behind thedetection area to allow the capillary flow to continue and complete the assay.
~IG. 4 is a further extension to this approach, whereby the presence of two or more fluid pathways allows ' l~a;~ to be carried out. Analyte assays mentioned herein, and Icnown in the art, can be used in ~ ~ with this type of test card design. It depicts the use of four fluid guiding pathways 14 a,b,c,d extending from the immersion area 13. This is an arbitrary number and any number or c.. 1.. -~;.. of fluid guidmg pathways could be employed with this device. Test solutions of a Icnown volume, which may or may not be aliquots from am individual sample, are spotted at the a~ ~ site on each of the fluid guidmg pathways.
Alternatively, the test solution, such as a sample, can be applied at a single liquid arrlir~tirn that leads to multiple fluid guidimg pathways for multiple analysis. The test solutions are guided over the dry reagent reservoirs to the detection areas. Each sample is monitored; L 1~ L ~ly. By selecting the ~ lo, pathway material and the geometric shape of the individual fluid guidmg pathways, the assay can be tailored to suit the, ~ of each analyte monitoring system. Beyond the detection areas, a common or mdiYidual waste reservoir system will be deposited. Flow stops when the respective reservoir is saturated and the reactions are completed. Therefore, the area, material, depth and ~ , of this reservoir will affect the successful operation of each of the mdividual assays.
~IGS. 5 and 6 illustrate a further refinement of this mvention. With this approach, a single assay is performed using two or more fluid pathways. This allows wos6/osslo 21~7061 ~ ~ h, ~' r~"
20.
for a sequential, timed delivery of a range of assay c.~ to a single site (tbe detection area). Each pathway can be: u~.t~ using a selected material or materials and defined geometry and thickness (or thicknesses). Hence, the fluid flow and the retention of individual assay l r ' can be effectively controlled.
EIGS. ~5 and 6 show devices havmg three fluid guiding pathways 24a,b,c/34a,b,c leading from the immersion area 13 and coalescing at tbe detection area or zone that is also a merged zone 15. Beyond this, there is a reseNoir 16. In both examples, the central pathway 24b/34b has a spotting pomt 11 for a sample, and the outer fluid guidmg pathways have dry reagent reservoirs 12a, 12c. F.ll lh.,l~
the fluid guiding pathways are such that liquid passes along all tbree fluid guiding pathways from the immersion area 13 towards the reservoir 16. Flow along the central pathway 24b/34b will convey sample from the spotting point 11 to the detection area 15 some time before reagent reaches that area from one outer pathway, and reagent from the other outer pathway arrives after a further delay. In FIG. 5, this sequential delivery is achieved by making the three fluid guiding pathways differ in length. In ~G. 6, the outer fluid guiding pathways are equal m length and similar rn length to the central pathway, but they differ in c~ a;.... at least in part (particularly, all or part of the pathway portions downstream of the dry reagent resenoirs should permit fast flow relative to upstream of the reservoirs 12 a,c). Thus, the outer fluid guiding pathways may include portions comprismg ~fi~lvoellulv~, while the oentral pathway is formed of cellulose. One outer pathway may have a greater proportion of its length which contains luLIl " ' and/or be formed of a mixture containing a higher proportion thereof. Fine tuning of delivery .l.--,-~ of the individual fluid guiding pathways, by means of varying the material, its thickness and geometry, can be readily ~-c~ using printing techniques.
An example of the use of such a device is a "smdwich type~ ~.
One outer pathway 24a/34a is composed of a single material with one dry resenoirlocated at 12a. The central pathway 24b/34b has a spotting point 11 located at astrategic point. The detection area 15 contains; .1. ~ l antibody. The ovher outer pathway 24c/34c has one dry reservoir (sited at 12c) located along its length.
A known sample volume is applied to the spotting point (which can be a hole, island or strip described herein or a hole lined with an inert wicking material (e.g., .~ W096105510 ~ 06'1 P~ ~ S /~

sponge) to prevent the sample from binding to the dry prinoed pathway) on the central pathway 24b/34b, and the buffer solution is introduced to the immersion area end of the device, e.g., by placing it in a trough of suitable buffer. The sample solution is carried up the guidance pathway to the deoection area. Antigen present in the sample is "captured" by the ~ ~ ' ' antibodies on the deoection area. The continuing flow of buffer along the central pathway will remove unbound conjugaoes and other substances that may inoerfere with the assay. During this time, buffer will be contir~ally guided along the first ouoer pathway 24a/34a. As a result of this flow, th ~: , of its dry resenoir 12a (in tbis example, a second antibody labelled with an ~,ulul,, ~ enzyme) will be taken up by the buffer stream and carried towards the detection area 15. The geometry and/or nature of the ~h.. ~ A~ ~ maoerial will influence the timing of the arrival of reagents at the deoection area. Labelled antibody (the conjugate) will bind with the antigen already bound on the deoection area to form a complex. Continued flow will remove excess unbound, labelled antibody.
Concurrent flow of buffer m the other outer pathway 24c/34c will eventually reach the drv reservoir sited at 12c, and the contents (m this example a suitable ~ul.~; s;.
substrate) will be taken up into the liquid flow. This pathway will usually be longest in terms of retention times. Finally, the substrate will reach the amtibody-antigen-antibody complex at the detection area and produce a detectable reaction indicating presence of the analyte of interest.
~IGS. 7 and 8 show a single pathway device which mcludes an elèctrode assembly. A backing sheet 101 of insulating material (e.g., PVC film) has the form of a square with a tab 102 extending from the center of one edge. The sheet has been screen-printed m a region adjacent to the tabbed edge with an electrode assembly, m this example, having two electrodes 105 connected by conductive strips 105a to contact pads 104 on the tab 102. 8~ . .sly~ a pattern of ~,h.~ O ,' ~ material (e.g., silica or cellulose) was printed, including a portion overlying (and hence, in intimate contact with) the electrodes 105. The electrodes are designed to effect an L.u~L.,ll~ical reaction m the presence of reactant. The two-electrode ~
consists of a working electrode amd a reference electrode. In this example, the reference electrode (composed of Ag/AgCl ink) will operate as a coumter electrode, acting as either a source or a sink for electrons. (A similar device could have a three-219706113. i",~
WO 96/05510 r~

