CA2179309A1 - Diagnostic flow cell device - Google Patents
Diagnostic flow cell deviceInfo
- Publication number
- CA2179309A1 CA2179309A1 CA002179309A CA2179309A CA2179309A1 CA 2179309 A1 CA2179309 A1 CA 2179309A1 CA 002179309 A CA002179309 A CA 002179309A CA 2179309 A CA2179309 A CA 2179309A CA 2179309 A1 CA2179309 A1 CA 2179309A1
- Authority
- CA
- Canada
- Prior art keywords
- flow cell
- flow
- flow channel
- silver
- reagent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0689—Sealing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0636—Integrated biosensor, microarrays
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0645—Electrodes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0654—Lenses; Optical fibres
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0877—Flow chambers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/817—Enzyme or microbe electrode
Abstract
The present invention provides a diagnostic flow cell for determining the presence or amount of an analyte which may be contained in a test sample. The flow cell comprises a spacing layer having a longitudinal void disposed between a pair of opposed substrates. The spacing layer and the opposed substrates define a flow channel wherein reagent means can be immobilized. When the immobilized reagent means is contacted with an analyte, the reagent means can produce an electrically, optically, or electrically and optically detectableresponse to the analyte. Hence, the reagent means that is immobilized within the flow channel can comprise (i) a counter electrode, a reference electrode anda working electrode, (ii) an optically sensitive dye or (iii) a counter electrode, a reference electrode and a working electrode and an optically sensitive dye. The flow cell can be interfaced with means for introducing a test sample into and out of the flow cell's flow channel and detection means for detecting a signal generated by the immobilized reagent means. The present invention also provides methods for detecting the presence or amount of an analyte which may be contained in a test sample.
Description
WO 95/22051 ~ 1. 7 9 3 0 3 F~,l/o~ S.tlS00 DIArT~OSTIC FLOW CELL DEVICE
Field of thP Tnvention The present invention relates to a flow cell device for 5 detecting the presence or amount of an analyte in a test sample.
In particular, the present invention relates to a flow cell device having immnhi1i7pd reagent means which produce an Pl~c~T~ lly or optically riPtecTe~hlp response to an analyte which may be contained in a test sample.
F~A l~ v ~ u~ l . .ri of thP Invention Tmmnhili7Pd catalytically-active-m~lpc~llp~ such as enzymes have been used as binding participants to ~iPtPrminP the presence or amount of the immnhili7ed enzyme's substrate that may be 15 present in a test sample. CU--~ UC~-~A1 ILt:l Ui~y analyzers use enzymes, which have been bound to porous surfaces, for the . Ull~ io.l of enzyme ~,uI,,,i~c.~s to optically or electrorhP7nir~11y riPt~C~ hlP products. For example, Cl~t1U 1I~ 1 sensors utilize the ability of an immnhili7.pd enzyme to form an ~ ~u.1l....;. ~lly 2 0 active molecule as a result of t,he action of the enzyme on its substrate. Such sensors employ a pu~ rl~l and ~LU,U~.ulUtl,llC
electrodes that typically consist of an enzyme or working electrode, a reference electrode and a counter electrode. The enzyme electrode is ty-pically made of platinum and is supplied with an 2 5 overlaying oxidase enzyme layer. When the surface of an eleckode is i_mersed in a sample c.,..i~;.,;..g an ~x;.1; .A1~1r substrate and mn1Pcn1slr oxygen, both mnlPcl~lP.~ diffuse into the enzyme layer where the substrate reacts with the enzyme resulting in reduction of the enzyme. The reduced enzyme is oxidized by the luc~ u 3 0 oxygen which, in turn, is reduced to perûxide. At a !~. . Ir.. :~. . I.ly high electrode potential (m~int~inPd via the reference electrode), the platinum portion of the enzyme electrode oxidizes the peroxide to l~ c.ie o~ygen and transfer two electrons to the counter electrode. The pu~ . measures the current generated by the 3 5 transferred electrons and the amount of current is related to the amount of n i ii7~hlP substrate in the sample. Hence, the presence WO 9512~051 ~ 3 ~ 9 ~ J C ~oo ` .
and/or amount of an n~i-li7uhlP. 6ubstrate in the sample can be .1~.1~ . .,.;-.PA
Prior to the present invention, devices which L u l.~.";rully or optically detect the presence or amount of an 5 analyte which may be present in a test sample are generally single use 1;O~ devices which are incapable of analy_ing a plurality of circulating test samples.
S~mmurv of the Jnvention According to the present invention, a ~lju~nnc1~r flow cell for ~- ' ....;..;..~ the presence or amount of an analyte in a test sample is provided. The ~iu~nrsti~ flow cell CGI~l,u~ B (i) a spacing layer disposed between a first and a second opposed substrate, wherein the spacing layer has a 1....L;I,...1;-.A1 void and wherein the spacing 15 layer and opposed substrates define a flow channel; (ii) fastening means for coupling the spacing layer and the opposed O~OI.l~.leO, (iii) inlet means for p-.. ;I ~ .... ~ a sample to enter the flow channel;
(iv) outlet means for ~... ; ~ ': .. ~ the sample to exit the flow channel; and (v) immnhili7pd reagent means for ,UlUdU~l~lg a 20 .lptpctDhl~p signal, wherein the reagent means is at least partial~y contained within the flow channel. Using methods well known in the art, the flow cell can be intPrfurPd with detection means for detecting a signal 6~ L~d by the immnhili7Pd reagent means.
A-1tli*~nully, mPtlln(lnlngiP.c are well known in the art for placing 25 the flow cell in fluid cnmmllniruti~n with flow means for --Llu~u~lg a test sample into the flow cell'6 flow channel.
The immnhili7Pd reagent means can compri6e (i) a counter electrode, a reference electrode and a working electrode or (ii) an optically sensitive dye. Ad~ L~6~uoly~ rt.nnhinu*~mc ofthe 3 0 reagent means can be employed.
The present invention also provides methods for detecting the presence or amount of an analyte which may be contained in a test sample. According to one Pmho-liml~nt of the invention, the flow cell device can generate an PlPrtrirully (1PtP~^l~lP response to 3 5 an analyte that may be present in a test sample.
wo 95122051 ~ 1 7 9 3 0 9 ~ Soo According to another Pmho~imPnt of the invention, the assay device can generate an optically tlPt~D~tshlP response to an analyte that may be present in a test sample.
According to still another .-.. h~.l;.. -.. l. of the invention, the 5 assay device can generate optically and el~ u. l.~.";. ~lly (~DfDrt 11" 1eVl~v~ 3 to analytes which may be present in a test sample.
Due to the lûw costs ~qqoris~tDd with the m~ .e of the flow cell, it can be used as a l;~l.v~ .l,ls assay unit. However the 10 present invention provides an assay device that can be used for multiple assay6 and is thereby reusable.
Description of the Drawin~q Figure 1 illustrates an expanded view of an C1~ ...;rsll flow cell.
Figvre 2 ill-l'tr~tRq a partially cnmrlDtDd electrorhPmir~l flow cell.
Figure 3 illustrates an ~qcpnnhlpd electrochemical flow cell which is intPrfslr~Pd with detection means.
Figure 4 illustrates a cross sectional view of an ~qqPmhlDd electrorhPmir~l flow cell.
Figvre 5 illll"r~tPq an expanded view of an optical flow cell.
Figure 6 illustrates an ~qqPmhl~Dd optical flow cell.
Figure 7 illustrates a cross sectional view of an ~ .lPd 2 5 optical flow cell.
Figure 8 graphically illustrates an elFv~ u~ ;r~l flow cell's working electrode response to varying concentrations of glucose.
Figure 9 graphically illustrates an optical flow cell's 30 response to varying .~ I;.- .q of dissolved oxygen.
WO 9S/22051 217 ~ 3 Q g r~ soo , !, ';:
Field of thP Tnvention The present invention relates to a flow cell device for 5 detecting the presence or amount of an analyte in a test sample.
In particular, the present invention relates to a flow cell device having immnhi1i7pd reagent means which produce an Pl~c~T~ lly or optically riPtecTe~hlp response to an analyte which may be contained in a test sample.
F~A l~ v ~ u~ l . .ri of thP Invention Tmmnhili7Pd catalytically-active-m~lpc~llp~ such as enzymes have been used as binding participants to ~iPtPrminP the presence or amount of the immnhili7ed enzyme's substrate that may be 15 present in a test sample. CU--~ UC~-~A1 ILt:l Ui~y analyzers use enzymes, which have been bound to porous surfaces, for the . Ull~ io.l of enzyme ~,uI,,,i~c.~s to optically or electrorhP7nir~11y riPt~C~ hlP products. For example, Cl~t1U 1I~ 1 sensors utilize the ability of an immnhili7.pd enzyme to form an ~ ~u.1l....;. ~lly 2 0 active molecule as a result of t,he action of the enzyme on its substrate. Such sensors employ a pu~ rl~l and ~LU,U~.ulUtl,llC
electrodes that typically consist of an enzyme or working electrode, a reference electrode and a counter electrode. The enzyme electrode is ty-pically made of platinum and is supplied with an 2 5 overlaying oxidase enzyme layer. When the surface of an eleckode is i_mersed in a sample c.,..i~;.,;..g an ~x;.1; .A1~1r substrate and mn1Pcn1slr oxygen, both mnlPcl~lP.~ diffuse into the enzyme layer where the substrate reacts with the enzyme resulting in reduction of the enzyme. The reduced enzyme is oxidized by the luc~ u 3 0 oxygen which, in turn, is reduced to perûxide. At a !~. . Ir.. :~. . I.ly high electrode potential (m~int~inPd via the reference electrode), the platinum portion of the enzyme electrode oxidizes the peroxide to l~ c.ie o~ygen and transfer two electrons to the counter electrode. The pu~ . measures the current generated by the 3 5 transferred electrons and the amount of current is related to the amount of n i ii7~hlP substrate in the sample. Hence, the presence WO 9512~051 ~ 3 ~ 9 ~ J C ~oo ` .
and/or amount of an n~i-li7uhlP. 6ubstrate in the sample can be .1~.1~ . .,.;-.PA
Prior to the present invention, devices which L u l.~.";rully or optically detect the presence or amount of an 5 analyte which may be present in a test sample are generally single use 1;O~ devices which are incapable of analy_ing a plurality of circulating test samples.
S~mmurv of the Jnvention According to the present invention, a ~lju~nnc1~r flow cell for ~- ' ....;..;..~ the presence or amount of an analyte in a test sample is provided. The ~iu~nrsti~ flow cell CGI~l,u~ B (i) a spacing layer disposed between a first and a second opposed substrate, wherein the spacing layer has a 1....L;I,...1;-.A1 void and wherein the spacing 15 layer and opposed substrates define a flow channel; (ii) fastening means for coupling the spacing layer and the opposed O~OI.l~.leO, (iii) inlet means for p-.. ;I ~ .... ~ a sample to enter the flow channel;
(iv) outlet means for ~... ; ~ ': .. ~ the sample to exit the flow channel; and (v) immnhili7pd reagent means for ,UlUdU~l~lg a 20 .lptpctDhl~p signal, wherein the reagent means is at least partial~y contained within the flow channel. Using methods well known in the art, the flow cell can be intPrfurPd with detection means for detecting a signal 6~ L~d by the immnhili7Pd reagent means.
A-1tli*~nully, mPtlln(lnlngiP.c are well known in the art for placing 25 the flow cell in fluid cnmmllniruti~n with flow means for --Llu~u~lg a test sample into the flow cell'6 flow channel.
The immnhili7Pd reagent means can compri6e (i) a counter electrode, a reference electrode and a working electrode or (ii) an optically sensitive dye. Ad~ L~6~uoly~ rt.nnhinu*~mc ofthe 3 0 reagent means can be employed.
The present invention also provides methods for detecting the presence or amount of an analyte which may be contained in a test sample. According to one Pmho-liml~nt of the invention, the flow cell device can generate an PlPrtrirully (1PtP~^l~lP response to 3 5 an analyte that may be present in a test sample.
wo 95122051 ~ 1 7 9 3 0 9 ~ Soo According to another Pmho~imPnt of the invention, the assay device can generate an optically tlPt~D~tshlP response to an analyte that may be present in a test sample.
According to still another .-.. h~.l;.. -.. l. of the invention, the 5 assay device can generate optically and el~ u. l.~.";. ~lly (~DfDrt 11" 1eVl~v~ 3 to analytes which may be present in a test sample.
Due to the lûw costs ~qqoris~tDd with the m~ .e of the flow cell, it can be used as a l;~l.v~ .l,ls assay unit. However the 10 present invention provides an assay device that can be used for multiple assay6 and is thereby reusable.
