WO2008043041A1 - Method and device for rapid parallel microfluidic molecular affinity assays - Google Patents

Method and device for rapid parallel microfluidic molecular affinity assays Download PDF

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Publication number
WO2008043041A1
WO2008043041A1 PCT/US2007/080479 US2007080479W WO2008043041A1 WO 2008043041 A1 WO2008043041 A1 WO 2008043041A1 US 2007080479 W US2007080479 W US 2007080479W WO 2008043041 A1 WO2008043041 A1 WO 2008043041A1
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WIPO (PCT)
Prior art keywords
reagent
sample
membrane
capture
inlet
Prior art date
Application number
PCT/US2007/080479
Other languages
French (fr)
Inventor
Paul Yager
Turgut Fettah Kosar
Michael Wai-Haung LOOK
Afshin Mashadi-Hossein
Katherine Mckenzie
Kjell E. Nelson
Paolo Spicar-Mihalic
Dean Stevens
Rahber Thariani
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University Of Washington
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Application filed by University Of Washington filed Critical University Of Washington
Priority to US12/444,385 priority Critical patent/US8101403B2/en
Publication of WO2008043041A1 publication Critical patent/WO2008043041A1/en
Priority to US13/344,408 priority patent/US9138743B2/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/16Reagents, handling or storing thereof
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/06Auxiliary integrated devices, integrated components
    • B01L2300/0627Sensor or part of a sensor is integrated
    • B01L2300/0636Integrated biosensor, microarrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0809Geometry, shape and general structure rectangular shaped
    • B01L2300/0816Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0887Laminated structure
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0475Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • This invention relates generally to methods and devices for rapid parallel molecular affinity assays performed in a microfluidic environment.
  • the invention exploits hydrodynamic addressing to provide simultaneous performance of multiple assays in parallel using a minima! sample volume flowing through a single channel
  • Immunoassays take advantage of the specific binding abilities of antibodies to be widely used in selective and sensitive measurement of small and large molecular anaiytes in complex samples.
  • the driving force behind developing new immunological assays is the constant need for simpler, more rapid, and less expensive ways to analyze the components of complex sample mixtures.
  • Current uses of immunoassays include therapeutic drug monitoring, screening for disease or infection with molecular markers, screening for toxic substances and illicit drugs, and monitoring for environmental contaminants.
  • the invention provides a method and assay device for detection of an anaiyte in a fiuidic sample.
  • the device comprises:
  • a microfluidic chamber having a first inlet;
  • a first surface in communication with the first inlet, wherein the first surface comprises a plurality of capture regions:
  • a plurality of capture agents immobilized on the capture regions wherein the capture agents specifically bind the anaiyte;
  • a reagent storage depot in communication via a single fiuidic channel with the first surface, wherein the storage depot comprises a plurality of reagent regions;
  • detection reagents that specifically bind the anaiyte and that become mobile upon contact with fluid, wherein the detection reagents are disposed within the reagent regions.
  • the first surface can comprise a porous carrier, such as a membrane or other porous structure, a fiat surface, or other structure to which the capture agents can be immobilized while retaining the ability to be brought into contact with analytes delivered via fluid passing over the first surface,
  • a porous carrier such as a membrane or other porous structure, a fiat surface, or other structure to which the capture agents can be immobilized while retaining the ability to be brought into contact with analytes delivered via fluid passing over the first surface
  • the reagent storage depot can comprise one or more cavities, and/or a polymeric compound immobilized on the device.
  • the storage depot is provided by stabilizing the reagents in a solid state using, for example, a porous matrix (e.g., a polymer, ge! or soluble salt) that either swells on contact with the fluid and releases the reagents or completely dissolves thereby delivering the reagent.
  • the storage depot can also be provided by locating the detection reagents, in dry form, in physical cavities, such that contact with fluid mobilizes the reagents.
  • the reagent(s) is immobile in its dry form and becomes mobilized upon contact with fluid such that the reagent is delivered, upon mobilization, to the first surface where it can react with the captured anaiyte.
  • the storage depot comprises a porous membrane that is aligned parallel to the first surface.