electrode ~ t, with a third electrode (counter electrode) e.g composed of graphite. In this case, the current flow would be through the working and counter electrodes.) The c~ of the working electrode material, which may be based on graphite printing ink, can be altered to suit l~yuu~u~llt~, e.g., to enhance the oxidation of a reaction product, catalytic materials can be added to the ink ~
Generally, the electrode ~ can be built up using a series of layers e.g conducting tracks (from the electrode face to a contact point), overlay pads (conducive to the electrode ~ uu~ ..l) and an insulation shroud ( to isolate the contacts from the solution).
The guidance pathway consists of a single track 110 with a spottmg point 112 and a d}y reservoir 113 deposited at a precise location. At one end of the pathway, the pattern expands to cover the entire width of the backing material. This is the immersion area 117, where the elution buffer is introduced, e.g., by placing this region of the device in a trough containing the elution buffer. The other end of the pathway broadens to cover the detection area 116 (which overlies the electrode ~ " L.' )and thereafter, to provide a waste fluid reservoir 118.
During operation, it is envisaged that the device can be used for a "one-shot"
y. A typical example is the detection of the human chorionic g~l~lu~ JlPill (hCG) hormone. With this assay, antigen labelled with an en7yme (e.g., glucose oxidase) is attached to ' ~i7P-I antibodies at the dry reservoir site 113 Substrate for tbe en7ymatic reaction in this example glucose is deposited, rn sufficient quantity, at the detection area. The electrodes are connected by the contact pads 104 to a p~Jt~ poised at a selected potential Sample solution is deposited on the spotting point 112 and buffer solution is introduced into the lower end of the device. Buffer is guided up the pathway 110 from the immersion area 117, by capillary action. The flow passes over the spotting pomt 112 from which it takes up the sample When the solution reaches the dry reservorr 113 containing the labelled antigen, a ,1;~l.l, ..,....~ reaction occurs Liberated labelled antigen is carried rn the buffer stream to the detection area 116f where the en7yme undergoes a reaction with the substrate present at this site The reaction of glucose oxidase with glucose is as follows:
glucose oxidase 21 ~ 7~6~ s ~j , glucose + 0z----~ glucorlic acid + H20z The product hydrogen peroxide can be detected ~ u~ .Hl,~lly using a two- or three-electrode system. Hydrogen peroxide will be oxidized at a sufficiently high potential, and, in c~ , a current will be generated. Ml _ of this current will indicate firstly, the presence of analyte in the sample solution and secondly, the amount of analyte present. If the sample solution contains a high of antigen, more .1.~ will occur and hence, a higher current will be generated. In contrast, a low . of antigen in the sample solution will produce a smaller current. Alternatively, a glucose oxidase assay system can be used in a "sandwich assay" where the conjugate, in this case glucose oxidase labelled antibody, that binds to the analyte is a non 5~ ' ' reagent.
~IG. 9 depicts an alternative ,.,., ~,. ,. .,~ for carrying out assays using this approach. Elements cul.c r ' ~ to those of the first ~ l ,o~ have ~ g reference numbers. A plurality of guidance pathways 121, 122, 123 (three in thisexample) are printed onto an inert backing material 101. They coalesce at a detection area 116. Each has a spotting point 112 or a dry reservoir 113. The fluid guiding pathways differ in lengths, so that ~ ~ .. l.. ,.. ~ from the reservoirs 113 and the spotting poimt 112 rcach the detection area 116 at different times. Again, either a two- or three-electrode system can be iu.,ull ' into the design, depending on the l~ UilClll.,lll:~
for the assay. EIG. 10 shows an equivalent device, m which the fluid guiding pathways 131, 132, 133 do not differ greatly im length but are composed, at least in part, of different materials, selected and disposed to give sequential delivery at the detection area. For example, llhl~ 5,, may be hl~u~ ' to give slower transit times for fluid guiding pathways or parts thereof; fibrous cellulose may be i .Ill, ,. .-~--1 to produce faster times.
The device of ~G. 9 or ~G. 10 c~m be used for a "sandwich type"
where the series of guidance pathways can be used to facilitate reagent delivery and washing steps.
For example, in the ~G. 9 ~ ' t, the central amd shortest pathway 121 has a spotting point 112, for sample deposition. Antibodies, specific to hCG, are i.,, -~1.-1;, ~l on the surface of the detection area 116. Guidance pathway 122 is longer and cor~ains a dry reservoir 113 where antibody labelled with enzyme (glucose WO 9610S510 2 i 9 7 0 6 1 1 ~1,~ . ,~
. 2~i.
oxidase) is deposited. The third and longest guidance pathway 123 has a dr,v reservoir 113 containing glucose (the substrate for the enzyme reaction). Agam, the electrodes arc connected to a p. . .i;-.J -l poised at a selected potential during the detection period.
Following application of the sample solution to the spotting point, the lower end S of the device is immersed mto a trough of buffer. The buffer travels up the fluid guiding pathways by capillary action. On the central pathway 121/131, the sample is carried along by the movmg buffer onto the detection area 116. Here, antigen present in the sample is captured by antibodies; .- .1.;1;~- d at this area. The contmuing flow of buffer will remove any sample ~."..j,.".. .,I~ that are not bound. During this period buffer will also track along the second longest pathway 122 (or the second slowest pathway 132), taking up the second (enzyme labelled) antibody from the reservoir 113.
This flow will reach the detection area after the sample, and the labelled antibody will form a sandwich complex with the amigen-antibody formation already i,. . ~ 1 at the detection area. Continued buffer flow from both guidance pathways will remove unbound labelled antibody from the detection area. Finally, the buffer flow in the longest, and often the slowest, pathway 123/133 will deliver the enzyme substrate (glucose) to the detection area, and the enzyme reaction will take place. Again, the current generated by the ~I~!~.IUIII.,.~;~, oxidation of the product, hydrogen peroxide, can be ~fPtP~ninP~i The magnitude of this current will be determined by the ~ of analyte in the sample and hence, the antibody-antigen-antibody complex formed at the detection area.
In addition to ~,~,.rulllfi~ ," the proposed device cam be used for carrymg out h~blidi~liull assays, based on the specific binding of nucleic acid sequences with o~ - y sequences. APhe design of the device allows different buffers to be used to alter the stringency of the hj11.;.1;,- ;.lll Traditional h~blidi~liulrbased detection systems are time-consuming and mvolve several steps, thus requiring a tMined operator The use of this device will be simpler and moreMpid. This will allow the d~ r ' of a range of assays with medical, food and ~IIV ilUIIIII'~ll l l A number of different nucleic acid assay formats can be envisaged. A label, such as the enzyme glucose oxidase, may be ill~,Ul,UUl~...d such that ele~ ul,h.,llliudl detection of the reaction product can be carried out as described for the i~