Description of the Drawin~q Figure 1 illustrates an expanded view of an C1~ ...;rsll flow cell.
Figvre 2 ill-l'tr~tRq a partially cnmrlDtDd electrorhPmir~l flow cell.
Figure 3 illustrates an ~qcpnnhlpd electrochemical flow cell which is intPrfslr~Pd with detection means.
Figure 4 illustrates a cross sectional view of an ~qqPmhlDd electrorhPmir~l flow cell.
Figvre 5 illll"r~tPq an expanded view of an optical flow cell.
Figure 6 illustrates an ~qqPmhl~Dd optical flow cell.
Figure 7 illustrates a cross sectional view of an ~ .lPd 2 5 optical flow cell.
Figure 8 graphically illustrates an elFv~ u~ ;r~l flow cell's working electrode response to varying concentrations of glucose.
Figure 9 graphically illustrates an optical flow cell's 30 response to varying .~ I;.- .q of dissolved oxygen.
WO 9S/22051 217 ~ 3 Q g r~ soo , !, ';:
DPt~ilpd DPcrr~Dtion nf rhr- InvPn~;~.n The following rlPfinitirnc are ~rplir~hlP to the invention:
I. DEFINITIONS
The term "analyte", as used herein, refers to the rrnnrolm or rrnnrrAit;~n to be detected or ~D~d and which initiates the generation of a ~ lf- response. Analytes include, but are not intended to be limited to, enzymes or enzyme substrates, metal ions, blood gases, toxins, organic ~ .u ...~lc, proteins, peptides, 10 a_ino acids, carbohydrates, nucleic acids, hrrmrnPc, steroids, vitamins, drugs (inrlll~in~ those ~lminil ~ d for therapeutic purposes a3 well as those ~minictDred for illicit purposes), and mPt"h(~litPC of any of the above substances. For example, such analytes include, but are not intended to be limited to, alanine 15 aminuLl~lDrel ~c (ALT), aspartate aminuL~llDr~ 3c (AS~), creatinine kinase (CK), creatinine kinase MB (CK-MB), lactate dellyLu6~as~ (LDH), garnma glutarnyl ~ vt-~uL ~l~rc (GGTP), alkaline rhr~ , glucose, fructose, g;~ r~ P sucrose, lactose, lactate, ;l 1 ul~ urea, creatinine, tri~l~.;deD, uric 20 acid, bilirubin, 1~.1u~ , p~ ..;- -.., sodium, chloride, calcium, cL;u..., lithium, oxygen, carbon dioxide, hydrogen ions (pH), h.. ~"~lVl.;.l, glycated hPmnglrhin (Gly. Hb), C-reactive protein, serum li~u~ul~ills~ serum albumin, deoxyrihrnllrlPir acid (DNA), rih~-mlrlPio acid (RNA), bile acids, salicylates, A~' ...;.. ~l.~.., 2 5 theophylline, p_t~ll.y Luhl and the like.
The term "test sample", as used herein, refers to a material sll~pected of con~inin~ the analyte. The test sample can be used directly as obtained from the source or following a pre-L~ L~ellL to modify the character of the sample. The test sample can be derived 3 0 from any biological source, such as a physiological fluid, inrlll~in~, blood, saliva, ocular lens fluid, cerebral spinal fluid, 6weat, urine, milk, ascites fluid, mucous, synovial fluid, pPrit~nP~l fluid, amniotic fluid and the like, and fPrmPntsltirn broths cell cultures, and chemical reaction mixtures and the like.
3 5 The test sample can be pretreated prior to use, such as ~UIt:~U illg plasma from blood, diluting viscous fluids, and the like. Methods WO 9S1~051 PCTIUS95101500 ~17~30g of i.~:~.L. ~ L can involve filtration, ~1iFt~ ti~-n, ~u~ .lL~Liu inactivation of i"~. r~.;"~ c~-mr~mPntC~ and the addition of reagents. In addition to biological or physiological fluids, other liquid samples can be used such as water, food products and the 5 like for the 1.. r,.. i.. ,. P of ~:.lvilu.. l.. ~.~Lidl or food production assays. In addition, a solid material sllcpertPd of ~ the analyte can be used as the test sample. In some inQt~nrPc, it may be beneficial to modify a solid test sample to form a liquid medium or to release the analyte.
II. FLOW CELLS
The present invention is directed to a lliA~n~ flow cell rt~mrricin~ two opposed substrate layers and a spacing or gasket layer disposed between the two opposed substrate layers. The 15 spacing or gasket layer has a lon~ibl-lin~l void which, in c.. l.;"--~.;on with the ~ub~L z~Les, defines the flow cell's flow channel. A substrate layer, or ~e substrate layers, can be supplied with apertures which, when the flow cell is slccPmhlP~i can serve as inlet and outlet means for illL u~u~hlg sample into the 2 0 flow channel and allowing sample to exit the flow channel.
Preferably, the apertures are supplied to one substrate and are located near the ends of the flow channel defined by the ~ub~L c.L~s and the spacing layer.
Reagent means that can generate a ~PtP~t~hlP signal when 2 5 contacted with an analyte can be imm~lhi1i7Pd on a substrate or the Sllhqt~At~C Typically, the immohi1i7Pd reagent means is sllhst~nti~lly insoluble. C~nnceq~lPnt1y, the reagent means can be contacted with multiple test samples thereby making the flow cell reusable. Depending upon the analyte to be detected, the opposed 3 0 ~..h~ c can have a single or multiple reagent means immtlhili7Pd thereon. Thus, by immllhili7inF the ~ v~l;aL~
reagent means, a single flow cell can, for example, electrically and optically detect analytes which may be present in a test Qsample.
A-l~itinn~lly, by immohili7in~ a mlllbrli~ity of the same reagent 3 5 means to a substrate or the substrates, a single flow cell can detect the presence or amoumt of an analyte in replicates.
WO 95/22051 PCI/I[JS95/01500 21~9309 The various layers that can comprise the flow cell herein provided can be applied to the opposed s~ f~AtPc~ and the substrates can then be coupled with a gasket layer to thereby form the flow cell taught herein. Due to the de6ign of the flow cell and 5 the manner in which it can be ~ rA~ the (liAl nf~;c flow cell i8 reusable, int:~dllS;ve: to produce, easily stored in a complete or inrnmrl~t~ form, capable of being patterned with immnbili reagent means, and, as compared to previous te~`hn~' )C.Y~ the number of mA~-hinPd parts is greatly reduced. Moreover, signal to 10 noise ratios which are ~_.lt:l.lLdd by the reagent means are increased and cl~_LI.~...A~..^';~ h.~ es are reduced.
The opposed substrates can be made of any chemically inert, non-culldu~ Liv~, and physically durable material which is capable of supporting the va~ious rnaterials applied thereon. P ~ ?v of 15 such materials include, but are not intended to be limited to film plastics such as polyester, poly~l,uuAAL~, pûly~Ly ~:ue, polyeth~rimirl~, and the like; molded plastics such as acrylic, phenolic, p~lyl 1 ~, and the like; ceramics such as alumina (Al203), ~irconia (ZrO2), magnesia (MgO), and tbe like; glass;
2 0 silicon wafers; and the like; preferably the substrate material comprises a polyester film.
The spacing or gasket layer materials are typically chemically and dc_LIu- 1,~,.,;. Ally inert as well as sllh~snt;slly non a~sulb~llL and impervious to water. ~YA~nrl~ of materials 2 5 that have these properties include, but are not intended to be limited to printing inks, painted inks, sprayed inks, late~es, urethanes, vinyls, polyesters, film plastics and the like, ~l~f~ ly the spacing layer ~ a dielectric printingink. The thickness of the flow cell's spacing layer is largely r~r-ln~ihl~ for 3 0 the volume of the flow channel. Thus, for e~ample, when it is desirable to have lar~e amounts of sample contained within the flow channel, the thickness of the spacing layer can be increased.
The amount of sample ~ ntAin~d in the flow channel, in relation to the exposed substrate surface area, is preferably in the range of 3 5 between about 1.0 ,u~/cm2 and about 500 ,ul/cm2, more ~u. ~ kly WO 95122051 _ 7 _ PCI/US95/01500 between about 2 ~Icm2 and about 250 Ill/cm2 and most ~!Jlerel~bly between about 2.6 ~l/cm2 and about 100 Ill/cm2.
The (1iA~nnqtic flow cell can further comprise a mask layer immnhili~ad between the two 6ubstrate layers. Similarly to the 5 spacing layer, the mask layer is typically chemically and electrnrh~smirAlly inert as well as non abDvll,ellL and impervious to water. ~ mrl~a~ of suitable mask layer materials have been described with reference to the spacing layer. When multiple analytes are to be detected, or rephcates of the same analyte are to 10 be detected, the mask layer is particularly preferred and serves as a barrier between the various imm-)hili~ad reagent means.
The opposed Dul)bLl~lLes7 and the various layers that can be disposed between the ~ Pc, can be secured or coupled to one another to form a flow cell using fastening means such as, for 15 example, lAminAt~ solvent bonding, nuts and bolts, rivets and the like, ~u~er~ Lly an a&esive layer secures the Ellh--~rAt~a~, and the various layers thereon, to each other. An adhesive layer is y~ere~c.hly inert chemically and ele~Llu 1.~ lly as well as being capaMe of retaining its adherent quality in saline solutionE.
2 0 l;~Ampl~s of such materials include, but are not limited to ultraviolet cured pressure sensitive adhes*es, heat cured pressure senDitive adhesives, vinyl based pressure sensitive adhesives, and the like, preferably a polyurethane based ultravioletly cured adhesive.
A. ELECTROCHEMICAL FLOW CELLS
In the case where the ~ .LII~ n flow cell is capable of lu 1,~... - ~lly detecting an analyte that may be contained in a test sample, the sllhri~At~ can have reagent means immnhili~`ad 3 0 thereon which form a reference electrode, a counter electrode and a working electrode. T_e reference electrode can comprise (i) a material or a romhinAti-m of materials capable of ~shili~7in~ a test sample's potential or otherv~ise providing a constant potential within a test solution; and (ii) a ~ udu~,~ve trace material which is 3 5 capable of being intPrf~rad with a detection means and which is D~ ally inert at the assay device's operating potential.
WO 9S/22051 E~ ,' vlS00 ~179309 Materials capable of d~eloui..~ a stable potential include oxidation/lGdu~ liull pairs (variably referred to as "redox couples") inr11lrlin~, but not intended tû be limited to, silver/silver chloridc/~ uLl,G blends, lU~ /lu~.. ~uu6 chloride blends, 5 silver/silver iodide blends and the like, preferably silver/silver chloride blends which can be ~ .Red u6ing screen printing tPrhniqllPR F. 1 of materials that can be used as WlldU~
trace material include, but are not intended to be limited to gold, carbon, nickel, silver, palladium, rllthPnillm, rhodium, tin o~ide, 10 indium tin ûxide and the like, ~l~rG~bly carbon that has been dispersed in a screen printing ink.
The counter electrode can comprise an electrûrhPmir~lly Culldu~ c material which is relatively inert at the a66ay device's operating potential. F. 1 of materials having these 1 5 properties include, but are not intended to be limited to gold, carbon, nickel, silver, r~ lillm, rllthPnillm, rhûdium, tin ûxide, indium tin oxide and the like, ~ bly carbon that has been dispersed in a screen printing ink.
The working electrode can comprise (i) a culldu~ G trace 20 material and ii) an enzyme or enzymes imm~ili7Pd to or in contact with the Culldu~ , trace. P~r_~Lly, the enzyme is imm~-hili7Pd on the conductive trace such that when the flow cell is ..... hlrrl, the enzyme will remain H~. ~ C for multiple u6es.
A particularly preferred method of immnhili~n~ an enzyme to the conductive trace material employs an immnhili7~t;~m medium disclosed in co-owned and co-pending ~rFlir~tinn Serial No.
(Atty Docket No. 5488.US.01), entitled BIOREAGENT
IMM(lRTT,T~ATION MEDIUM, filed on even date herewith and incorporated herein by reference.
The ~iUlGC.6~1~ immnhili7~tinn medium rn7nrriRPR i) an enzyme which is immnhili7Pd to a solid phase and ii) a binding reagent comprising a latex resin, wherein the immnhili7Pd enzyme is evenly dispersed. The binding reagent may also include optional ih~Gdi~llL~ which enhance the immnhili7sltinn medium's chemical and physical properties. The enzyme can be immnh~ pd to the solid phase by methods well knûwn in the art such as, for WO 9S/220S1 2 1 7 ~ 3 ~ 9 P .~ ISOO
~ .