  • the device is well-suited to an embodiment having a first surface in which the plurality of capture regions are arranged linearly and perpendicular to the long axis of the single fiuidic channel thai provides communication between the storage depot and the first surface.
  • the reagent regions are likewise arranged linearly and perpendicular to the long axis of the single fiuidic channel, such that the linear arrangement of reagent regions is parallel to the iinear arrangement of capture regions.
  • the device typically comprises a plurality of polymeric layers.
  • the polymeric layers can be used to devise the configuration of inlets, channels, cavities and surfaces suitable for a particular embodiment.
  • a second inlet is provided in communication with the storage depot The second iniet can be used to deliver fluid to effect mobilization of the reagents stored in the storage depot.
  • the same fluid stream that delivers analyte to the first surface can also serve to effect mobilization of the reagents stored in the storage depot.
  • an outlet is provided in communication with the first surface.
  • Such an outlet can be used, for example, to draw fluid away from the first surface if desired.
  • the outlet allows one to analyze the effluent or to draw off excess fluid prior to delivery of a subsequent fluid stream, in addition to other uses.
  • the device can further comprise one or more channels that provide communication between the first inlet and the first surface and/or between the second inlet and the storage depot.
  • 3 channels provide communication between the first inlet and the first surface.
  • Multiple channels from the inlet to the first surface can be used to deliver multiple analytes, or, in a typical embodiment, three channels are used to deliver one analyte sample and two control samples (e.g., positive and negative controls).
  • the invention further provides a method of detecting the presence of an analyte in a Ruidtc sample.
  • the method typically comprises;
  • step (b) contacting a single stream of fluid with the plurality of detection reagents under conditions effecting migration of the detection reagents to the first surface: (c) detecting the presence of detection reagent bound to anaiyte that is bound to the immobilized capture agents, whereby presence of detection reagent is indicative of the presence of the anaiyte, in a typical embodiment, the delivering of step (a) comprises pumpsng the flutdic sample into the first inlet. The method can further compnse delivering one or more control sampies via laminar flow into the first inlet.
  • step (a) comprises delivering one stream of a test fluidic sample, one stream of a positive control fluidic sample, and one stream of a negative control fluidic sample.
  • the streams of fluidic sample are delivered via a single channel, in another embodiment, the streams of fluidic sample are delivered via separate channels
  • a 3-channei embodiment can deliver test sample, positive control sample and negative control sample, each via a separate channel
  • the 3 streams can be delivered in one channel using controlled fluid pumping to avoid mixing of streams.
  • the contacting of step ⁇ ) comprises pumping fluid into a second inlet that is tn communication with the reagent storage depot.
  • the fluid is typically a buffer and serves to mobilize the reagent so that it can contact and bind anaiyte that has been immobilized on the first surface upon binding capture agent.
  • rinsing or washes can be used to clear out unbound reagents between steps of the method.
  • the delivering of step (a) provides the contacting of step (b), whereby the fluidic sample, upon contact with the detection reagents, effects migration of the detection reagents.
  • steps (a) and (b) can be accomplished with a single stream of fiuid ⁇ c sample.
  • the reagent regions can be positioned between the first inlet and the capture regions.
  • the capture agents and the detection reagents comprise antibodies and/or antigens.
  • the contacting of step (b) further comprises delivering to the first surface an amplification reagent that binds to the detection reagents.
  • the defection reagents are labeled, either directly or indirectly, and the detectable signal can be provided or amplified using known techniques and materials. Detection of signal can be achieved by a variety of means known in the art, including but not limited to, measuring an optica! property such as optica! absorbance, reflectivity, optica! transmission, chemiiumin ⁇ scence or fluorescence, in some embodiments, signal can be detected by eye. Optica! readers are preferred when a quantitative measurement is desired.
  • FiG. 1A Schematic design of version 1 of the polymeric disposabie in which the secondary reagents are contained within cavities in the disposable. Two sets of fluid inlets are located at the right and left ends of the disposable as well as a single outlet path beiow the embedded membrane (center), which is Soaded with molecules.
  • FIG. 1B Close-up of the centra! portion of the same image as in FigurelA.
  • FIG. 1C Cut-away view of the same device (central portion) as in Figures 1A and 1 B, showing the relative locations of the different layers 1 the capture membrane and the secondary reagent depots.