WO 96105510 21 9 7 ~ 6 :~ P~
25.
In some assay formats, it will be the sample nucleic acid which is labelled by the user.
Fo} example, a target single-stranded nucleic acid sequence is irr~tbili7~1 on the detection area. When tbe end of the device is immersed in the sample solution, tbe sample, labelled witb the enzyme, travels up the .,L., O ,' - fluid guiding patbways, and, as it flows over the detection area, the ~ L .. -: - y analyte sequence is captured. Using a two-track system (a simplified version of ~IGS. 9 or l0), the enzyme substrate is delivered, after a suitable time delay, for detection of the label now present on tbe detection area. Alternatively, the labelled sample could be applied to a spotting point, and a buffer would then transport the sample to the target and wash away any unbound sample before the enzyme substrate is delivered. In the another- "- .~.... ,I the sample is placed directly onto tbe target area over the electrodes. In tbis case, it may be necessary to i ~"l''.. ,r an i,.,~ r barrier around the sides of a sample well in order to prevent sample wicking away from tbe area before all of tbe analyte sequence has been captured. The use of a sample well also allows thepossibility of i,l~.. l.. ,,~i.~,, a membrane over the well. This could carry out some sample ~!lCLIl t, e.g., removing debris from analy_ed cell LIIC~ Liu In order to make the labelling step simpler for the user, a specific binding pair system such as the biotinl:~L c~L~ ' system could be used. In this assay, the user would carry out biotin labelling of the sample. This is a standard and ~L-~;oL~rulwd~d procedure, e.g., using the PCR technique. The sample would then be applied to a spotting point on a fast or short track or directly to the detection area which has an ~ 1i7~ target sequence - ,' y to the analyte sequence. The device will be dipped into a buffer which will travel rapidly up the first track, so as to deliver any sample applied to the spotting point to the target area and wash away any unbound sample. A second, slower track will deliver a ~Ic~,Llvidil-en7yme conjugate to the target area. The ~Ll~ r;dill binds to the biotin label and is thus trapped on the electrode with any sample of ~ sequence to the target. I~xcess conjugate continues to travel into the waste resenoir before the longest track delivers the enzyme substrate for el~LIu~L~ dl detection.
Further assay formats are possible in which the user does not carry out any labelling. The frrst of these is a d~ type assay. In a dry resenoir on the shorter track, there will be a double-stranded nucleic acid sequence. One of the strands wo s6/osslo 2 1 9 7 0 6 i ~ C ~

will be imm~hili7f-ri whereas the ~,,,,,I,L ." ..I ..y strand will be moblle and labelled with an enzgme. In general, this labelled slrand will be only partially c~ d Y, e.g., being shorter than, and/or containing a mismatch. The sample sequence (analyte) will be more c~ g. Thus, as the sample travels up the device, its ~ d~r sequences will displace the labelled strand. This will then continue to move up the track and will be recaptured by a target sequence ' ' ' on the detection area, thus trapping enzyme label on the electrode before the longer track delivers the substrate.
A further refmement is the use of a "sandwich" type hjh. ;.1;,~ In this two-target nucleic acid, sequences are used. These are ,".,~l.L ~- g to opposite ends of the sample sequence, thus, when all three sequences are present, the sample forms a link between the two targets. Target 1 could be labelled with an er~gme and placed in a dry reservoir on the shorter track. Target 2 will be i .~hili7~ ~i on the detection area. As the sample travels up the device, it first hybridi_es to Target 1 and therefore is labelled and then captured by Target 2 as it flows over the detection area.
Alternatively, a hyhl;d~liull between sample and Target 1 could be carried out in a tube prior to applying the mixture to a spotting point. As with the other assays, the enzyme substrate is delivered after a suitable interval to allow for removal of unbound sample by the buffer flow. This approach, which; ~ r ~ two hylJIidi~ Liu~l steps, will allow the d~ ,lu~ l of highiy specific assays.
A further d~ ,lo~ ,l.. of this type of assay is the hl~ul,uul~Liull of naturallyoccurring or ' protein peptide receptors, e.g., from cell surfaces, as the affinity agent. In addition to receptors purified from cells, synthetic peptidesmimicking the binding site of whole cell receptors or protein bnnding proteins could be used. These may be more stable and easier to manipulate than whole receptors. The receptor will be ;.. . ~1.;1; J on the detection area. Alternatively, peptides that bind receptors or other analytes could be ;--....l.;li,. i A l.;UIII~ iVt~ or a .i;~p .. .,~
assay forrnat could be used. In the first of these analyte conjugated to an enzyme label is present in dry reservoir and tbis competes for the receptor sites with the uniabelled analyte present in the sample as they low over the detection area. Thus the moreanalyte is that present in the sample the less label will be present on the electrode area when the en7yme substrate is later delivered by the longer track. In a 1;~l 1- . .1 ~ W096/05510 2197067 r~
27.
U~ . j type assay, receptor or synthetic peptide bound to labelled analyte will be in~nlnhili7P(7 on the electrode area. Sample analyte will displace the labelled analyte as the sample flows over the electrode area. The amount displaced wiL be ,uluyulliu~l to the amount of analyte in the sample. The remaining label is then detected S ele~ u~L~lll~lly when the longer track delivers the substrate.
In a third possibility a receptor is used as the mobile labelled element. The Lgand for the receptor is i1n~nhili7pd over the detection area and labelled receptor is placed in a dry reservoir on the ~LUlLl,ilL/Ll~DI track. Sample is applied to this track and as it is carried up the device it interacts with the labelled receptor. As this then flows over the detection area, receptor which has not bound ligand present in the sample becomes bound to the i" . h.l;,. d ligand. Thus the label trapped on the detection area is inversely l~lu~ul~io.l~l to the ligand . in the sample.
Receptor bound to sample ligand continues to flow into the waste reservoir. A
slower/longer track delivers the label substrate for detection of the receptor trapped in the detection area.
In a further variant a mobile receptor or binding protein is captured by antibody i~nlnhili7Pd over the detection area. The ligand for the receptor is ' ' ' on the same pathway but upstream of the reservoir containing labelled receptor. Sample is applied to this track and as it is carried up the device it interacts with the labelled 2û receptor. Receptor which has not interacted with ligand in the sample is trapped by the,, nl, 1;, .1 Iigand sited further along the pathway. Any receptor which has bound ligand from the sample passes over the ' " ' Lgand amd is captured at the detection area. Thus the label trapped on the detection area is l....l.n,l;n.,~l to the Lgand . - o~l;"" in the sample.
It is envisaged that other en_yme systems can be used to produce a signal that could be detected cl~ u L, lli~lly. A further refinement of the assay would be to iIII.,UI~ ' a trapping layer to cover the electrode area, with the aim of ~. ~ .1.,-';.
the ~.I.,.,IIu~L~ lly active product from the enzyme reaction (e.g, H2OI or mediator, such as ferrocene or L~ y ~ ) by specifically retarding its removal from the detection area, leading to an euhanced signal at the electrode. This approach could ~;~;Il;fl~y improve the detection of low . of aralyte in a given sample solution.