_ 9 _ example, covalent, ionic or ads~l~iiv. bonding of the enzyme to the solid phase. By way of example and not of limitsltinn, the various enzymes, solid phases, resins and optional ingredients that can be employed in the bioreagent immnhili7~nn medium can be found below in Table 1.
OPTIONAL
ENZYMES SOLID PH~SES RESINS INGREDIENTS
glucose oxidase, agarose and acrylic latex, pl~o~;ri7Prc, film ~l~.l.,.. ,.~e d~.;vaLivGs styrene acrylic forming agents, oxidase, lactate thereof, latexes, vinyl thickeners, oxidase, glycerol polyacrylamide acetatelatex and stabilizers, rhnBph~tP and dGl;v~ s polyulGlllane dispersing oxidase, thereof, silicas, latex agents, and cholesterol aluminosilicates, dPfo~min~
oxidase, aluminum agents cholesterol oxides, carbon or esterase, lipase, graphite glycerol kinase, particles, and ~lllt.~m~t.P platinum group dehydL ug~.lase, metal oxides creatinine rlPslmin~cP, and uricase 1 0 In order to allow easy interface with detection means for detecting a signal ~G~ led by the immnhili7Pd reagent means, it is preferred that a portion of the immnhili~Pd reagent means is rnnt:RinPd within the flow channel and a portion of the immnhili7Pd reagent means extends out of the flow cell's ~ow channel.
1 5 It will be ulldGl~uûd~ of course, that the present invention is not limited to flow cells having three electrode systems, and that two electrode systems are ~ For example, a two WO 9S/22051 r~ IS00 ~17930~
electrode ~ullfl~ul~Lull can comprise a working electrode and a cnmhinslt;nn rt~r~ ~ce/~uullLtl electrode.
B. OPTICAL FLOW CELLS
In cases where the rliA~n~ ;c flow cell ûptically detects the presence or amûunt of an analyte which may be contained in a test sample, the immnhili7~d reagent means generates an optically A~t~rtSIh1f~ signal when it is contacted with an aualyte which may be contained in a test sample. The i nn~-hili7.0d reagent means that can be used in optically based flow cells are generally cu~yuullds or mixtures of r~mrûllnl1~ that, when contacted with an analyte, emit a signal which is optically APt~r~ Examples of such immnhi1i7~sd reagent means include, but are not intended to be limited to pH sensitive dyes; oxygen sensitive dyes; dyes or chelating agents which are sensitive to ions such as calcium and ;-,-.. ions; and the like; lul~r~.Ably platinum tetra(~ n ~ uht llylkorphyrin which is an oxygen sensitive dye that changes its fluorescence lifetime in the presence of dissolved oxygen. Such an optically sensitive dye is disclosed in 2 0 U.S. Pat. No. 4,810,65~ and U.S. Pat. No. ~,043,286 both of which are herein i~ uu~ 3d by reference.
Optical flow cell devices ~ f~ ly have the imrnnhili7~d reagent means entirely rnn~sin~d within the flow cell's flow channel, and such a flow cell is cullG~ d to be ;..~ r...~d with a 2 5 detectiûn means using optical fibers, optical wave g udes, incident beams of light, and the like.
C. MULTIPLE APPLICATION FLOW CELLS
As it will be u~ d~ Luod by one skilled in the art, multiple 3 0 reagent means can be immnhili7~A to the substrate or ~ub~LI~L~s.
Consequently, a single flow cell can optically and el~lu l.-...; .lly detect the presence or amount of an analyte or multiple analytes which may be contained in a test sample.
WO 95n2051 2 17 9 3 0 3 PCT/US95101500 III. FLOW CELL PRODUCTION
The flow cell can be m~mlf~t-t.lred by layering the oppo6ed Dv.lvYL~ d~ with the various layers that may comprise the cell. The various layers applied to the opposed ~ R are largely 5 .1_~ . upon the type of flow cell desired (i.e.. eldv.,~u 1.~...;. _1, optical or ~ ;llnl nn ~ v .1~ 1 and optical). ~fter the various layers have been applied, the YUI,~ ids can be coupled to each other to form the flow cell which then can be ;..l_. r, ~1 with detection means for detecting a signal generated by the 10 immnhili7~d reagent means and flow means for introducing sample into the flow cell's flow channel. The ~ulv~ri-Lds can be layered with the various layers described herein using any means capable of applying a ~ ly thicklayer. ~.Y~lmpl of such means include, but are not intended to be limited to Rt~-" ilinE~
15 spray painting, tampo printing, rhn~-lil.l.n~ y, and the like, preferably screen printing. For example, in the case of an el~vv~u~l-. ---;c~-l flow cell, it has been found avlv~l~6vv~us to screen print the following successive layers to one ~ulv~Ll2lL~v. a reference electrode, a working electrode, a mask layer, a spacing layer and 2 0 an adhesive layer; while applying a counter electrode layer to a second substrate. The two ~ulv~LLnLds and their r~ Iayers can then be, for example, l-...;..nled to each other to form the flow cell. It will be ....~ --o~l of course, that many variations, in ter~ns of the possible order of layers and ~nmhin~t;~-nR of layers, 2 5 are possible.
Advantageously, the flow cell can be mass produced by layering, for example, sheets or rolls of substrate material with the various layers described herein. The sheets or rolls of layered substrate can be stored at any of the stages of the layering process.
3 0 For example, in the case of an clcvllv 1-_ ";. ~1 flow cell, sheets of substrate material can be layered with a reference electrode and a working electrode and stored before ~ 5~ --1, layers, for exvsmple a mask layer and an adhesive layer, are applied thereon. After all of the desired layers have been applied to the substrate material, 3 5 they can be, for example, cut from the rolls or sheets of substrate WO 9512~051 217 9 3 ~ 9 PCTNS95/01500 material and AccPmhl~' to form the ~liA~nrcti~ flow cell herein provided.
IV. DETECTIO~ MEiANS
Detection means for detecting an electrorhPmirAl response to the presence of an analyte that may be rrntAinPd in a test sample include, but are not intended to be limited to pot~ntjo~ 9, pot ~ - . p, and the like. Such detection means can be placed in ~-rmmllnir~t.irn with the flow cell using ...~ b,~Pfi well 10 known in the art. For example, an ~ l flow cell can be placed in .. i.. l: .. , or ;.,l~. r. ~, with the detection mean6 using electrical connectors such as wires and clamps.
Detection means for detecting an optical response to the presence of an analyte that may be ~nntsinPd in a test sample 15 include, but are not intended to be limited to lllminr, nPtPrS~
spectrorh.-l- ..~ . ," and the like. Such detection means can be . . r~-~d with suitable detection means using ...~ ,, well known in the art. For example, an optically based flow cell can be d with detection means using fiber optic cables.
2 0 It will be . . . ,~ "0~, of course, that the detection means employed is largely (1-1J'~ upon the analyte being detected and therefore, the immnhili7Pd reagent means employed. It will also be 1ln~rrFt~)od that multiple detection means can be i..l- r~. ~ d with the flow cell.
V. FLOW MEANS
A~iti~nA1ly, it will be obvious to one of skill in the art, that the flow cell can be rtmnPriPd to flow means which can transport samples into and out of the flow cell's flow channel. ~YAmp1Pc of 3 0 suitable flow means include, but are not intended to be limited to syringes, syringe pumps, ~ lillg pumps, Lc~
pumps, pressure or vacuum sources and the like, ~ c ~bly pPriFts1tir pumps.
~ WO 95122051 ~ ~ 7 9 3 o 9 PCTIUS9SlolSoO
VI. EMBODIMENTS
While many types of devices fall within the æcope of the present invention, particularly preferred ~mhorlimPnt~ will be described in rnnj11nrt;r~n with the drawings. Referring now to the 5 drawings, Figure 1 shows an expanded view of an electrorhP.mir~1 flow cell. Figure 1 shows substrate 10 and the several layers that can be applied thereon to form one part of an cle~L.u. ' 1 flow cell. Specifically, the layers that can be applied to the substrate 10 include: the reference electrode including redox couple 22 and 10 cuu~u. li~ trace 32; the conductive traces 30 which form part of the working ~ ud63, a mask layer 40; a spacing layer 60; and an adhes*e layer 60. Figure 1 also shows a second substrate 70 and a counter electrode 80 which cdn be layered on substrate 70 to form the other half of an el~ Llu~ 1 flow cell. To assist in coupling 1 5 the 3uh3Lld~e3 to form the clc~l.l.. 1~...:. .1 flow cell, opposed substrates 10 and 70 are provided with ~ nmPnt apertures 12 and 14, and 72 and 74; the mask layer 40 is provided with ~ nmPnt apertures 42 and 44; the spacing layer is provided with s~ nm,ont apertures 52 and 54; the adhesive layer is provided with ~ nmPnt 20 apertures 62 and 64; and the counter electrode is provided with n n~nt. apertureS 82 and 84.
Adhesive layer 60 and spacing layer 50 are supplied with ....1;"~1 voids 66 and ~6 which partially define the flow cell's flow channel when the adhesive and spacing layers are 2 5 sandwiched between 3uL~ 3 10 and 70. Substrate 70 and counter electrode layer 80 are supplied with port apertures 76, 78, 86 and 88. When the 3uh31~.c.l,e3, and their l~3lu~ ~iv~ layers, are II~RPmhlPd or coupled to each other, the port apertures align near the ends of the 1,...~1...1;";~1 voids 56 and 66, and serve as inlet and 3 0 outlet ports for the flow channel.
As seen best in Figure 1, mask layer 40 is supplied with a plurality of apertures 46 equal to the number of conductive traces.
When the flow cell is ~PmhlPtl, these apertures allow portions of the culldu~liv~: traces 30 and redox couple 22 to remain exposed.
3 5 Prior to coupling the 3u1,31~ s, an enzyme can be immnhili7Pd on WO 95/22O51 217 ~ 3 0 9 P~,11~J..,' _1500 , the exposed portions of the conductive traces 30 to complete the working electrodes.
Figure 2 shows sub6trats 10 with the layers .. 1. ;A;........ e the reference electrode (22 and 32) and the conductive traces 30 5 immnhili7Pd thereon. A portion of the redox couple 22 is exposed such that when the flow cell is a~^APmhled and a test sample is contained within the flow cha~nel, the exposed portion of the redox couple can contact the test sample and maintain its potential.
Figure 3 shows an h`` ...klrd electrorhA~ni~^al flow cell 1 0 which is i.,i~ d with potA-Atin~at 400. As shown in Figure 3, the flow cell is i ll ll . r^.~.~d with the pu~. . l ;o~ ~ . l via a series of electrical .^nnnPr~;.^nR leading from the counter electrode, working el~,Lludes and reference electrode to the potA~t;^-^~st Sperifira-lly, cnnnA~^t;-^nA 330 and wires 340 interface the counter electrode and 1 5 the po~^^t;^,~st, ~^nnnPrt;^nA 350 and wires S60 interface the working el~ udes and the potA^.~ at~ and ",~ . 380 and wire 370 interface the reference electrode and the po~^^tin~tat All . .. Ill~l~ IA are made with the conductive traces comprising the various elc~,lA.ud~.
2 0 Figure 3 alsû shows a complete working electrode rnmrriAin~ im nnhili7Pd en_yme 100 and CUll~U~.iV~ trace 30.
AMiti/mAlly inlet and outlet ports 300 and 310 are shown near the ends of flow channel 3ao.
Figure 4 shows a cross-sectional view of an Cl~,Llu. l~l..,.;rAl 2 5 flow cell as taken through segment A-A of Figure 3, As shown from that view, several of the layers which c9n form An ~ .. 1,1';1 cle~,Llu. l.~-..;ral flow cell ca9n be seen. Included in the layers observable from this view are the D~lLDL AL~D 10 and 70; redox couple 22; the working electrode including cull.lu~Li~ traces 30 3 0 and the en_yme immAhili7Pd thereon 100; and the counter electrode 80. The flow chai nel 320 is also illustrated by Figure 4.
The various layers that can comprise an optical flow cell can be seen best in Figure 5. Figure 5 shows two substrate layers 110 and 140 and the layers that c. n be placed th~. ~t ' . . ~ . . Substrate 3 5 110 is supplied with apertures 112 which, when the flow cell is ,A,r ~, . . .hlPtl are located near the ends of lrn~itll-linal voids 122 and WO 9sl22051 ,~ ~ 7 ~ ~ o 9 F~l/v~ SoO
1~2, and serve as inlet and outlet ports for the F~llC ...hlFd optical flow cell's flow channel. Adhesive layer 120 is shown between substrate 110 and spacing layer 130 but would be equally effective between substrate 140 and spacing layer 130. The adhesive layer 120 and the spacing layer 130 have lnnEit~ in~l voids, 122 and 132, 6~ ly which help define the flow cell's flow channel when sandwiched between ~ubtiLL~LLVh 110 and 140. Also shown in Figure 5 are a plurality of dye spots 142 which are immnhili7pd on substrate 140. The substrates 110 and 140, the adhesive layer 120 1 0 and the spacing layer 130 are also supplied with ~liEnmPnt apertures 114, 144, 124 and 134 which assist in aligning the flow cell during assembly.