  • the exit port for the device is below the membrane, and fluid exits to the right,
  • FiG. 2 Schematic of the minimal set of structural layers required to assemble version 1 of the immunoassay device.
  • FIG. 3 Schematic of assembled immunoassay device with three inlet holes on the right, one on the left, and one outlet hole invisible below the porous membrane. Secondary antibodies are printed on a membrane (left column of dots) with three cycles of the same set. Capture membrane (right column of dots) is spotted or striped with capture antibody and blocked. The relative locations of 4 valves are indicated along the bottom of the figure.
  • Step 1 involves closing valves 2 and 3, opening valves 1 and 4, Buffer is pumped from the right (valve 4 to valve 1) to wet out both membranes. Valve at left is closed and pumping stopped.
  • FIG. 5 First version of sample load, in version 1, step 2 comprises pumping in sample from the right, with valves 1 and 2 closed. Sample exits below the membrane via an outlet not shown here, No flow over the secondary antibody membrane, which antibodies do not diffuse away because of high molecular weight.
  • FIG. 8 Illustration of how, in the second version of the sample load, everything is the same as in the previous version, except that iaminar flow is used to flow 2 or 3 different solutions across the capture reagent membrane. With valves 1 and 2 closed, three solutions are pumped in: sample, positive control (all anaSytes at high levels), and negative control (no sample antigens). No flow over the secondary antibody membrane, which antibodies do not diffuse away because of high molecular weight,
  • FiG. 7 illustrates the rinse. Vaives 1 and 2 are closed; 3 and 4 are open. Rinse with buffer to remove excess sample from membrane.
  • FiG. 8 Illustrates the ioading of secondary antibodies. Close valves 1 and 4; pump buffer from vaive 2 to 3, pushing 2" antibody from left membrane through the one at the right. Continue until sufficient 2" antibody is transferred to capture zones.
  • FIG. 9 illustrates the rinsing of secondary antibodies. Using fluids from either vaive 1 or 2 (with valve 3 open and 4 closed), flush until ail excess secondary antibody is pushed through capture membrane Detect (if this is Au-iabeled antibody, for example) by measuring optical density of spots. Assay is complete.
  • FIG. 10 illustrates a detection step. This and further steps are only necessary if using B ⁇ amplification step. Pump secondary reagent from right at slow rate, Postsve controls and positive sample spots darken over a few seconds to minutes.
  • FIG. 11 Schematic of version 2 of the device and system.
  • FIG. 12 Schematic of the minimal set of structural layers required to assemble version 2 of the immunoassay device as shown in Figure 11.
  • FIG. 13 Assay results showing the decrease in signal (from left to right) seen as the anaiyte concentration in the sample decreases.
  • the analyte is Plasmodium falciparum Histidine-Rich Protein II, or PfHRP2.
  • the red spots (upper 6 rows) show the results generated using an antibody-conjugated gold particle as a detection molecule; the blue spots (lower 2 rows) use an enzyme-conjugated antibody as the detection molecule, followed by an enzyme substrate that becomes a blue precipitate in the presence of the enzyme,
  • FiG. 15A-B A self-contained microfiuidic format, consisting of a laminate device in which connecting fluidic channels are formed, a membrane patterned with capture molecules, a porous pad containing dried detection reagent, and an external fluid- pumping and imaging system.
  • the multiple fluid inlets are each fed by separate pumps in this preliminary design, sidestepping the need for valves.
  • the device is pictured as a diagram ( Figure 15A) and photograph ( Figure 15B) of two revisions of the design.
  • FiG. 16A-B Functional schematic (FiG. 16A) and CAD design (FIG. 16B) for assay card with single fluid inlet to the reaction chamber (the location of the assay membrane) .
  • FiG. 17B CAD design of assay card with multiple inlets to reaction chamber
  • FiG. 18 Two capture reagents patterned in two 4x4 arrays on a membrane. On the left, a PfHR P2 capture molecule is patterned; on the right, an aldolase capture molecule.
  • FiG. 19 Five sequential frames from a video of a dry-reagent pad being rehydrated.
  • FiG. 20 Three frames from a video of an assay showing (1) sample introduction to membrane; (2) rehydrated conjugate introduced to membrane; and (3) capture spot labeled by conjugate.