2lg7o6J
WO96/05510 r~.,~,S,~

Any of the designs outlined in P~GS. 1, 5, 6, 8, 9 and 10 could form the basis of a multi-analyte and/or multi-sample analysls'-~ whereby the designs can be multiplied to form an integral analysis system. ~he analysis system can be printed as an array of dedicated fluid guiding pathways and detectors on a single strip of suitable backing S material. The cu.. lUl,~iuu would permit for each analyte individual optimized designs of cll., , ,' ~ materials, thicknesses and geometries of fluid guiding pathways,dry reservoir sites and ~ ~ of electrodes. ruHI~ lllvlc any ~ of designs can be used to form an optimized integral analysis system. For example, the designs outlined m IIGS. 8 and 10 may be printed on a single strip of backing material.

E~ IP ~ 1: SCREEN P~UNTED G~DANOE PATHWAYS FOR Or~CAL DETEC~ON
Assay devices were prepared for the detection of human chorionic gv~dvll~,' (hCG), as shown in l~G. 5. Each has a backing sheet on which a pattern of ~,lu~ material has been deposited to defme an immersion area 13, a reservoir 16, and three fluid guiding pathways 24a,b,c of different lengths extending from the immersion area 13 to the resenoir 16. The fluid guiding pathways coalesce at a junction region or merged zone 15 adjacent the reservoir to which they are connected by a single pathway.
PROnSION OF PATHWAY PATTERN
The c L..II ~ material or matrix was silica, of a grade used as a separation matrix for HPLC (5~ Spherisorb, available from Phase S~p ~-~tit)nc~ Clwyd, GB). It was mixed with dry powder of a bmder (calcium phosphate or calcium sulphate) (5% by weight). The mrxture was slurried with 2-lJULU~ l acetate.
A backing sheet (P.V.C.) was cut to shape and coated on one side with matt emulsion paint prior to printing the silica. It was dried at 40~ for 1 hour.
A cuu~lRiu.~l screen printing apparatus was provided with a screen on which the desired pattern was defined in a cu..~. ' manner. For the frst printing step, this was the entire pattern shown in FIG.5 except for the junction region 15. The prepared backing sheet as located umder the screen. The slurry was applied to the screen by means of a rubber squeegee. Slurry passed tbrough the screen to provide the pattern on the painted surface of the backing sheet. The sheet was removed and . . r ~ wo 96/OS~ilO 2 1 9 7 0 6 ~ . ~1/~. ,~

allowed to dry at 40~ for 1 hour. In a second printing step using the same procedure, the junction region was printed using fimrt~ li7P~I Spherisorb (also available from Phase SPp~ti~
PR~D~R~TIn1~1 OF ANALYTICAL DEVICE
Various reagents were applied to patterned sheets from step (a). In the example this was done manually, but for mass production it could be carried out by further printing steps, e.g., screen printing, ink jet printing or air brush printing.
A solution of amtibodies specific to hCG was deposited over the junction region,to form a detection region 15. This was foilowed by mcubation at 4~C for 12h. The detection area was washed several times with distilled water and allowed to dry in air at a ~ ,.c not greater than 45~. (Either room I , or 37~ was generally used.) Bovine serum albumin was added to block any active sites which had not reacted with the amtibodies specific to hCG, and the sheet was incubated at 4~C for 1 hour. The detection area was washed several times with distilled water amd allowed to dry.
Labelled antibodies to hCG were deposited at a reservoir site 12c on the pathway 24c of '~ length. Labelling involved ~ c~ to glucose oxidase.
The labelled antibodies were applied in a buffer solution which was allowed to dry under the same conditions as the bound antibodies. The boumd antibodies and the mobile antibodies in the reservoir both bind to hCG but they recognize differentepitopes in this example.
A dry deposit of a colour-producing substrate (o-dianisidine and peroxidase) forthe enzyme was formed at reservoir site 12a on the longest pathway 24a.
A spotting point 11 was mdicated on the shortest pathway 24b by marking with a w.l~.,.. t;u~l non-aqueous ink on the surface of the pathway or on the adjacent portion of the backing sheet.
USE
Two devices were tested. A test sample of buffer containing hCG was applied to the spotting point 11 of one, "test", device, while a Cull~ r ~ volume of buffer was applied to the spottmg point 11 of the other, ~control", device. The devices were placed in a tank of buffer so that their immersion areas 13 were subst~mtially immersed. The passage of solvent fronts up the fluid guidmg pathways could be _ _ _ _ _ _ _ _ _ _ _ _ _ WO 96/05510 1 9 7~ 61 - ; . t . ~_I/lL~ ~ IL5 ' ''3d;
observed. In less than 10 minutes, all fluid guiding pathways had been traversed. The detection region 15 of the control device had developed strong colour, due to peroxidase-catalyzed oxidation, by hydrogen peroxide, of 0-dianisidine to a brown product. The detection region 1~ of the test device was almost lmrolo EXA~PLE 2: SCREEN I~UNTED G~DANOE PATHWAYS FOR ELECT~UCAL
DETECTION OF ANALYTES
Devices as shown in ~IG. 9 were prepared. PVC sheet was cut to shape and painted (as in Example 1) to provide the backing sheets 101. Several screen-printing steps were then performed in register: (a) with Ag/AgCI ink to form the reference electrode; (b) with graphite ink to form the working electrode, the conductive strips 105a and the contact pads 104; (c) with a resin ink to insulate the conductive strips;
and (d) with a silica slurry as in Example 1, to form the pathway pattern, including the detection area 116 applied over the electrodes 10~. Antibodies specific to hCG were ;., .1.;1;, . .1 in the detection area. Bovine serum albumin was then inkjet printed over the detection area to infill between bound antibody molecules and thus prevent non-specific binding. GOD-labelled amtibody is deposited as a dry reservoir on pathway 122. Glucose is deposited as a dr,v reservoir on the longest pathway 123. A spotting pomt 112 is marked on the shortest track 121.
For use, a r.,......... .~ was coupled to the contact pads 104 of one device. Asample containing hCG was applied to the spotting point. The immersion area 117 was immersed in buffer. The current passed by the p.,' ~ ' was monitored. The initial low baselme level rose rapidly once the buffer had conveyed the sample, the labelled GOD and the glucose to the detection area. Current was plotted against time.
Successive ~l,.. ;., .. s ~ using identical devices with different amounts of HCG in the applied sample showed that the height of the peak in the plot correlated with the amount of hCG in the sample.