Figure 6 illustrates an ~cqpmhlpd optical flow cell includirlg substrate 110; dye spots 142; and apertures 112 which serve as inlet 1 5 and outlet ports for the flow channel 160.
Figure 7 shows a cross sectional view, as taken through section B-B of Figure 6, of an optical flow cell that is;,.l. . r~.~, d with detection means 600. As shown by Figure 7, the dye spots 142 are cnn~oinPd in the flow channel 160 which is defined by ~ul ?~-2 0 110 and 140; and the 1~; l 1; ~1 void in the spacing layer (not observable from this p~ c. liv~). As shown in Figure 7, the detection means 600 can comprise a light source 610, a light detector 620 and a intensity reader 630.
2 5 VII. ASSAY METHODS
The flow cell device can be used to detect the presence or amomnt of an analyte which may be present in a test sample. A
test sample can be introduced into the flow cell's flow channel wherein it contacts the im~r~hili7Pd reagent means. The presence 3 0 or amount of the analyte can be tlPtprmin~d as a result of the analyte ~ e the immnhili7Pd reagent means. When contacted with the test sample, the immnhili7Pd reagent means generates a d~ t 1 1 - signal that is detected by detection means and is ulL~liv~ of the presence or amount of analyte that may be 3 5 rnn~oin~d in the test sample. It will be -- .~ i looA of course, that the test sample can be, for example, il~ du~ ~d into the flow cell's WO 95/22051 F~ ~ 1500 21~93~9 flow channel and stopped therein~ it can be cnntim1n11c1y flowed through the flow channel or it can be recirculated through the flow channel.
The following examples are not intended to limit the invention herein provided but are intended to illustrate the invention.
T1 ~Amp1a 1 Prnrl11rt;~m of An RiorF~TFnt Tmmnhili~Atinn MFAium Usin~
(~T1~1rrcP O~ Re ~ nrbed to PlAtini7F~i CArbon The enzyme binding resin (see below) was 6elected from the commercial paint industry fnrmlllAt~ designed to be fast drying, adherent and able to emulsify pigments. The mixing ed-L~t was designed to generate high shear rates, thus 15 producing a very highly dispersed pigment and small particle size.
In order to obtain this smooth emulsion, the fnrm111Atinn C ..~ were mixed in a WIG-L-BUG (Crescent Dental M:lllllr~.. .II;IIg" Lyons, IL) which is a device designed to mix dental ~mAl~mc The mixer l..F. 1.7...;~ ~lly agitates the 2 0 fnrm111 Atinn in a 2 ml sWnless steel vial rnnt~inin~ a ball bearing, thus allowing 0.5 to 1 ml coating ~ -...c to be blended.
MAt.F'ri Al C
1. Platinized V3l1can XC 72 (carbon black) cnntAinin~ 10%
2 5 platinum 2. Glucose Oxidase (GOD Grade I from Aspergillus niger, Boehringer M~nnhPim ~3jorhpmi~Al~ Tntli InArnli~, IN) 3. PL~ Le Buffered Saline (PBS: l00 _M sodium PllO~La~, l00 mM NaCl, pH 6.0) 4. Acrylic resin mix (31.6% non-volatile solids fnrm111At.F!d as shown below) WO 95122051 217 ~ 3 0 ~ r~ ;c Arrvlic RPRin ~i~ Cnmnnn~nt %bv ~izht Joncryl 537 acrylic emulsion (Jobnson Wax, Racine, WI) 54 Joncryl 56 acrylic resin (Johnson Wax, Racine, WI) 2 DMAMP 80 (Angus Chemical Co., Northbrook, IL) Ektasolve EP ( Eastman l~h_mi~Alc, Kingsport, TN) 9 Distilled water 9 Pl ùc~l " . ,~
En_yme was adsorbed to ~ ": -~l carbon by adding a solution of GOD in PBS buffer to sllcr~ncinnc of rl~tini7Ad carbon in PBS buffer. One A"`1J..IA....I contained 101 mg of rl -l ;"; -d carbon in 0.44 rnl of PBS plus 0.2 rnl of a 60 mg/ml solution of GOD, while the other ,CI-cr_ncinn contained 51 mg of rl~ l carbon in 1 5 0.2 ml of PBS plus 0.1 ml of a 50 mg/rnl solution of GOD. The mi~tures were allowed to statically incubate for 1.5 hours at an ambient tt:lu~ Ult before they were ~ iL6~d at 2000 rpm.
The bll~ IIA were discarded and the resulting wet GOD/carbon pellets were l~ ~,ut~uded in 0.8 gm of the rehin mix 20 and blended in a WIG-L-BUG for 10 minutes. A WIG-L-BUG
(Crescent Dental M~m1fAri.~rin~, Lyons, IL) is a device designed to mix dental AmAlFAmc The mixer ",~ "; Ally agitates the formulation in a 2 ml stainless steel vial cnnt~inin~ a ball bearing, thus allowing 0.5 to 1 ml coating ~ lA to be blended. The 25 resulting coating solutions were hand .];hl,~ d onto separate Cl~ udeh and tested ~lc~.lv l,_-": lly The fnr~mll~tinn for the coating solution is shown below in Table 2.
Table 2 Formula mg carbon mg Resin mg Resin Resin solids/
Mix Solids Carbon ratio 101 800 253 2.50 WO 95/22051 PCI/US9~/01~00 ~17930~`
FYAmrl~ 2 T~ rtro~h~mirAl Flow C~ll MAt.f'riAlC' 5 1. ICI ST505 Heat ~t ~ d Polyester Film (Tekra Gu~u~ iull, Milwaukee, WI) 2. Acheson SS24950 Silver/Silver Chloride Ink (Acheson Colloids, Port Huron, MI) 3. Acheson 423SS Carbon Ink (Acheson Colloids, Port Huron, 1 0 MI) 4. Acheson ML25198 TnclllAtinæ T)i~l~ctrir (Acheson Colloids, Port Huron, MI) 5. Acheson W 8002 Adhesive (Acheson Colloids, Port Huron, MI) 15 6. Bioreagent Tmmnl~ Ati-~n Medium (from Exanple 1) 7. Pot~t;ol~3t - an eight channel pul -.~.;n~ was Acc~n~hl~d at Abbott Labu~ -.;c3 (Abbott Park, IL) to ~.. I.. n~ the flow cell described herein 2 0MAnl1fArtllrin~ Prorrl11lre:
A 0.007 inch thick polyester film was used as substrate support material for both halves of the fiow cell. A working electrode, reference electrode, mask layer, spacing layer and adhesive were appbed to one substrate and a counter electrode was 2 5 applied to the other sllh~ At~ The mAmlfArt-lrin~ process wiU be explained with ~ ~ f~ 3 to Figure 1. A layer of silver/silver chloride ink 22 was screen printed in a pattern to form the redox couple portion of a single reference electrode. Next, a layer of carbon ink was screen printed on the redo~ couple and substrate in 3 0 a pattern to form eight working electrode conductive traces 30 and the conductive trace portion 32 of the reference electrode. The redox couple layer and conductive trace layers were printed with quantities of the It~u~ . materials that were sufficient to provide an end to end ~ .e of less than 100 ohms.
3 5 On top of both the silver/silver chloride ink and c~rbon ink layers a layer of dielectric 40 was screen printed in a pattern to ~ wo gs/220s~ 7 9 3 ~ 9 Pf'TNS95/01500 mask all of the working conductive traces $0 and redox couple 22 except for a small circular area 46 which was 0.066 inches in diameter. The masking layer of dielectric was applied in a quantity sufficient to become water i...l. ...F ~hlF~. A layer of 5 dielectric was then applied to the mask layer in a pattern to form the spacing layer 60 having 1....~; l -1; ..~1 void 56. The spacing layer of dielectric was applied using a quantity sufficient to give the flow cell's flow channel a 10 ~/cm2 volume. A 0.001 inch layer of a&es*e 60 was then printed on top of the spacing layer. The 10 adhes*e layer also had 1r.n~ihl~in~1 void 66. A paper release liner was added to the surface of the adhesive to protect it during handling. A steel ruled dye was then used to cut this part of the device from the sheet of polyester film. A steel ruled dye was also used to cim~ cly cut ~ nmPnt. apertures 12,14, 42, 44, 52, 1 5 54, 62 and 64 from all layers printed on the polyester film.
Again, with reference to Figure 1, the second substrate of the flow cell was prepared by screen printing a layer of carbon ink 80 (counter electrode) onto a sheet of polyester film. The quantity of carbon ink used was sufficient to provide an end to end lG~ ,G
2 0 of less than 100 ohms. The outline of this part of the flow ceU, the nmFnt. apertures and the fluid inlet and outlet ports were cut from the sheet of polyester film with a steel-ruled die.
After the gub~Lltlles and the layers immnhili7sd thereon were cut from the polyester film, 0.36 111 of 1 J~ G~b.,.ll.
25 imm~lhili~lti~m medium (from Example 1) was .l;~ ed onto the circular area of the eight working electrode traces 30. The bioreagent was allowed to cure at room L~ GILL~UlG for one hour.
Using a jig and ~ nmsnt pins, the two 1~ were alibned and li--..;..~d together.
Te9i;nF P1Uff.1~IIG
The testing IJLUI~GdU G will be described with reference to Figure 3. The ~RC'smhlsd flow cell was placed on a stand and ....... r~ to a psri~slti-~ pump (not shown) via fluid crnns~innc to 3 5 the inlet and outlet ports 300 and 310. Electrical ~r.nns~ti~nc (between the f~ow cell and por ntio~g~ 400) to all eight of the WO 95/22051 ? 9 PCT/US9S/01500 2 0 `
working electrodes 30, the counter electrode (not shown) and the reference electrode ~4 were made using clamps and wires 3~0 and 860; 330 and 340; and 360 and 370 l~,U~ iVtly. Liquids were drawn t_rough the cell with a peristaltic pump and the potential of the 5 ~ dudes relative to one another was controlled by a three electrode pu'~ n '-l with eight separate current ~ -. ;l.r rh l~nn~sl c The working electrode potential was set at 350mV versus the on board Ag/AgCl reference electrode. Solutions of differing 10 glucose rnnr~ l : ll lc were then ~ ,c~ vely drawn through the cell and the current which passed at each of the eight working electrodes wa6 Illu...Lul~d and recorded. The graph shown in Figure 8 illustrates the current response of one of the working clc~ ~udes to 0 mM, 2.5 mM, 6.5 mM and 11.4 mM glucose 15 solutions in pH 7.5 aqueous pl.n;~l.h~ buffer (PBS - 40.5 mM
Na2HPO4, 9.5 mM NaH2PO4, 50 mM NaCl). Also shown in Figure 8 is the linear regression t'~rough the four data points.
As shown by Figure 8 the electrode response varies linearly with the glucose cvl~ lion in the test sample.
F~Y~mnlf. 3 Pl~,u. -l a~iull of an Oxv~en ,~Pncibve Dve Free base tetra(ppnt~fl1lnrophenyl)~lus~llylill [H2(TFPP)]
was made 2 5 by adding 2.0 ml of p ~ n .. -~ Phyde, 1.5 ml pyrole and 2.0 ml of boron triflllnri-le etherate to 1500 ml dichlul - l". .P to form a re~rfi~)n mi~rblre All materials cn~nrricin~ the reaction mixture are available from Aldrich Chemical Co., Inc., Milwaukee, WI. The reaction mixture was stirred for 1 hour 30 before 2.5 grams of 2,3-dichloro-5,6-dicyano-1,4-b~l~u~luillulle (Sigma, ST. Louis, MO) was added a~d the resulting mixture was heated to 40C for 1 hour. Then, the solvent was flash dried arsd the crude solid H2(TFPP) product was ~ u~lsluclc~hed over silica gel using dichlol. "~ P as the eluting solvent. 1.0 gram of the 3 5 purified H2(TFPP) and a 10 times molar excess of PtCl2 (Aldrich Chemical Co.Irsc.) were refluxed for 24 hours in 500 ml of WO95/22051 P~,l/ll,, lS00 ~17g3~9 7.~ ;le to synthesize crude platinum tetra(p~ ..... v~uh~llyl)l,ul~lly~ [Pt(TFPP)]. The product was purified on a neutral alumina column using CH2Cl2 as the eluant.