  • FiG. 21 images indicating steps of automated optical measurement. On the left, four separate registration marks are identified in an image; on the right, the analyzed image (captured by a flatbed scanner, 48-bit RGB, 3200 dpi) with simulated blue registration marks and red assay spots, the location of each marked with an "X.”
  • the invention relates to a method and device for performing rapid molecular binding assays, including immunoassays, and in particular, sandwich immunoassays.
  • the method invoives binding a plurality of primary capture reagents to a plurality of locations on a porous membrane, placing a matched set of secondary (or detection) binding molecuies in a line of cavities or on a porous membrane aligned parallel to the reagent storage locations, but separated by a gap, and a method for sandwiching the anaiyte in question between them using laminar flow in a microfiuidic device.
  • the sample is loaded onto the first membrane by pumping it through said first membrane, where sample anaiyte molecuies become bound to the capture molecules immobilized on that membrane. Fluid is then pushed past the storage depot line or through the second membrane to release the secondary capture molecules and transport them to the first membrane to "sandwich" the anaiyte molecules.
  • Detection is then possible by either directly (if the secondary capture molecule is directly observable (such as a fiuorescently- or Au-!ab ⁇ ied secondary antibody) or indirectly (using for example, secondary antibodies labeled with enzymes such as horseradish peroxidase (HRP) followed by flow over the first membrane of a solution producing an observable signal, such as precipitatable tetramethylbenzidine (TMB).
  • HRP horseradish peroxidase
  • TMB precipitatable tetramethylbenzidine
  • the device allows the simultaneous performance of dozens of immunoassays (as well as positive and negative control reactions) in a minimum of time using a minimum of sample volume and in a minima! space. Reading the results of the immunoassays may either be made directly (by eye), or with the aid or a quantitative optical reader. Conventional off-the-shelf reagents can be used to minimize cost. It is particularly well adapted for performance of multiple immunoassays on an inexpensive polymeric disposable device that may be read out directly or using an optical reader.
  • the invention disclosed herein is a design for a molecular binding assay (and a method of using that design).
  • This assay system is well suited to use as the basis of immunoassays such as "sandwich immunoassays".
  • immunoassay reagents and immunoassays are referred to herein as immunoassay reagents and immunoassays, respectively, it is understood by those skilled in the art that a device that could perform any other assay (based on proteins, aptamers, nucleic acids, or other molecules) that involves molecules capable of binding to each other would fall under the scope of this invention.
  • the device is fabricated from inexpensive polymeric components combined with porous membranes capable of binding to and immobilizing capture reagents such as capture immunoassays or target antigens, depending on the format of the immunoassay.
  • capture reagents such as capture immunoassays or target antigens, depending on the format of the immunoassay.
  • the arrangement allows for storage of both capture reagents and secondary reagents in dry form on the polymeric rnicroflutdic device, thereby creating a self-contained disposable that can be used with or without a reader technology.
  • the disposable can be made to perform multiple immunoassays in parallel, as well as perform measurements of multiple anaiyte concentrations in samples, positive control solutions, and negative control solutions simultaneously.
  • the assay assumes laminar flow conditions in all components, and microfluidic dimensions.
  • the immunoassay format can be manufactured very inexpensively, such that a polymeric disposable is suitable for use in point-of-care assays.
  • Optical detection methods opticalca! absorption, diffuse reflectance absorption, or fluorescence
  • the assays can operate in a simple qualitative yes/no fashion, or in a quantitative manner (using, for example, a quantitative optica! reader).
  • Detection of the optical signal indicating the binding of the anaiyte can be performed in either of two well-understood ways;
  • One version involves the use of an optically detectable secondary antibody, such as an antibody bound (covIERiy or noncovIERiy) to colored microspheres, fluorescent molecules or nanoparticies, or strongiy absorbing dyes of nanoparticles (such as gold nanoparticies).
  • the assay is an ELiSA assay, in which the secondary antibody is labeled with an enzyme, and the final step after binding of the secondary antibody to the anaiyte is exposure of the enzyme-loaded capture membrane with a "developing solution"; examples are to be taken from the list of a!l known ELISA systems, including any of several commercially available horseradish peroxidase/ precipitating tetramethyibenzidine systems.