EXA~PLE 3: SCREEN PR~D G~DANCE PATHWAYS FOR ELECT~UCAL
DETECTION OF HCG
Screened printed test cards were prepared with guidance pathways for the detection of hCG anElyte as shown in ~ G.11. Test cErds were ~ ur~ul~:d using ~ WO96/05510 2197~61 r~ 3 PVC backing sheets (400,u) a~ 45mm x 30mm in size, which are suitable for rmsertion into â portable, hand-held reader.
Test cards were screen printed using a screen printer from DEK Prmting Machines Ltd. (model 247). Each region of a test card was printed using a defmedpattern dictated by the screen used for printing, as shown m ~IG. 11. Screens were selected based on the particulate and solvent c~ ;.." of the inlcs used for printing.
For printing of Ag conduction traclcs to electrodes 200, Ag/AgCI electrodes 210,carbon cul~dn~LiyiLy strips 220, worlcing electrode base pad 230, insulation shroud 240, working electrode 250, cellulose acetate membr~me 260, stainless steel screens of 200 counts per imch mesh size were used with an emulsion thiclcness of 23 ~ amd an angle of orientation of 45~. For printing of silica gel fluid guiding pathways 270 and 275, large phenyl silica fluid guiding pathways 280, and small phenyl silica fluid guiding pathways 290, stainless steel screen of 125 counts per inch mesh size were used with an emulsion thickness of 23 ~ and an angle of orientation of 45~.
Test cards with printed regions were made as follows:
(a) PVC backing sheets (400,um thickness) were sized (45mm x 30mm) using a guillotine to form test cards.
(b) Matt vinyl emulsion paint (crown vinyl matt, Crown Decorative Products Ltd., United Kingdom) painted three times over the entire area of a test card and dried for 30 minutes at 40~C between each coat.
(c) Two Ag conduction tracks 200 (1 layer) (silver ink electrodag 477SS
RFU, Acheson Co!loids, Prairie Rock, Plymouth, United Kingdom) were printed for the working amd l~ ../CUI~ electrodes.
Following deposition the test cards were left to dry for 30 minutes at 40~C.
(d) An Ag/AgCI reference electrode 210 (I layer) (1070 Ag, Materials ~.1.- ". ~. .,,-~;...1 & Analysis Services, Melbourne Science Park, Moat Lane, Cambs, United Kingdom) was printed onto the vinyl painted PVC
test card m an orientation parallel to the guidamce path flow. Following deposition the cards were left to dry for 30 minutes at 40~C.
(e) A carbon base pad 230 (graphite ink electrodag 423SS, Acheson Colloids) of the working electrode and two c~lldu~livi~y strips (graphite wo 96/05510 ~ 7 0 ¢ 1 P~
32.
ink electrodag 423SS~ r~u;~mg the full length of the card were , v ~ly printed using the same screen.
(f) An insulation shroud 240 (1 layer) was printed usmg a organic solvent (matt vinyl white, Apollo Colours Ltd, Plumstead, London, United S Kingdom) and allowed to dry for one hour at 40~C. Using the same screen as for the insulation shroud, a matt vinyl emulsion paint (2 layers) (same paint as Step b) was printed over the insulation layer and allowed to dry for 30 minutes at 40~C.
(g) A working electrode 250 (2 layers) was printed usimg a working electrode ink prepared as 5mg of MCA4 (a catalytic carbon powder based on rhodinized carbon, supplied by Materials, ~I . - - ,.. ;, I ;1 l.. &
Analysis Services) in 5ml of a 3 % (w/v in distilled water) h~d~u~.,;hyll,~,lh~lu~c solution. The working electrode was printed p..l,.-..~...~1_. to the fluid flow m the fluid guiding pathway. The solution was stirred for 1 hour on a rotary stirrer to achieve a il""".t. ~." ' solution. After each application the ink was allowed to dry for 30 minutes at 40~C.
(h) The electrical continuity of both the working and ~ ,/counter electrodes was tested after the printing of working electrode to ensure good contact between the electrode faces and their respective conduction tracks.
(i) A main channel 270 (four layers) and glucose feeder pathway 275 (four layers) were printed using a silica gel paste solution prepared as 7g of high purity grade silica gel without binder (average particle size 5-25 and average pore diauneter 60A), 3g calcium phosphate (dibasic) bmder and 14g of a 3 % (w/v in water) h.~ u~ ,cllulose solution (ambient ;, at 20~C). The solution was stirred by hand using a metal spatula until a uniform . y was achieved. After each printing the four layers, cards were dried for 45 minutes at 40~C.
~v) A cellulose acetate membrane 260 (2 layers) was printed using a cellulose acetate solution prepared as 4 % (w/v) cellulose acetate powder (acetyl content was ~ 1y 40%) m a 1:1 (v/v) solution of 2197061 ~
wo 96/05510 acetone and ~y~ 1. i. ~- .... ~ After each printing the cards were dried for 30 minutes at room t- 1.~ ., r c, (k) The phenyl silica (Phase Scp~tilmc Ltd, Deeside Industrial Park, Clwyd, UK) region >uLlu~ d~g the electrode 280 (4 layers) was printed usrng a phenyl silica solution prepared as phenyl silica paste mixed with a 10% L.~ lL~ ~ylccllulose solution m water in a 2:1 (w/v) ratio.
Hy~Lu.~;lLrl~llulose acts as an organic bmder to allow the paste to be printed. The phenyl silica solution was strrred by hand usrng a glass rod to achieve a uniform c~ y. After each printrng the cards were t dried for 45 mrnutes at room tLl~ c. The phenyl silica paste was prepared as follows:
(1) Phenyl silica particles (2g) of 5~L were weighed in a glass bottle, washed twice with PBS (phosphate buffered salrne) buffer and spun down usrng a centrifuge (5 minutes, at 4000G).
(2) The phenyl silica particles were then incubated in a 5% BSA
rn PBS buffer solution at 4~C on rotary stirrer for 12 to 24 hours.
(3) The resulting BSA blocked phenyl silica was washed three tirnes with PBS buffer to remove unattached BSA, then , l l in a solution of goat serum and PBS buffer (1:1 ratio). Goat serum was rncluded in order to block all of the non-specific binding sites not occupied by the BSA protein.