An oxygen sensitive dye solution was prepared by dissolving 100 5 mg of the purified Pt(TFPP) in a 25 ml of a silicone polymer stock solution. The polymer stock solution was made by dissolving 10.0 grams ûf a dimethylsilo~ane-hicrhpnnl (General Electric Inc., Waterford, NY) in 100 ml tetra~lyLurul~l.
~YslmnlP 4 Opti~l Flow Cell M~t.Pri~lc 1. ICI ST505 Heat .s~hili7pd Polyester Film (Tekra Corporation, Milwaukee, WI) 2. Acheson ML25198 rnc~ tin~ Dielectric (Acheson Colloids, Port Huron, MI) 3. Acheson SU459 Adhesive (Acheson Colloids, Port Huron, MI) 20 4. Platinum tetra(p~ . n...~ uull~ .lyl)~uullJLy ;1l from EYample 3.
~ l;llF P~U~6~1UI~
The - ~ JlU~,6dUl~ will be described with 2 5 reference to Figure 5. A layer of dielectric ink 130 was screen printed onto a substrate 140, which ~ a sheet of polyester film, to form the spacing layer of the nOw cell. The spacing layer of dielectric was applied using a quantity sufficient to give the flow cell's flow channel a 10 ~llcm2 volurne. A .001 inch layer of 3 0 adhesive 120 was then screen printed on top of the dielectric layer in the pattern shown in Figure 5. A release liner was then applied onto the surface of the adhesive to protect it during handling. The outhne of the cell and ~ nmPnt apertures 114, 134 and 144 were then cut from the polyester sheet using a steel-ruled die. The other 3 5 substrate was fo~med by cutting the cell outline, ~lignnnPnt i i: ~ \ j WO 95/22051 r~.,., . Isoo ~1~93~9 aperture6 114 and inlet and outlet ports 112 from a 0.007 inch thick sheet of polyester film After removing the release liner, 1.0 ,ul aliquots of the oxygen sensitive dye 142 (from Example 3) were tben applied to 5 substrate 140 such that the dye spots were aligned within the lnn{~itll(lin~l voids 122 and 1$2. rlAhe dye was allowed to dry for one hour at room l~...l.~ . ,. I.... t and the polyester ~uL~ L~i were then pQcitinnPd uging an ~ nmPnt. jig and ~liEnmPnt pins before being 1 imin~tPd together.
T ~ P~
The ~cRPmhlPd flow cell was fixed on a stand and fluid tubes were (-nnnPr~ql1 to tbe iDlet and outlet ports. Tonometered solutions were then moved into and out of tbe cell using a 15 peristaltic pump. Tests were p~. f.,. .--Pd by pnCitirlninE a 2~0 Ilm optical fiber (Ensign-Bickford Optics Co.; Avon, CT) above the dye spots 142 and illllmin~tinE the dye with light emitted from a pulsed light emitting diode (Hewlet Packard Co., Cupertino, CA) while a solution of l.. h~ d oxygen buffer was flowing t_rough the 20 flow channel. Using an avalanche phul~ rl~
Corp., Bl;d~,~..dL~-, NJ), ph~ u~is~ L intensity LUea~UIt~lu~.Lb were recorded in 2 time regions (3-17 llseconds and 3-200 1l6econd6) after each P~ritstirm pulse. The pho~lJhu,e,Act".L inten6ity ratio was rAl(~--l, tPd and plotted as a function of oxygen Cull~LLidLion.
2 5 As shown by Figure 9, a ~ udu~le non-linear rPl~ltinn.A~hip between the inten6ity ratio and oxygen partial pressure is observed.
I. DEFINITIONS
The term "analyte", as used herein, refers to the rrnnrolm or rrnnrrAit;~n to be detected or ~D~d and which initiates the generation of a ~ lf- response. Analytes include, but are not intended to be limited to, enzymes or enzyme substrates, metal ions, blood gases, toxins, organic ~ .u ...~lc, proteins, peptides, 10 a_ino acids, carbohydrates, nucleic acids, hrrmrnPc, steroids, vitamins, drugs (inrlll~in~ those ~lminil ~ d for therapeutic purposes a3 well as those ~minictDred for illicit purposes), and mPt"h(~litPC of any of the above substances. For example, such analytes include, but are not intended to be limited to, alanine 15 aminuLl~lDrel ~c (ALT), aspartate aminuL~llDr~ 3c (AS~), creatinine kinase (CK), creatinine kinase MB (CK-MB), lactate dellyLu6~as~ (LDH), garnma glutarnyl ~ vt-~uL ~l~rc (GGTP), alkaline rhr~ , glucose, fructose, g;~ r~ P sucrose, lactose, lactate, ;l 1 ul~ urea, creatinine, tri~l~.;deD, uric 20 acid, bilirubin, 1~.1u~ , p~ ..;- -.., sodium, chloride, calcium, cL;u..., lithium, oxygen, carbon dioxide, hydrogen ions (pH), h.. ~"~lVl.;.l, glycated hPmnglrhin (Gly. Hb), C-reactive protein, serum li~u~ul~ills~ serum albumin, deoxyrihrnllrlPir acid (DNA), rih~-mlrlPio acid (RNA), bile acids, salicylates, A~' ...;.. ~l.~.., 2 5 theophylline, p_t~ll.y Luhl and the like.
The term "test sample", as used herein, refers to a material sll~pected of con~inin~ the analyte. The test sample can be used directly as obtained from the source or following a pre-L~ L~ellL to modify the character of the sample. The test sample can be derived 3 0 from any biological source, such as a physiological fluid, inrlll~in~, blood, saliva, ocular lens fluid, cerebral spinal fluid, 6weat, urine, milk, ascites fluid, mucous, synovial fluid, pPrit~nP~l fluid, amniotic fluid and the like, and fPrmPntsltirn broths cell cultures, and chemical reaction mixtures and the like.
3 5 The test sample can be pretreated prior to use, such as ~UIt:~U illg plasma from blood, diluting viscous fluids, and the like. Methods WO 9S1~051 PCTIUS95101500 ~17~30g of i.~:~.L. ~ L can involve filtration, ~1iFt~ ti~-n, ~u~ .lL~Liu inactivation of i"~. r~.;"~ c~-mr~mPntC~ and the addition of reagents. In addition to biological or physiological fluids, other liquid samples can be used such as water, food products and the 5 like for the 1.. r,.. i.. ,. P of ~:.lvilu.. l.. ~.~Lidl or food production assays. In addition, a solid material sllcpertPd of ~ the analyte can be used as the test sample. In some inQt~nrPc, it may be beneficial to modify a solid test sample to form a liquid medium or to release the analyte.
II. FLOW CELLS
The present invention is directed to a lliA~n~ flow cell rt~mrricin~ two opposed substrate layers and a spacing or gasket layer disposed between the two opposed substrate layers. The 15 spacing or gasket layer has a lon~ibl-lin~l void which, in c.. l.;"--~.;on with the ~ub~L z~Les, defines the flow cell's flow channel. A substrate layer, or ~e substrate layers, can be supplied with apertures which, when the flow cell is slccPmhlP~i can serve as inlet and outlet means for illL u~u~hlg sample into the 2 0 flow channel and allowing sample to exit the flow channel.
Preferably, the apertures are supplied to one substrate and are located near the ends of the flow channel defined by the ~ub~L c.L~s and the spacing layer.
Reagent means that can generate a ~PtP~t~hlP signal when 2 5 contacted with an analyte can be imm~lhi1i7Pd on a substrate or the Sllhqt~At~C Typically, the immohi1i7Pd reagent means is sllhst~nti~lly insoluble. C~nnceq~lPnt1y, the reagent means can be contacted with multiple test samples thereby making the flow cell reusable. Depending upon the analyte to be detected, the opposed 3 0 ~..h~ c can have a single or multiple reagent means immtlhili7Pd thereon. Thus, by immllhili7inF the ~ v~l;aL~
reagent means, a single flow cell can, for example, electrically and optically detect analytes which may be present in a test Qsample.
A-l~itinn~lly, by immohili7in~ a mlllbrli~ity of the same reagent 3 5 means to a substrate or the substrates, a single flow cell can detect the presence or amoumt of an analyte in replicates.
WO 95/22051 PCI/I[JS95/01500 21~9309 The various layers that can comprise the flow cell herein provided can be applied to the opposed s~ f~AtPc~ and the substrates can then be coupled with a gasket layer to thereby form the flow cell taught herein. Due to the de6ign of the flow cell and 5 the manner in which it can be ~ rA~ the (liAl nf~;c flow cell i8 reusable, int:~dllS;ve: to produce, easily stored in a complete or inrnmrl~t~ form, capable of being patterned with immnbili reagent means, and, as compared to previous te~`hn~' )C.Y~ the number of mA~-hinPd parts is greatly reduced. Moreover, signal to 10 noise ratios which are ~_.lt:l.lLdd by the reagent means are increased and cl~_LI.~...A~..^';~ h.~ es are reduced.
The opposed substrates can be made of any chemically inert, non-culldu~ Liv~, and physically durable material which is capable of supporting the va~ious rnaterials applied thereon. P ~ ?v of 15 such materials include, but are not intended to be limited to film plastics such as polyester, poly~l,uuAAL~, pûly~Ly ~:ue, polyeth~rimirl~, and the like; molded plastics such as acrylic, phenolic, p~lyl 1 ~, and the like; ceramics such as alumina (Al203), ~irconia (ZrO2), magnesia (MgO), and tbe like; glass;
2 0 silicon wafers; and the like; preferably the substrate material comprises a polyester film.
The spacing or gasket layer materials are typically chemically and dc_LIu- 1,~,.,;. Ally inert as well as sllh~snt;slly non a~sulb~llL and impervious to water. ~YA~nrl~ of materials 2 5 that have these properties include, but are not intended to be limited to printing inks, painted inks, sprayed inks, late~es, urethanes, vinyls, polyesters, film plastics and the like, ~l~f~ ly the spacing layer ~ a dielectric printingink. The thickness of the flow cell's spacing layer is largely r~r-ln~ihl~ for 3 0 the volume of the flow channel. Thus, for e~ample, when it is desirable to have lar~e amounts of sample contained within the flow channel, the thickness of the spacing layer can be increased.
The amount of sample ~ ntAin~d in the flow channel, in relation to the exposed substrate surface area, is preferably in the range of 3 5 between about 1.0 ,u~/cm2 and about 500 ,ul/cm2, more ~u. ~ kly WO 95122051 _ 7 _ PCI/US95/01500 between about 2 ~Icm2 and about 250 Ill/cm2 and most ~!Jlerel~bly between about 2.6 ~l/cm2 and about 100 Ill/cm2.
The (1iA~nnqtic flow cell can further comprise a mask layer immnhili~ad between the two 6ubstrate layers. Similarly to the 5 spacing layer, the mask layer is typically chemically and electrnrh~smirAlly inert as well as non abDvll,ellL and impervious to water. ~ mrl~a~ of suitable mask layer materials have been described with reference to the spacing layer. When multiple analytes are to be detected, or rephcates of the same analyte are to 10 be detected, the mask layer is particularly preferred and serves as a barrier between the various imm-)hili~ad reagent means.
The opposed Dul)bLl~lLes7 and the various layers that can be disposed between the ~ Pc, can be secured or coupled to one another to form a flow cell using fastening means such as, for 15 example, lAminAt~ solvent bonding, nuts and bolts, rivets and the like, ~u~er~ Lly an a&esive layer secures the Ellh--~rAt~a~, and the various layers thereon, to each other. An adhesive layer is y~ere~c.hly inert chemically and ele~Llu 1.~ lly as well as being capaMe of retaining its adherent quality in saline solutionE.
2 0 l;~Ampl~s of such materials include, but are not limited to ultraviolet cured pressure sensitive adhes*es, heat cured pressure senDitive adhesives, vinyl based pressure sensitive adhesives, and the like, preferably a polyurethane based ultravioletly cured adhesive.
A. ELECTROCHEMICAL FLOW CELLS
In the case where the ~ .LII~ n flow cell is capable of lu 1,~... - ~lly detecting an analyte that may be contained in a test sample, the sllhri~At~ can have reagent means immnhili~`ad 3 0 thereon which form a reference electrode, a counter electrode and a working electrode. T_e reference electrode can comprise (i) a material or a romhinAti-m of materials capable of ~shili~7in~ a test sample's potential or otherv~ise providing a constant potential within a test solution; and (ii) a ~ udu~,~ve trace material which is 3 5 capable of being intPrf~rad with a detection means and which is D~ ally inert at the assay device's operating potential.