  • a likeiy appiication for such a disposabie is a point-of-care immunoassay system for use in the developing world, although use as an inexpensive point-of-care diagnostic system is also possible.
  • the disposable polymeric immunoassay system can be coupled to other types of assays in a single integrated device.
  • the device can be fabricated from seven polymeric layers.
  • a representative example of a multiple polymeric layered device is shown in Figure 2,
  • FIG. 2 A schematic of the minima! set of structural layers required to assemble version 1 of the immunoassay device (of Figure 1A) is shown in Figure 2, The layers are numbered in order of assembly.
  • Layers 1, 4 and 8 are just C (carrier) layers (plain sheets of an appropriate polymer such as Mylar, PMMA, or others), whereas layers 2 and 7 are ACA (adhesive, carrier, adhesive) layers.
  • Layers 3 and 6 are AC layers, with adhesive on one side that serve to seal layer 5 « the membrane, in place.
  • Layer 1 is the top cover of the device, and must consist of a clear (optically transparent) material to allow optica! observation of the !ayer 5
  • Layer 2 is the main fluid cavity.
  • Layer 3 is the "floor" of the main fluid cavity, which contains a (here large and rectangular) hole for fluid outflow, as we!! as a multiplicity of storage depots for storage of secondary reagents. These secondary reagents can be placed into the storage depots as one of the last steps of assembly of the polymeric device.
  • Layer 4 is the cavity that localizes the permeable membrane.
  • Layer 5 consists of a permeable membrane onto which capture molecules are immobilized prior to final assembly of the device, and which is placed within the rectangular cavity in layer 4, The deposition of the different capture molecules onto layer 5 can be in any form, but are shown here as circular spots.
  • Layer 6 supports the permeable membrane.
  • Layer 7 collects all flow through the membrane to a single port.
  • Layer 8 is the floor of the device and couples to inlets and outlets for the device. Note that, in this schematic, the right side of all layers ⁇ but 5) are shown with two holes. In the schematic below either one hole or three are used, as explained below. Note that further embodiments of the device can be assembled in part using injection molded parts to reduce the part count and reduce fabrication costs.
  • FIG. 3 Shown in Figure 3 is an operation sequence for version 1 of the device as shown in Figures 1A and 2, in this schematic, the assembled device has three inlet holes on the right, one on the left, and one outlet hole invisible below the porous membrane, The cavity is designed In such a way that fluid entering the main cavity is "fully developed “, and, therefore, flowing almost exclusively horizontally and at the same horizontal velocity top to bottom (as shown in this figure) by the time it reached either the membrane from the right, or the secondary reagent storage depots from the ieft
  • buffer is used to wet out the device from the right. Such a process proceeds with the exit below the device closed, so that almost all fluid flows from right to left.
  • the high molecular weight of the secondary reagents prevents them from diffusing appreciably in the vertical direction (as shown in this figure) during the complete operation of the device.
  • the capping layer layer 1 in Figure 2 can be manufactured with ftns that fit between the secondary reagent storage depots. The wet-out pushes minimum fluid through the membrane.
  • Figure 5 illustrates a first version of sample load
  • the valves "below" the left side of the device are closed and the sample is pumped in through a single inlet from the right forcing the sample to flow through the semi-permeabie membrane.
  • the second version of the sample load Figure 6 everything is the same as in the previous version, except that laminar flow is used to flow 2 or 3 different solutions across the capture reagent membrane.
  • buffer is flushed from the right (with the vaive under the ieft side closed) to clear excess (free) anaiyte from the device and flush the capture membrane.
  • the secondary reagent (2° Ab, for example) is then loaded onto the anaiyte moiecuSes that are bound to the capture membrane (via the capture molecules) by pumping buffer from the ieft iniet (with ail the right inlet valves closed; see Figure 8). This continues until aii of the 2° Abs are transferred. Laminar flow (or channels or fins, if necessary) wili ensure that the appropriate T Abs are transported to the appropriate capture molecule regions on the membrane.