(4) The phenyl silica paste in goat serum was rncubated at 4~C
for 15 hours then washed three times in PBS buffer and stored at 4~C until required.
(I) A phenyl silica/capture antibody region 290 (2 layers) was printed using a phenyl silica capture antibody solution prepared as follows:
(1) Phenyl silica particles (2g) (5 micron) were weighed in a srnall glass bottle, washed twice with PBS buffer and spun down usmg a centrifuge (5 mmutes, at 4000G).
(2) The silica particles were then suspended in 5ml of PBS
buffer and antibody solution (l.Oml, 10.1 mg per ml, goat 21~7~
W0 96/OSS10 ; ~ ~ ~; r~
34.
polyclonal an~t~ d~' was added to the silica. Followrng a complete mixm~g, the solution was incubated for 12 to 24 hours at 4~C on a rotary stirrer.
(3) The phenyl silica/antibody mixture was spun down usmg a centrifuge (5 minutes, 4000G) and washed three times in PBS
buffer to remove unattached antibodies.
(4) Following washing the mixture was rncubated with a 5%
BSA in PBS buffer solution for 12 to 24 hours at 4~C, on a rotary stirrer.

(5) The BSA blocked phenyl silica was washed three times with PBS buffer to remove unattached BSA, then ~ ;I m a PBS buffer/goat serum (1: 1 ratio) solution for 16 hours at 4GC.

(6) After this final blocking stage, the BSA/goat serum blocked silica was washed three times with PBS buffer and stored at 4~C
un~il required.
After each printing the cards were allowed to dry for 45 minutes at room t.~
(m) Glucose solution (2 ~1 of 0. lM solution) was applied halfway 300 along the glucose delivery pathway and dried for 45 minutes at room ~lu~la ul~. The O.lM glucose solution was prepared in O.lM sodium phosphate pH 6.8 containing O.lM potassium chloride and stored at 4~C
for 18 hours to allow for Illu~u~ldli~
(n) Blocked antibody/conjugate 320 (1.5 ~1) was applied onto the larger coversheet (30mm x lOmm) and dried for 1 hour at room 1 ~l~ ."t...;-Blocked amtibody/conjugate (g~ld~ol.~.,lo~l anti-~-hCG/glucose oxidase mixed 1:1 with 2% BSA in water) solution was prepared by mixmg antibody conjugate with BSA to give a fmal blocking protein of 1 %. Following drying, a narrow strip of masking tape fixed the larger coversheet over the marn (phenyl silica) pathway such that the dried conjugate was ~ ,., t~ 5-7mm upstream of the working electrode area. The second, smaller, coversheet (2 x 15mm) is an option and cam be positioned over the secondary glucose delivery WO 96/OSSIO 2 1 9 7 0 6 1 P~
3S.
pathway. Its function is, if required, to accelerate the flow of fluid through this pathway.
(o) The completed test card was stored at 4~C ur~il use.
FIG. 12 shows a three ~' ~ ' cross-section of the detection zone in FIG.
S 11 showing the PVC backing sheet 310, emulsion paint 320, carbon base pad 330, catalytic carbon 340, cellulose acetate membrane 350, phenyl silica-antibody layer 360, PVC cover-sheet 370, and conjuage 320.
Steps a through n are the preferred order for making such test cards. The order of the steps may be adapted to meet specific needs of other assay reagents. Only some steps may be completed to produce some of the most simplied; ' ~ ~ of the invention, i.e., printing of a fluid guiding pathway with a detection zone.
Test cards were tested using a buffer solution comprising of O.lM sodium phosphate and O.lM potassium chloride pH 7.0 with or without antigen (l,OOOmlU of hCG (final c~ -)). In order to ~l. ",I ~l~,r aprlir~llility of the test to lS operation in complex media, tests were also performed in diluted urine from a nu~ female with and without antigen. The urine was diluted 1:1 with a buffer solution composed of O.lM sodium phosphate containing 0.1 potassium chloride pH
6.8. This sample solution was used as the negative control. To prepare a positive control, the urine/buffer solution was spiked with hCG to give a final ~ of 500rnIU. Tests were performed at an applied potential of +350mV (versus an Ag/AgCI electrode). Presence of an analyte was indicated by an increase in current due to oxidation of H2Ol generated by the captured iromuno-c~ yte complex.
FIG. 13 shows the ele~,llu~ l;~l response of a negative control test card. No antigen (false positive) was detected, as the current maintained a steady-state for over 140 seconds. No antigen was detected m the negative control urine samples as well.
FIG. 14 shows the ele~ u~ response of a positive control test card.
Antigen (true positive) was detected within 120 seconds, as the current linearlyrncreased for over 140 seconds. Antigen was detected in the positive control urine samples as well.
E~AMPLE 4: ~FI.Inn~ AOETATE SL~ EXCLU~ION MEMBRANE FOR THE
DETECr}ON ZONE

WO 96/05510 ~19 ~ 0 6 i r~
36.
Cellulose acetate ",. " 1""", were~tf~sted for their ability to reduce the loss of a detectable signal by reducrng non-specific binding in the detection zone. Test cards were prepared as described in Example 3, except for changes to the method of test card ~ LiUII stated herein. l~f ...1..,- .. ~ were printed on top of test card worlcing electrodes as mixtures of u~, ' ' and acetone with a range of cellulose acetate u... ~....1...1 i....~ (from 0.5 % to 6 % w/v) and were tested using the glucose oxidase assay system discussed herein. t~ of cellulose acetate from 3 % to 5 % (wlv in c f~ and acetone 1:1) reduced ba.,k~lUUUli noise and improved 1U~ludu~ iliLy, and accuracy of test card assays. Cellulose acetate c~ . - ~ ~ less than 3% (w/v) (.5% to ~u,ul~ , 3%) were not suitable for printing inks, as the viscosity did not permit printing Phenyl silica could be printed directly over the cellulose acetate membrane without disrupting the structure of the membrane.
Cellulose acetate .. ... l ,.. prepared from a solution of cellulose acetate (4 % in a 1:1 mixture of acetone and .,y. 1. .' ~ . .f ) produced the best I~ k~lUUIId signals, response times and printing ~1 r ,~ , as well as improving the ~~,u-udu~.il~ili~y, and accuracy of test card assays.