WO 9S/22051 E~ ,' vlS00 ~179309 Materials capable of d~eloui..~ a stable potential include oxidation/lGdu~ liull pairs (variably referred to as "redox couples") inr11lrlin~, but not intended tû be limited to, silver/silver chloridc/~ uLl,G blends, lU~ /lu~.. ~uu6 chloride blends, 5 silver/silver iodide blends and the like, preferably silver/silver chloride blends which can be ~ .Red u6ing screen printing tPrhniqllPR F. 1 of materials that can be used as WlldU~
trace material include, but are not intended to be limited to gold, carbon, nickel, silver, palladium, rllthPnillm, rhodium, tin o~ide, 10 indium tin ûxide and the like, ~l~rG~bly carbon that has been dispersed in a screen printing ink.
The counter electrode can comprise an electrûrhPmir~lly Culldu~ c material which is relatively inert at the a66ay device's operating potential. F. 1 of materials having these 1 5 properties include, but are not intended to be limited to gold, carbon, nickel, silver, r~ lillm, rllthPnillm, rhûdium, tin ûxide, indium tin oxide and the like, ~ bly carbon that has been dispersed in a screen printing ink.
The working electrode can comprise (i) a culldu~ G trace 20 material and ii) an enzyme or enzymes imm~ili7Pd to or in contact with the Culldu~ , trace. P~r_~Lly, the enzyme is imm~-hili7Pd on the conductive trace such that when the flow cell is ..... hlrrl, the enzyme will remain H~. ~ C for multiple u6es.
A particularly preferred method of immnhili~n~ an enzyme to the conductive trace material employs an immnhili7~t;~m medium disclosed in co-owned and co-pending ~rFlir~tinn Serial No.
(Atty Docket No. 5488.US.01), entitled BIOREAGENT
IMM(lRTT,T~ATION MEDIUM, filed on even date herewith and incorporated herein by reference.
The ~iUlGC.6~1~ immnhili7~tinn medium rn7nrriRPR i) an enzyme which is immnhili7Pd to a solid phase and ii) a binding reagent comprising a latex resin, wherein the immnhili7Pd enzyme is evenly dispersed. The binding reagent may also include optional ih~Gdi~llL~ which enhance the immnhili7sltinn medium's chemical and physical properties. The enzyme can be immnh~ pd to the solid phase by methods well knûwn in the art such as, for WO 9S/220S1 2 1 7 ~ 3 ~ 9 P .~ ISOO
~ .
_ 9 _ example, covalent, ionic or ads~l~iiv. bonding of the enzyme to the solid phase. By way of example and not of limitsltinn, the various enzymes, solid phases, resins and optional ingredients that can be employed in the bioreagent immnhili7~nn medium can be found below in Table 1.
OPTIONAL
ENZYMES SOLID PH~SES RESINS INGREDIENTS
glucose oxidase, agarose and acrylic latex, pl~o~;ri7Prc, film ~l~.l.,.. ,.~e d~.;vaLivGs styrene acrylic forming agents, oxidase, lactate thereof, latexes, vinyl thickeners, oxidase, glycerol polyacrylamide acetatelatex and stabilizers, rhnBph~tP and dGl;v~ s polyulGlllane dispersing oxidase, thereof, silicas, latex agents, and cholesterol aluminosilicates, dPfo~min~
oxidase, aluminum agents cholesterol oxides, carbon or esterase, lipase, graphite glycerol kinase, particles, and ~lllt.~m~t.P platinum group dehydL ug~.lase, metal oxides creatinine rlPslmin~cP, and uricase 1 0 In order to allow easy interface with detection means for detecting a signal ~G~ led by the immnhili7Pd reagent means, it is preferred that a portion of the immnhili~Pd reagent means is rnnt:RinPd within the flow channel and a portion of the immnhili7Pd reagent means extends out of the flow cell's ~ow channel.
1 5 It will be ulldGl~uûd~ of course, that the present invention is not limited to flow cells having three electrode systems, and that two electrode systems are ~ For example, a two WO 9S/22051 r~ IS00 ~17930~
electrode ~ullfl~ul~Lull can comprise a working electrode and a cnmhinslt;nn rt~r~ ~ce/~uullLtl electrode.
B. OPTICAL FLOW CELLS
In cases where the rliA~n~ ;c flow cell ûptically detects the presence or amûunt of an analyte which may be contained in a test sample, the immnhili7~d reagent means generates an optically A~t~rtSIh1f~ signal when it is contacted with an aualyte which may be contained in a test sample. The i nn~-hili7.0d reagent means that can be used in optically based flow cells are generally cu~yuullds or mixtures of r~mrûllnl1~ that, when contacted with an analyte, emit a signal which is optically APt~r~ Examples of such immnhi1i7~sd reagent means include, but are not intended to be limited to pH sensitive dyes; oxygen sensitive dyes; dyes or chelating agents which are sensitive to ions such as calcium and ;-,-.. ions; and the like; lul~r~.Ably platinum tetra(~ n ~ uht llylkorphyrin which is an oxygen sensitive dye that changes its fluorescence lifetime in the presence of dissolved oxygen. Such an optically sensitive dye is disclosed in 2 0 U.S. Pat. No. 4,810,65~ and U.S. Pat. No. ~,043,286 both of which are herein i~ uu~ 3d by reference.
Optical flow cell devices ~ f~ ly have the imrnnhili7~d reagent means entirely rnn~sin~d within the flow cell's flow channel, and such a flow cell is cullG~ d to be ;..~ r...~d with a 2 5 detectiûn means using optical fibers, optical wave g udes, incident beams of light, and the like.
C. MULTIPLE APPLICATION FLOW CELLS
As it will be u~ d~ Luod by one skilled in the art, multiple 3 0 reagent means can be immnhili7~A to the substrate or ~ub~LI~L~s.
Consequently, a single flow cell can optically and el~lu l.-...; .lly detect the presence or amount of an analyte or multiple analytes which may be contained in a test sample.
WO 95n2051 2 17 9 3 0 3 PCT/US95101500 III. FLOW CELL PRODUCTION
The flow cell can be m~mlf~t-t.lred by layering the oppo6ed Dv.lvYL~ d~ with the various layers that may comprise the cell. The various layers applied to the opposed ~ R are largely 5 .1_~ . upon the type of flow cell desired (i.e.. eldv.,~u 1.~...;. _1, optical or ~ ;llnl nn ~ v .1~ 1 and optical). ~fter the various layers have been applied, the YUI,~ ids can be coupled to each other to form the flow cell which then can be ;..l_. r, ~1 with detection means for detecting a signal generated by the 10 immnhili7~d reagent means and flow means for introducing sample into the flow cell's flow channel. The ~ulv~ri-Lds can be layered with the various layers described herein using any means capable of applying a ~ ly thicklayer. ~.Y~lmpl of such means include, but are not intended to be limited to Rt~-" ilinE~
15 spray painting, tampo printing, rhn~-lil.l.n~ y, and the like, preferably screen printing. For example, in the case of an el~vv~u~l-. ---;c~-l flow cell, it has been found avlv~l~6vv~us to screen print the following successive layers to one ~ulv~Ll2lL~v. a reference electrode, a working electrode, a mask layer, a spacing layer and 2 0 an adhesive layer; while applying a counter electrode layer to a second substrate. The two ~ulv~LLnLds and their r~ Iayers can then be, for example, l-...;..nled to each other to form the flow cell. It will be ....~ --o~l of course, that many variations, in ter~ns of the possible order of layers and ~nmhin~t;~-nR of layers, 2 5 are possible.
Advantageously, the flow cell can be mass produced by layering, for example, sheets or rolls of substrate material with the various layers described herein. The sheets or rolls of layered substrate can be stored at any of the stages of the layering process.
3 0 For example, in the case of an clcvllv 1-_ ";. ~1 flow cell, sheets of substrate material can be layered with a reference electrode and a working electrode and stored before ~ 5~ --1, layers, for exvsmple a mask layer and an adhesive layer, are applied thereon. After all of the desired layers have been applied to the substrate material, 3 5 they can be, for example, cut from the rolls or sheets of substrate WO 9512~051 217 9 3 ~ 9 PCTNS95/01500 material and AccPmhl~' to form the ~liA~nrcti~ flow cell herein provided.
IV. DETECTIO~ MEiANS
Detection means for detecting an electrorhPmirAl response to the presence of an analyte that may be rrntAinPd in a test sample include, but are not intended to be limited to pot~ntjo~ 9, pot ~ - . p, and the like. Such detection means can be placed in ~-rmmllnir~t.irn with the flow cell using ...~ b,~Pfi well 10 known in the art. For example, an ~ l flow cell can be placed in .. i.. l: .. , or ;.,l~. r. ~, with the detection mean6 using electrical connectors such as wires and clamps.
Detection means for detecting an optical response to the presence of an analyte that may be ~nntsinPd in a test sample 15 include, but are not intended to be limited to lllminr, nPtPrS~
spectrorh.-l- ..~ . ," and the like. Such detection means can be . . r~-~d with suitable detection means using ...~ ,, well known in the art. For example, an optically based flow cell can be d with detection means using fiber optic cables.
2 0 It will be . . . ,~ "0~, of course, that the detection means employed is largely (1-1J'~ upon the analyte being detected and therefore, the immnhili7Pd reagent means employed. It will also be 1ln~rrFt~)od that multiple detection means can be i..l- r~. ~ d with the flow cell.
V. FLOW MEANS
A~iti~nA1ly, it will be obvious to one of skill in the art, that the flow cell can be rtmnPriPd to flow means which can transport samples into and out of the flow cell's flow channel. ~YAmp1Pc of 3 0 suitable flow means include, but are not intended to be limited to syringes, syringe pumps, ~ lillg pumps, Lc~
pumps, pressure or vacuum sources and the like, ~ c ~bly pPriFts1tir pumps.
~ WO 95122051 ~ ~ 7 9 3 o 9 PCTIUS9SlolSoO
VI. EMBODIMENTS
While many types of devices fall within the æcope of the present invention, particularly preferred ~mhorlimPnt~ will be described in rnnj11nrt;r~n with the drawings. Referring now to the 5 drawings, Figure 1 shows an expanded view of an electrorhP.mir~1 flow cell. Figure 1 shows substrate 10 and the several layers that can be applied thereon to form one part of an cle~L.u. ' 1 flow cell. Specifically, the layers that can be applied to the substrate 10 include: the reference electrode including redox couple 22 and 10 cuu~u. li~ trace 32; the conductive traces 30 which form part of the working ~ ud63, a mask layer 40; a spacing layer 60; and an adhes*e layer 60. Figure 1 also shows a second substrate 70 and a counter electrode 80 which cdn be layered on substrate 70 to form the other half of an el~ Llu~ 1 flow cell. To assist in coupling 1 5 the 3uh3Lld~e3 to form the clc~l.l.. 1~...:. .1 flow cell, opposed substrates 10 and 70 are provided with ~ nmPnt apertures 12 and 14, and 72 and 74; the mask layer 40 is provided with ~ nmPnt apertures 42 and 44; the spacing layer is provided with s~ nm,ont apertures 52 and 54; the adhesive layer is provided with ~ nmPnt 20 apertures 62 and 64; and the counter electrode is provided with n n~nt. apertureS 82 and 84.
Adhesive layer 60 and spacing layer 50 are supplied with ....1;"~1 voids 66 and ~6 which partially define the flow cell's flow channel when the adhesive and spacing layers are 2 5 sandwiched between 3uL~ 3 10 and 70. Substrate 70 and counter electrode layer 80 are supplied with port apertures 76, 78, 86 and 88. When the 3uh31~.c.l,e3, and their l~3lu~ ~iv~ layers, are II~RPmhlPd or coupled to each other, the port apertures align near the ends of the 1,...~1...1;";~1 voids 56 and 66, and serve as inlet and 3 0 outlet ports for the flow channel.
As seen best in Figure 1, mask layer 40 is supplied with a plurality of apertures 46 equal to the number of conductive traces.
When the flow cell is ~PmhlPtl, these apertures allow portions of the culldu~liv~: traces 30 and redox couple 22 to remain exposed.
3 5 Prior to coupling the 3u1,31~ s, an enzyme can be immnhili7Pd on WO 95/22O51 217 ~ 3 0 9 P~,11~J..,' _1500 , the exposed portions of the conductive traces 30 to complete the working electrodes.
Figure 2 shows sub6trats 10 with the layers .. 1. ;A;........ e the reference electrode (22 and 32) and the conductive traces 30 5 immnhili7Pd thereon. A portion of the redox couple 22 is exposed such that when the flow cell is a~^APmhled and a test sample is contained within the flow cha~nel, the exposed portion of the redox couple can contact the test sample and maintain its potential.