  • Those that produce a precipitated product are preferred because of the buiid-up of signal possible on the membrane over time and pushing of reagents through the membrane, but non-precipitating systems can aiso be used.
  • other detection reagents can be stacked on top of the 2 a Ab layer to produce strong signals using fluorescence or optical absorbance.
  • the above-mentioned scheme relies on the deposition of the secondary reagents onto an impermeabie surface to form depots for subsequent movement to the capture membrane.
  • An alternative that allows the use of technology demonstrated tn other types of assays is to use a second permeable membrane as the depot for the 2 C Abs, allowing these reagents to be preloaded into a membrane before assembly of the card, and washed out of this membrane by flowing buffer up through the membrane.
  • the preliminary design is shown in Figure 11. This design allows all the reagents to be printed onto large sheets of membrane using commercial printing mechanisms for great simplification of manufacturing and. thereby, cost savings.
  • the secondary reagent membrane can be prepared in the same way as the secondary reagents are in lateral flow immunoassay devices (immunochxomatographic test strips),
  • FIG. 1 1 Shown in Figure 1 1 is a schematic of version 2 of the device and system; tt is very similar to that shown in Figure 1A, except that the 2 n Ab storage is now on a permeable membrane that sits in a cavity like that for the capture membrane, there is a second channel below the second membrane (which is an inlet, not an outlet) and the 2° Ab spots are deposited (in a matrix of preserving chemicals) on the second membrane (at left).
  • the second membrane is of a type with no or very low protein retention.
  • FIG. 11 Schematic of the minimal set of structural layers required to assemble version 2 of the immunoassay device as shown In Figure 11.
  • the layers are numbered in order of assembiy and have the same characteristics as those mentioned in version 1 above.
  • Layer 3 is the "floor" of the main fluid cavity, which contains 2 (here large and rectangular) holes for fluid passage.
  • Layer 4 contains the cavities that localize the permeable membranes.
  • Layer 5 consists of a two separate (and different) permeable membranes. The one onto which capture molecules are immobilized prior to final assembiy of the device is identical to that described in version 1 ( Figures 1A and 2), The one at the left is for storage of the 2 s reagents (e.g., Abs).
  • 2 s reagents e.g., Abs
  • Both sets of reagents am “spotted” or “striped” onto the membranes and dried prior to insertion into their respective cavities in layer 4.
  • Layer 6 supports the permeable membranes.
  • Layer 7 now has two separate cavities for controiling flow in the vicinity of the membranes. The one at right is identical to that in version 1 , and collects all flow through the membrane to a single port. The new cavity at left delivers fluid flow to the 2° reagent storage membrane at left, as described befow.
  • Layer 8 is the floor of the device and couples to inlets and outlets for the device.
  • FIG. 3-10 Reference is made to Figures 3-10 for a usage sequence for version 2 that is similar to that described above for version 1.
  • step 4 (2" Ab ioading) of version 2 ( Figure 8)
  • the flow of fluid is up through valve 1 and the 2" Ab storage membrane and over to and down through the capture membrane.
  • Using fluids from either valve 1 or 2, (with valve 3 open and 4 closed) flush until ail excess secondary Ab is pushed through capture membrane is shown in Figure 9.
  • the next step is to detect (if this is Au- labeied Ab, for example) by measuring optical density of spots.
  • the BioDot vacuum manifold is suitable for testing of the flow-through immunoassays of the invention, it consists of 96 individual open-bottom wells and a vacuum plenum that applies a low pressure beiow each well. Between the welis and the plenum is placed a porous membrane, patterned with capture molecules against analytes of interest. Reagents such as the sample, washing buffers, and detection molecule are added sequentially to the wells and drawn through by the applied vacuum. Pictured is an example of the assay results. Each circle in the grid lies underneath a single well and represents a unique set of assay conditions.
  • the assay results presented in Figure 13 show the decrease in signal (from left to right) seen as the analyte concentration in the sample decreases.
  • the analyte is Plasmodium falciparum Histidine-Rich Protein II, or PfHRP2.
  • the red spots (first 6 rows) show the results generated using an antibody-conjugated gold particle as a detection molecule; the blue spots (last 2 rows) use an enzyme-conjugated antibody as the detection molecule, followed by an enzyme substrate that becomes a biue precipitate in the presence of the enzyme.