EXAIMPLE 5: FLOW A~'~'FIFR~TOR5 To P~Uf;3, CONJUGAT~ RETEr~TION
The use of flow - ---' to reduce undesired conjugate retention was tested using conjugate releasing reservoirs applied to PVC cover-sheets. Two types of flow a~ffl. ..~ were tested to decrease undesired conjugate retention in the guidancepathways and detection zone: 1) conjugates released from synthetic sponges, such as dir' ..~ ' 6 sponges bonded to PVC coverslips and 2) conjugates released from deposits on the PVC cover-sheets.
Synthetic sponges were bonded to PVC cover-sheets using glue. Several types of sponge materials were tested for their ability to completely release dried conjugate upon hydration. The sponges were cut using a scalpel to a uniform size (15mm long, lOmm wide and 3mm thick) and bonded to a PVC coversheet (30mm long, lOmm wide and 400~m thick) using a UUltUU~.Il,iv.l glue, such as bostik Antibody conjugate (1.5~1) .6 mg per ml was centrally deposited on the sponge surface at a central location upon completion of the bonding of the sponge to the PVC. The cover-sheet was positioned 5-7mm upstream of the electrode detection zone and in capillary contact with the main 219706~, .
wo 96/05510 . ~ ~,J. _ /~S
37.
pathway, in this case ~ " 'y over the main phenyl silica pathway. Masking tape affixed the cover-sheet in place.
Alternatively, conjugate was dried directly on PVC cover-sheets. Conjugate was centrally deposited as 1.5~L1 on the PVC cover-sheets and left to dry for 1 hour at S room i r ' Following drying, the cover-sheet was positioned 5-7mm upstream of the electrode detection zone and in capillary contact with the main pathway, in this case ' ' "~ over the main phenyl silica pathway.
Test cards, with either conjugate releasing sponge cover-sheets or conjugate releasing PVC cover-sheets, were tested by immersmg the test cards in a buffer solution amd assaying for hCG using the sandwich glucose oxidase system described herein. Negative control solutions contamed no antigen; positive control solutions contained a standard (SOOmlU) hCG c~ .--- .,u,-~ Following complete washing (until complete saturation of the silica gel on the test card), the cover-sheet was removed and 2~1 of a O.lM glucose solution directly pipetted onto the surface of the electrode area lS m either conjugate releasing sponge cover-sheet or conjugate releasing PVC cover-sheet test cards. Negative controls for both types of cards clearly showed no el~ .u.,L.,I..i..al response from the immuno-complex mdicatmg tbat unboumd conjugate has been removed from the electrode site. In contrast, positive controls for both types of cards clearly showed an .~Ie~LIU~h~ aI response from a sample solution containing hCG.

Claims

Claims 1. A fluid transport device comprising:
a backing sheet, and at least one fluid guiding pathway defined by a pattern of material through which fluid can flow and printed onto said backing sheet.
2. A device according to claim 1 wherein said pattern provides at least two fluid guiding pathways extending from a liquid application zone on said backing sheet, wherein liquid passes into each of said at least two fluid guiding pathways during use, said at least two fluid pathways form a merged zone, wherein flows that have passed along the two pathways are combined; and wherein said fluid guiding pathways aresuch that transit times taken for liquid or solutes to flow from said liquid application zone to said merged zone is different depending on the pathway.
3. A device according to claim 2 further comprising at least two different fluid guiding pathways with regions that differ in chemical or physical composition to produce different flow rates for liquids or regions that differ in RF values for solutes carried by a liquid, wherein transit times for liquids or solutes through said at least two different fluid guiding pathways are different.
4. A device according to claim 3 wherein at least one fluid guiding pathway has at least a portion comprising nitrocellulose to retard flow of a solute along it.
5. A device according to claim 3 wherein at least one fluid guiding pathway has at least a portion comprising fibrous cellulose to facilitate rapid flow along it.
6. A device according claim 3 wherein different pathways differ in length.
7. A device according claim 6 wherein at least some of said fluid guiding pathways have intermediate portions constituting sites at which components are adsorbed or sites adapted for the application of components or samples, wherein fluid flow along the pathways transports components or sample from said sites.
8. A device according claim 7 wherein said backing sheet comprises a sheet of plastic material or glass.
9. A device according to claim 8 wherein said plastic material comprises a coated layer that enhances the binding of fluid guiding pathway pattern.
10. A device according to claim 9 wherein said coated layer comprises an emulsion paint.

39.
11. A device according to claim 10 wherein said coated layer comprises a carbon-loaded resin.
12. A device according claim 2 wherein said at least two fluid guiding pathways fluidly communicate with a detection zone; and the device further comprises an electrode assembly printed on said backing sheet and disposed in relation to the detection zone to enable the detection of a chemical species.
13. A device according to claim 12 wherein the electrode assembly provides a working electrode and a reference electrode.
14. A device according to claim 13 wherein the reference electrode comprises Ag/AgCl ink.
15. A device according claim 12 comprising at least one reagent immobilized at said detection zone, said at least one reagent is adapted to interact with one or more assay components that are conveyed to the detection zone along one or more fluidguiding pathways, wherein said interaction is measured by said electrode assembly.
16. A device according to claim 15 wherein said at least one immobolized reagents is a natural or synthetic peptide receptor molecule or a fragment thereof having binding activity.
17. A device according to claim 15 wherein said at least one immobilized reagent isantibody or a fragment thereof having binding activity.
18. A device according to claim 16 wherein a said reagent immobilized at the detection zone is a single-stranded nucleic acid.
19. A device according to claim 18 wherein a said reagent immobilized at the detection zone is a member of a specific binding pair.
20. A device according to claim 12 wherein at least one pathway has a reservoir zone spaced from the detection zone, said reservoir zone comprises a component that is adapted to be transported to the detection zone by fluid flow through said fluid guiding pathway.
21. A device according to claim 20 wherein the reservoir region bears an immobilized nucleic acid strand to which a labelled strand of nucleic acid which is partially complementary is bound, so as to be displaceable by a more complementary nucleic acid sample and transported to the detection zone.

40.

22. A device according to claim 21 wherein said reservoir zone comprises a transportable component with a first binding portion adapted to bind to a first portion of an analyte and a second signal portion; and said detection zone comprises an immobilized reagent adapted to bind to a second portion of the analyte and not prevented by said first portion of an analyte from being bound to said first binding portion of the transportable components.
23. A device according to claim 22 wherein the analyte comprises a nucleic acid sequence and said first and second portions are discrete portions of said sequence;
said first binding portion and immobilized reagent comprising nucleic acid sequences complementary to said first and second analyte portions, respectively.
24. A device according to claim 23 wherein said transportable component is an enzyme-streptavidin conjugate; and said detection zone comprises an immobilized component for binding biotinylated analyte.
25. A device according claim 22 wherein there is a detection zone where analyte which has been applied to the device gives rise to a detectable phenomenon, and wherein said detection zone has means for tending to retain, or reduce the loss of, species responsible for the detectable phenomenon.
26. A device according to claim 25 wherein said detection zone extends over only a part of the width of a pathway, and is delimited at one or both sides by barriers to lateral diffusion.
27. A device according to claim 26 having a trapping zone provided in the region of the detection area.
28. A device according to claim 27 wherein the trapping zone is provided by a charged layer or by a material having size exclusion properties.
29. A method of manufacturing a fluid transport device comprising:
providing a backing sheet, and printing on to said backing sheet a material thatprovides at least one fluid guiding pathway.
30. A method according to claim 29 wherein said printing comprises applying a slurry of a material in an organic solvent.
31. A method according to claim 30 wherein said material comprises silica, cellulose, a silica derivative or a cellulose derivative.