Figure 3 shows an h`` ...klrd electrorhA~ni~^al flow cell 1 0 which is i.,i~ d with potA-Atin~at 400. As shown in Figure 3, the flow cell is i ll ll . r^.~.~d with the pu~. . l ;o~ ~ . l via a series of electrical .^nnnPr~;.^nR leading from the counter electrode, working el~,Lludes and reference electrode to the potA~t;^-^~st Sperifira-lly, cnnnA~^t;-^nA 330 and wires 340 interface the counter electrode and 1 5 the po~^^t;^,~st, ~^nnnPrt;^nA 350 and wires S60 interface the working el~ udes and the potA^.~ at~ and ",~ . 380 and wire 370 interface the reference electrode and the po~^^tin~tat All . .. Ill~l~ IA are made with the conductive traces comprising the various elc~,lA.ud~.
2 0 Figure 3 alsû shows a complete working electrode rnmrriAin~ im nnhili7Pd en_yme 100 and CUll~U~.iV~ trace 30.
AMiti/mAlly inlet and outlet ports 300 and 310 are shown near the ends of flow channel 3ao.
Figure 4 shows a cross-sectional view of an Cl~,Llu. l~l..,.;rAl 2 5 flow cell as taken through segment A-A of Figure 3, As shown from that view, several of the layers which c9n form An ~ .. 1,1';1 cle~,Llu. l.~-..;ral flow cell ca9n be seen. Included in the layers observable from this view are the D~lLDL AL~D 10 and 70; redox couple 22; the working electrode including cull.lu~Li~ traces 30 3 0 and the en_yme immAhili7Pd thereon 100; and the counter electrode 80. The flow chai nel 320 is also illustrated by Figure 4.
The various layers that can comprise an optical flow cell can be seen best in Figure 5. Figure 5 shows two substrate layers 110 and 140 and the layers that c. n be placed th~. ~t ' . . ~ . . Substrate 3 5 110 is supplied with apertures 112 which, when the flow cell is ,A,r ~, . . .hlPtl are located near the ends of lrn~itll-linal voids 122 and WO 9sl22051 ,~ ~ 7 ~ ~ o 9 F~l/v~ SoO
1~2, and serve as inlet and outlet ports for the F~llC ...hlFd optical flow cell's flow channel. Adhesive layer 120 is shown between substrate 110 and spacing layer 130 but would be equally effective between substrate 140 and spacing layer 130. The adhesive layer 120 and the spacing layer 130 have lnnEit~ in~l voids, 122 and 132, 6~ ly which help define the flow cell's flow channel when sandwiched between ~ubtiLL~LLVh 110 and 140. Also shown in Figure 5 are a plurality of dye spots 142 which are immnhili7pd on substrate 140. The substrates 110 and 140, the adhesive layer 120 1 0 and the spacing layer 130 are also supplied with ~liEnmPnt apertures 114, 144, 124 and 134 which assist in aligning the flow cell during assembly.
Figure 6 illustrates an ~cqpmhlpd optical flow cell includirlg substrate 110; dye spots 142; and apertures 112 which serve as inlet 1 5 and outlet ports for the flow channel 160.
Figure 7 shows a cross sectional view, as taken through section B-B of Figure 6, of an optical flow cell that is;,.l. . r~.~, d with detection means 600. As shown by Figure 7, the dye spots 142 are cnn~oinPd in the flow channel 160 which is defined by ~ul ?~-2 0 110 and 140; and the 1~; l 1; ~1 void in the spacing layer (not observable from this p~ c. liv~). As shown in Figure 7, the detection means 600 can comprise a light source 610, a light detector 620 and a intensity reader 630.
2 5 VII. ASSAY METHODS
The flow cell device can be used to detect the presence or amomnt of an analyte which may be present in a test sample. A
test sample can be introduced into the flow cell's flow channel wherein it contacts the im~r~hili7Pd reagent means. The presence 3 0 or amount of the analyte can be tlPtprmin~d as a result of the analyte ~ e the immnhili7Pd reagent means. When contacted with the test sample, the immnhili7Pd reagent means generates a d~ t 1 1 - signal that is detected by detection means and is ulL~liv~ of the presence or amount of analyte that may be 3 5 rnn~oin~d in the test sample. It will be -- .~ i looA of course, that the test sample can be, for example, il~ du~ ~d into the flow cell's WO 95/22051 F~ ~ 1500 21~93~9 flow channel and stopped therein~ it can be cnntim1n11c1y flowed through the flow channel or it can be recirculated through the flow channel.
The following examples are not intended to limit the invention herein provided but are intended to illustrate the invention.
T1 ~Amp1a 1 Prnrl11rt;~m of An RiorF~TFnt Tmmnhili~Atinn MFAium Usin~
(~T1~1rrcP O~ Re ~ nrbed to PlAtini7F~i CArbon The enzyme binding resin (see below) was 6elected from the commercial paint industry fnrmlllAt~ designed to be fast drying, adherent and able to emulsify pigments. The mixing ed-L~t was designed to generate high shear rates, thus 15 producing a very highly dispersed pigment and small particle size.
In order to obtain this smooth emulsion, the fnrm111Atinn C ..~ were mixed in a WIG-L-BUG (Crescent Dental M:lllllr~.. .II;IIg" Lyons, IL) which is a device designed to mix dental ~mAl~mc The mixer l..F. 1.7...;~ ~lly agitates the 2 0 fnrm111 Atinn in a 2 ml sWnless steel vial rnnt~inin~ a ball bearing, thus allowing 0.5 to 1 ml coating ~ -...c to be blended.
MAt.F'ri Al C
1. Platinized V3l1can XC 72 (carbon black) cnntAinin~ 10%
2 5 platinum 2. Glucose Oxidase (GOD Grade I from Aspergillus niger, Boehringer M~nnhPim ~3jorhpmi~Al~ Tntli InArnli~, IN) 3. PL~ Le Buffered Saline (PBS: l00 _M sodium PllO~La~, l00 mM NaCl, pH 6.0) 4. Acrylic resin mix (31.6% non-volatile solids fnrm111At.F!d as shown below) WO 95122051 217 ~ 3 0 ~ r~ ;c Arrvlic RPRin ~i~ Cnmnnn~nt %bv ~izht Joncryl 537 acrylic emulsion (Jobnson Wax, Racine, WI) 54 Joncryl 56 acrylic resin (Johnson Wax, Racine, WI) 2 DMAMP 80 (Angus Chemical Co., Northbrook, IL) Ektasolve EP ( Eastman l~h_mi~Alc, Kingsport, TN) 9 Distilled water 9 Pl ùc~l " . ,~
En_yme was adsorbed to ~ ": -~l carbon by adding a solution of GOD in PBS buffer to sllcr~ncinnc of rl~tini7Ad carbon in PBS buffer. One A"`1J..IA....I contained 101 mg of rl -l ;"; -d carbon in 0.44 rnl of PBS plus 0.2 rnl of a 60 mg/ml solution of GOD, while the other ,CI-cr_ncinn contained 51 mg of rl~ l carbon in 1 5 0.2 ml of PBS plus 0.1 ml of a 50 mg/rnl solution of GOD. The mi~tures were allowed to statically incubate for 1.5 hours at an ambient tt:lu~ Ult before they were ~ iL6~d at 2000 rpm.
The bll~ IIA were discarded and the resulting wet GOD/carbon pellets were l~ ~,ut~uded in 0.8 gm of the rehin mix 20 and blended in a WIG-L-BUG for 10 minutes. A WIG-L-BUG
(Crescent Dental M~m1fAri.~rin~, Lyons, IL) is a device designed to mix dental AmAlFAmc The mixer ",~ "; Ally agitates the formulation in a 2 ml stainless steel vial cnnt~inin~ a ball bearing, thus allowing 0.5 to 1 ml coating ~ lA to be blended. The 25 resulting coating solutions were hand .];hl,~ d onto separate Cl~ udeh and tested ~lc~.lv l,_-": lly The fnr~mll~tinn for the coating solution is shown below in Table 2.
Table 2 Formula mg carbon mg Resin mg Resin Resin solids/
Mix Solids Carbon ratio 101 800 253 2.50 WO 95/22051 PCI/US9~/01~00 ~17930~`
FYAmrl~ 2 T~ rtro~h~mirAl Flow C~ll MAt.f'riAlC' 5 1. ICI ST505 Heat ~t ~ d Polyester Film (Tekra Gu~u~ iull, Milwaukee, WI) 2. Acheson SS24950 Silver/Silver Chloride Ink (Acheson Colloids, Port Huron, MI) 3. Acheson 423SS Carbon Ink (Acheson Colloids, Port Huron, 1 0 MI) 4. Acheson ML25198 TnclllAtinæ T)i~l~ctrir (Acheson Colloids, Port Huron, MI) 5. Acheson W 8002 Adhesive (Acheson Colloids, Port Huron, MI) 15 6. Bioreagent Tmmnl~ Ati-~n Medium (from Exanple 1) 7. Pot~t;ol~3t - an eight channel pul -.~.;n~ was Acc~n~hl~d at Abbott Labu~ -.;c3 (Abbott Park, IL) to ~.. I.. n~ the flow cell described herein 2 0MAnl1fArtllrin~ Prorrl11lre:
A 0.007 inch thick polyester film was used as substrate support material for both halves of the fiow cell. A working electrode, reference electrode, mask layer, spacing layer and adhesive were appbed to one substrate and a counter electrode was 2 5 applied to the other sllh~ At~ The mAmlfArt-lrin~ process wiU be explained with ~ ~ f~ 3 to Figure 1. A layer of silver/silver chloride ink 22 was screen printed in a pattern to form the redox couple portion of a single reference electrode. Next, a layer of carbon ink was screen printed on the redo~ couple and substrate in 3 0 a pattern to form eight working electrode conductive traces 30 and the conductive trace portion 32 of the reference electrode. The redox couple layer and conductive trace layers were printed with quantities of the It~u~ . materials that were sufficient to provide an end to end ~ .e of less than 100 ohms.
3 5 On top of both the silver/silver chloride ink and c~rbon ink layers a layer of dielectric 40 was screen printed in a pattern to ~ wo gs/220s~ 7 9 3 ~ 9 Pf'TNS95/01500 mask all of the working conductive traces $0 and redox couple 22 except for a small circular area 46 which was 0.066 inches in diameter. The masking layer of dielectric was applied in a quantity sufficient to become water i...l. ...F ~hlF~. A layer of 5 dielectric was then applied to the mask layer in a pattern to form the spacing layer 60 having 1....~; l -1; ..~1 void 56. The spacing layer of dielectric was applied using a quantity sufficient to give the flow cell's flow channel a 10 ~/cm2 volume. A 0.001 inch layer of a&es*e 60 was then printed on top of the spacing layer. The 10 adhes*e layer also had 1r.n~ihl~in~1 void 66. A paper release liner was added to the surface of the adhesive to protect it during handling. A steel ruled dye was then used to cut this part of the device from the sheet of polyester film. A steel ruled dye was also used to cim~ cly cut ~ nmPnt. apertures 12,14, 42, 44, 52, 1 5 54, 62 and 64 from all layers printed on the polyester film.
Again, with reference to Figure 1, the second substrate of the flow cell was prepared by screen printing a layer of carbon ink 80 (counter electrode) onto a sheet of polyester film. The quantity of carbon ink used was sufficient to provide an end to end lG~ ,G
2 0 of less than 100 ohms. The outline of this part of the flow ceU, the nmFnt. apertures and the fluid inlet and outlet ports were cut from the sheet of polyester film with a steel-ruled die.
After the gub~Lltlles and the layers immnhili7sd thereon were cut from the polyester film, 0.36 111 of 1 J~ G~b.,.ll.
25 imm~lhili~lti~m medium (from Example 1) was .l;~ ed onto the circular area of the eight working electrode traces 30. The bioreagent was allowed to cure at room L~ GILL~UlG for one hour.
Using a jig and ~ nmsnt pins, the two 1~ were alibned and li--..;..~d together.