41.

32. A method according claim 31 wherein the printing technique is screen printing.
33. A method according to claim 32 as applied to the production of a device according to any of claims 14 to 29.
34. A method according to any of claims 33 comprising a further step of applying to an intermediate portion of at least one pathway a reagent substance.35. A method according to claim 34 wherein said further step employs a printing technique.
36. A method according to claim 29 wherein at least one pathway portion was produced by applying by a printing technique a mixture of material for providing a fluid pathway and a reagent substance.
37. A method according to claim 36 wherein either before or after said application of material for providing at least one fluid pathway, an electrode assembly is applied to the backing sheet by a printing technique; said at least one fluid pathway having a detection zone which overlies or underlies at least part of the electrode assembly.
38. A method according to claim 38 wherein the electrode assembly is applied by screen printing.
39. A method of claim 29 where said printing comprises applying a slurry of a material in an aqueous solvent.
40. A printing composition for printing fluid guiding pathways on test cards comprising:
phenyl silica paste mixed with a hydroxyethylcellulose aqueous solution at a ratio of .2:1 to 9:1 (w/w) of said phenyl silica paste to said hydroxyethylcellulose aqueous solution, respectively.
41. The printing of claim 40 wherein said hydroxyethylcellulose aqueous solution is a 1% to 25% (w/v) solution of hydroxyethylcellulose in water and a binder.
42. The printing composition of claim 41 wherein said hydroxyethylcellulose aqueous solution is a 5% to 15% (w/v) solution of hydroxyethylcellulose in water.
43. The printing composition of claim 41 wherein said ratio of said phenyl silica paste to hydroxyethylcellulose aqueous solution is a .5:1 to 2:1 (v/v) ratio.

42.

44. The printing according of claim 43 wherein said to hydroxyethylcellulose aqueous solution is a 10% (w/v) solution of hydroxyethylcellulose in water.
45. The printing composition of claim 44 wherein said ratio of said phenyl silica paste to hydroxyethylcellulose aqueous solution is a 1:1 (w/w) ratio.
46. The printing composition of claim 40 wherein said phenyl silica paste is hydrated with phosphate buffered saline and further comprises bovine serum albumin or goat serum.
47. The printing composition of claim 40 wherein said phenyl silica paste further comprises a capture antibody.
48. The printing composition of claim 47 wherein said conjugate is selected form the group consisting of antibody/glucose oxidase, peptide/glucose oxidase, antibody/alkaline phosphatase, peptide/alkaline phosphatase, antibody/lactate otidase, peptide/lactate oxidase, antibody/glutamate oxidase, antibody/glutaminase-glutamine oxidase, peptide/glutaminase-glutamine oxidase, antibody/alcohol oxidase, and peptide/alcohol oxidase.
49. The printing composition of claim 41 wherein said binder is calcium phosphate.
50. The printing composition of claim 41 wherein said paste is 30 to 60%
(w/v) phenyl silica.
51. The printing composition for fluid guiding pathways on test cards of claim 48 wherein said printing composition is 1 to 27 poise at 25°C.
52. A printing composition for printing fluid guiding pathways on test cards comprising:
cellulose acetate at a concentration of 3% to 10% (w/v) in an organic mixture further comprising a first organic solvent of having a boiling point between 100 to 175°C and a second organic solvent having a boiling point between 45 to 65°C, wherein said first organic solvent and said second organic solvent are mixed in at least a .25:1 ratio.
53. The printing composition of claim 52 wherein said cellulose acetate has an acetyl content of at least 40%.

43.

54. The printing composition of claim 53 wherein said cellulose acetate is dissolved in said organic mixture at a concentration of 3.5 to 8% (w/v).
55. The printing composition of claim 54 wherein said cellulose acetate is dissolved in said organic mixture at a concentration of 4 to 5% (w/v).
56. The printing composition of claim 54 wherein said first organic solvent is cyclohexanone and second organic solvent is acetone.
57. The printing composition of claim 56 wherein said cyclohexanone to said acetone ratio is .5:1 to 2:1, respectively.
58. The printing composition of claim 57 wherein said cyclohexanone to said acetone ratio is 1:1, respectively.
59. The printing composition of claim 58 wherein said cellulose acetate concentration is 4% (w/v) in said organic mixture.
60. The printing composition of claim 53 wherein said paste is 30 to 60%
(w/v) phenyl silica in water.
61. The printing composition of claim 60 wherein said printing composition has 1 to 27 poise at 25°C.
62. A printed fluid guiding pathway with a detection zone comprising:
a backing sheet suitable for printing, a printed electrode assembly or optical assembly, and a printed fluid guiding pathway to provide directional fluid transport through said printed detection zone during detection.
63. The printed detection zone of claim 62 wherein said backing sheet is coated with an emulsion paint.
64. The printed detection zone of claim 62 wherein said backing sheet has a carbon base pad.
65. The printed detection zone of claim 62 comprising said electrode assembly and wherein said fluid guiding pathway further comprises silica having an aromatic ring.
66. The printed detection zone of claim 65 wherein said fluid guiding pathway further comprises phenyl silica.
67. The printed detection zone of claim 66 wherein said fluid guiding pathway further comprises immobilized protein.

44.
68. The printed detection zone of claim 67 wherein said immobilized protein captures an analyte for one of the following assays, HIV, hepatitis, gonorrhea, syphilis, salmonella, campylobacter and herpes.
69. The printed detection zone of claim 67 wherein said immobilized protein captures an analyte for one of the following assays hCG, urease, cholesterol, bilirubin, glucose or lactate dehydrogenase.
70. The printed detection zone of claim 67 further comprising a flow accelerator.
71. The printed detection zone of claim 67 wherein said flow accelerator is located upstream of said electrode assembly.
72. The printed detection zone of claim 67 further comprising an exclusion membrane 73. The printed detection zone of claim 71 wherein said flow accelerator is a cover-sheet comprising a dried protein conjugate.
74. The printed detection zone of claim 67 wherein said conjugate is selected from the group consisting of antibody/glucose oxidase, and peptide/glucose oxidase 75. The printed detection zone of claim 74 wherein said electrode assembly is a two electrode system with a catalytic carbon electrode.