Te9i;nF P1Uff.1~IIG
The testing IJLUI~GdU G will be described with reference to Figure 3. The ~RC'smhlsd flow cell was placed on a stand and ....... r~ to a psri~slti-~ pump (not shown) via fluid crnns~innc to 3 5 the inlet and outlet ports 300 and 310. Electrical ~r.nns~ti~nc (between the f~ow cell and por ntio~g~ 400) to all eight of the WO 95/22051 ? 9 PCT/US9S/01500 2 0 `
working electrodes 30, the counter electrode (not shown) and the reference electrode ~4 were made using clamps and wires 3~0 and 860; 330 and 340; and 360 and 370 l~,U~ iVtly. Liquids were drawn t_rough the cell with a peristaltic pump and the potential of the 5 ~ dudes relative to one another was controlled by a three electrode pu'~ n '-l with eight separate current ~ -. ;l.r rh l~nn~sl c The working electrode potential was set at 350mV versus the on board Ag/AgCl reference electrode. Solutions of differing 10 glucose rnnr~ l : ll lc were then ~ ,c~ vely drawn through the cell and the current which passed at each of the eight working electrodes wa6 Illu...Lul~d and recorded. The graph shown in Figure 8 illustrates the current response of one of the working clc~ ~udes to 0 mM, 2.5 mM, 6.5 mM and 11.4 mM glucose 15 solutions in pH 7.5 aqueous pl.n;~l.h~ buffer (PBS - 40.5 mM
Na2HPO4, 9.5 mM NaH2PO4, 50 mM NaCl). Also shown in Figure 8 is the linear regression t'~rough the four data points.
As shown by Figure 8 the electrode response varies linearly with the glucose cvl~ lion in the test sample.
F~Y~mnlf. 3 Pl~,u. -l a~iull of an Oxv~en ,~Pncibve Dve Free base tetra(ppnt~fl1lnrophenyl)~lus~llylill [H2(TFPP)]
was made 2 5 by adding 2.0 ml of p ~ n .. -~ Phyde, 1.5 ml pyrole and 2.0 ml of boron triflllnri-le etherate to 1500 ml dichlul - l". .P to form a re~rfi~)n mi~rblre All materials cn~nrricin~ the reaction mixture are available from Aldrich Chemical Co., Inc., Milwaukee, WI. The reaction mixture was stirred for 1 hour 30 before 2.5 grams of 2,3-dichloro-5,6-dicyano-1,4-b~l~u~luillulle (Sigma, ST. Louis, MO) was added a~d the resulting mixture was heated to 40C for 1 hour. Then, the solvent was flash dried arsd the crude solid H2(TFPP) product was ~ u~lsluclc~hed over silica gel using dichlol. "~ P as the eluting solvent. 1.0 gram of the 3 5 purified H2(TFPP) and a 10 times molar excess of PtCl2 (Aldrich Chemical Co.Irsc.) were refluxed for 24 hours in 500 ml of WO95/22051 P~,l/ll,, lS00 ~17g3~9 7.~ ;le to synthesize crude platinum tetra(p~ ..... v~uh~llyl)l,ul~lly~ [Pt(TFPP)]. The product was purified on a neutral alumina column using CH2Cl2 as the eluant.
An oxygen sensitive dye solution was prepared by dissolving 100 5 mg of the purified Pt(TFPP) in a 25 ml of a silicone polymer stock solution. The polymer stock solution was made by dissolving 10.0 grams ûf a dimethylsilo~ane-hicrhpnnl (General Electric Inc., Waterford, NY) in 100 ml tetra~lyLurul~l.
~YslmnlP 4 Opti~l Flow Cell M~t.Pri~lc 1. ICI ST505 Heat .s~hili7pd Polyester Film (Tekra Corporation, Milwaukee, WI) 2. Acheson ML25198 rnc~ tin~ Dielectric (Acheson Colloids, Port Huron, MI) 3. Acheson SU459 Adhesive (Acheson Colloids, Port Huron, MI) 20 4. Platinum tetra(p~ . n...~ uull~ .lyl)~uullJLy ;1l from EYample 3.
~ l;llF P~U~6~1UI~
The - ~ JlU~,6dUl~ will be described with 2 5 reference to Figure 5. A layer of dielectric ink 130 was screen printed onto a substrate 140, which ~ a sheet of polyester film, to form the spacing layer of the nOw cell. The spacing layer of dielectric was applied using a quantity sufficient to give the flow cell's flow channel a 10 ~llcm2 volurne. A .001 inch layer of 3 0 adhesive 120 was then screen printed on top of the dielectric layer in the pattern shown in Figure 5. A release liner was then applied onto the surface of the adhesive to protect it during handling. The outhne of the cell and ~ nmPnt apertures 114, 134 and 144 were then cut from the polyester sheet using a steel-ruled die. The other 3 5 substrate was fo~med by cutting the cell outline, ~lignnnPnt i i: ~ \ j WO 95/22051 r~.,., . Isoo ~1~93~9 aperture6 114 and inlet and outlet ports 112 from a 0.007 inch thick sheet of polyester film After removing the release liner, 1.0 ,ul aliquots of the oxygen sensitive dye 142 (from Example 3) were tben applied to 5 substrate 140 such that the dye spots were aligned within the lnn{~itll(lin~l voids 122 and 1$2. rlAhe dye was allowed to dry for one hour at room l~...l.~ . ,. I.... t and the polyester ~uL~ L~i were then pQcitinnPd uging an ~ nmPnt. jig and ~liEnmPnt pins before being 1 imin~tPd together.
T ~ P~
The ~cRPmhlPd flow cell was fixed on a stand and fluid tubes were (-nnnPr~ql1 to tbe iDlet and outlet ports. Tonometered solutions were then moved into and out of tbe cell using a 15 peristaltic pump. Tests were p~. f.,. .--Pd by pnCitirlninE a 2~0 Ilm optical fiber (Ensign-Bickford Optics Co.; Avon, CT) above the dye spots 142 and illllmin~tinE the dye with light emitted from a pulsed light emitting diode (Hewlet Packard Co., Cupertino, CA) while a solution of l.. h~ d oxygen buffer was flowing t_rough the 20 flow channel. Using an avalanche phul~ rl~
Corp., Bl;d~,~..dL~-, NJ), ph~ u~is~ L intensity LUea~UIt~lu~.Lb were recorded in 2 time regions (3-17 llseconds and 3-200 1l6econd6) after each P~ritstirm pulse. The pho~lJhu,e,Act".L inten6ity ratio was rAl(~--l, tPd and plotted as a function of oxygen Cull~LLidLion.
2 5 As shown by Figure 9, a ~ udu~le non-linear rPl~ltinn.A~hip between the inten6ity ratio and oxygen partial pressure is observed.
Claims (19)
1. A diagnostic flow cell for determining the presence or amount of an analyte in a test sample, said flow cell comprises:
i) a spacing layer disposed between a first and a second opposed substrate, wherein said spacing layer has a longitudinal void and wherein said spacing layer and said opposed substrates define a flow channel;
ii) fastening means for coupling said spacing layer and said opposed substrates;
iii) inlet means for permitting said sample to enter said flow channel;
iv) outlet means for permitting said sample to exit said flow channel; and iv) immobilized reagent means for producing a detectable signal, wherein said reagent means is at least partially contained within said flow channel.
i) a spacing layer disposed between a first and a second opposed substrate, wherein said spacing layer has a longitudinal void and wherein said spacing layer and said opposed substrates define a flow channel;
ii) fastening means for coupling said spacing layer and said opposed substrates;
iii) inlet means for permitting said sample to enter said flow channel;
iv) outlet means for permitting said sample to exit said flow channel; and iv) immobilized reagent means for producing a detectable signal, wherein said reagent means is at least partially contained within said flow channel.
2. The flow cell of claim 1 further comprising a mask layer disposed between said substrates.
3. The flow cell of claim 1 wherein said fastening means is selected from the group consisting of a rivet, a nut and a bolt, a sonic weld and an adhesive.
4. The flow cell of claim 1 wherein said reagent means comprises a reference electrode, a counter electrode and a working electrode.
5. The flow cell of claim 4 wherein said reference electrode comprises:
(a) a redox couple selected from the group consisting of silver/silver chloride/graphite, mercury/mercurous chloride, silver/silver iodide and silver/silver chloride; and (b) a conductive trace comprising graphite and screen printed ink.
(a) a redox couple selected from the group consisting of silver/silver chloride/graphite, mercury/mercurous chloride, silver/silver iodide and silver/silver chloride; and (b) a conductive trace comprising graphite and screen printed ink.
6. The flow cell of claim 4 wherein said counter electrode comprises graphite and screen printed ink.
7. The flow cell of claim 4 wherein said working electrode comprises a conductive trace and an enzyme immobilized to said trace.
8. The flow cell of claim 7 wherein said enzyme comprises a bioreagent immobilization medium.
9. The flow cell of claim 1 wherein said reagent means comprises an optically active dye selected from the group consisting of a pH sensitive dye, an oxygen sensitive dye and an ion sensitive dye.
10. The flow cell of Claim 4 wherein said reagent means further comprises an optically active dye selected from the group consisting of a pH sensitive dye, an oxygen sensitive dye and an ion sensitive dye.
11. The flow cell of claim 1 wherein said flow cell further comprises flow means for introducing said test sample into said flow channel wherein said flow means is interfaced with said inlet and outlet means; and detection means for detecting a detactable signal wherein said detection means is interfaced with said immobilized reagent means.
12. A method of determining the presence or amount of an analyte in a test sample, said method comprising the steps of:
I) contacting an assay device with said test sample, wherein said assay device comprises a) a flow cell wherein said flow cell comprises i) a spacing layer disposed between a first and a second opposed substrate, wherein said spacing layer has a longitudinal void and wherein said spacing layer and said opposed substrates define a flow channel, ii) fastening means for coupling said spacing layer and said opposed substrates, iii) inlet means for permitting said sample to enter said flow channel, iv) outlet means for permitting said sample to exit said flow channel, and iv) immobilized reagent means for producing a detactable signal, wherein said reagent means is at least partially contained within said flow channel, b) detection means for detecting said detactable signal wherein said detection means is interfaced with said immobilized reagent means, and c) flow means for introducing said test sample into said flow channel wherein said flow means is interfaced with said inlet and outlet means;
and II) detecting said measurable signal.
I) contacting an assay device with said test sample, wherein said assay device comprises a) a flow cell wherein said flow cell comprises i) a spacing layer disposed between a first and a second opposed substrate, wherein said spacing layer has a longitudinal void and wherein said spacing layer and said opposed substrates define a flow channel, ii) fastening means for coupling said spacing layer and said opposed substrates, iii) inlet means for permitting said sample to enter said flow channel, iv) outlet means for permitting said sample to exit said flow channel, and iv) immobilized reagent means for producing a detactable signal, wherein said reagent means is at least partially contained within said flow channel, b) detection means for detecting said detactable signal wherein said detection means is interfaced with said immobilized reagent means, and c) flow means for introducing said test sample into said flow channel wherein said flow means is interfaced with said inlet and outlet means;
and II) detecting said measurable signal.
13. The method of claim 12 further comprising a mask layer disposed between said substrates.
14. The method of claim 12 wherein said reagent means comprises a reference electrode, a counter electrode and a working electrode.
15. The flow cell of claim 14 wherein said reference electrode comprises:
(a) a redox couple selected from the group consisting of silver/silver chloride/graphite, mercury/mercurous chloride, silver/silver iodide and silver/silver chloride; and (b) a conductive trace comprising graphite and screen printed ink.
(a) a redox couple selected from the group consisting of silver/silver chloride/graphite, mercury/mercurous chloride, silver/silver iodide and silver/silver chloride; and (b) a conductive trace comprising graphite and screen printed ink.
16. The method of claim 14 wherein said counter electrode comprises graphite and screen printed ink.
17. The method of claim 14, wherein said working electrode comprises a conductive trace and a bioreagent immobilization medium.
18. The method of claim 12 wherein said reagent means comprises an optically active dye selected from the group consisting of a pH sensitive dye, an oxygen sensitive dye, and an ion sensitive dye.
19. The method of claim 14 wherein said reagent means further comprises an optically active dye.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US19465294A | 1994-02-09 | 1994-02-09 | |
US08/194,652 | 1994-02-09 |
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CA2179309A1 true CA2179309A1 (en) | 1995-08-17 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CA002179309A Abandoned CA2179309A1 (en) | 1994-02-09 | 1995-02-06 | Diagnostic flow cell device |
Country Status (6)
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US (1) | US5520787A (en) |
EP (1) | EP0752099A1 (en) |
JP (1) | JPH09509485A (en) |
AU (1) | AU1911795A (en) |
CA (1) | CA2179309A1 (en) |
WO (1) | WO1995022051A1 (en) |
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- 1995-02-06 AU AU19117/95A patent/AU1911795A/en not_active Abandoned
- 1995-02-06 EP EP95911617A patent/EP0752099A1/en not_active Withdrawn
- 1995-02-06 CA CA002179309A patent/CA2179309A1/en not_active Abandoned
- 1995-02-06 JP JP7521277A patent/JPH09509485A/en active Pending
- 1995-02-06 WO PCT/US1995/001500 patent/WO1995022051A1/en not_active Application Discontinuation
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US5520787A (en) | 1996-05-28 |
JPH09509485A (en) | 1997-09-22 |
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