  • a similar format to the 96-weii plate is the mini-vacuum or "minivac" format, it also uses an applied vacuum to draw fluid from a reservoir through a membrane.
  • the reservoir in this case addresses a larger area of membrane, and the membrane is supported by a metal mesh.
  • Pictured in Figure 14 is a diagram of the format The mesh is depicted in the inset
  • the assay can be run in a self-contained microfiuidic format, consisting of a iaminate device in which connecting fluidic channels are formed, a membrane patterned with capture moiecuies, a porous pad containing dried detection reagent, and an external fluid-pumping and imaging system.
  • the muitipie fluid inlets are each fed by separate pumps in this design, sidestepping the need for valves.
  • the device is pictured in Figure 15A-B as a diagram (15A) and photograph (15B) of the design.
  • the self-contained microfiuidic format consists of a laminate device 150 in which connecting fl ⁇ idic channels are formed by a sample ioop 152 that ts met by a second channel 155 delivering mobilized reagents. Their contents combine into a single channel 130 through the membrane 153.
  • the device 150 aiso includes air vents 180, a membrane 153 patterned with capture molecules, a porous pad 156 containing dried detection reagent, and an external fluid-pumping and imaging system (not shown; representative example is microFlowTM System available from Micronics, Redmond, WA).
  • the muitipie fluid inlets include a sample inlet 151 and a second iniet 154, each fed by separate pumps in this design, sidestepping the need for valves.
  • the second inlet 154 is used to introduce fluid that is directed to the conjugate pad 156 via second channel 155 that feeds into the sample loop 152 before it enters reaction chamber 169 and contacts the membrane 153.
  • a bubble vent 157 can withdraw bubbles from the sample ioop 152 and an outlet 158 exits the reaction chamber 169 via waste line 159.
  • FIG. 16B and 17B are two alternate designs for the assay cards. They include reagent reservoirs for liquid reagents instead of the dried reagent pads described in part C above.
  • Figure 16A- B depicts a functional schematic (18A) and CAD design (16B) for assay card with single fluid iniet to the reaction chamber (the location of the assay membrane).
  • air vents 160 are positioned in waste reservoirs 161, 162, and a bubble vent 163 is provided for priming.
  • Valves 170 disposed throughout provide control points, such as between pipette loading vents 184 and reagent reservoirs 165-188, between pipette loading points 172 and reagent reservoirs 165-168, and between reagent reservoirs 185-168 and reaction chamber 169, as well as between pumps 174, 176 and reaction chamber 169.
  • Figure 17B depicts a CAD design of assay card with multiple inlets to the reaction chamber.
  • Pictured in Figure 18 is an example of two capture reagents patterned in two 4x4 arrays on a membrane.
  • a PIHRP2 capture molecule On the left, a PIHRP2 capture molecule is patterned; on the right, an aldolase capture molecule.
  • Both PfH RP2 and aldolase were introduced to the system, followed by a gold-conjugated antibody against PfHRP2, an enzyme- conjugated antibody against aldolase, and an enzyme substrate.
  • the PIHRP2 capture regions thus can be seen in red (left array) whiie the aldolase capture regions appear blue (right array). This assay was run in a simplified wet-reagent on-card assay.
  • Pictured in Figure 19 are five frames from a video of a dry-reagent pad being rehydrated. Fluid moves from left to right. Apparent is the lightening of the pad to its original white color as red fluid - the dried gold-antibody conjugate - passes out the channel.
  • the reagent's functionality is seen in the following section C.
  • the rehydrated gold-antibody conjugate is used in an on-card assay, using the card design pictured in Figure 15B. In this assay, the following steps are performed:
  • Analyte-containing sample is injected into the sample loop.
  • Buffer fluid pushes the sample from the sample loop through the membrane.
  • Gold-antibody conjugate is passed through the membrane, binding to the captured analyte.
  • Optica! measurement of assay results has been performed using several methods, images have been captured by both a flatbed scanner (48-bit RGB, 3200 dpi) and a USB "webcam.”
  • the assay results from captured images can be quantified by measuring the pixel count in one or more of the color channels. This measurement has been assisted by a semi-automated measurement process that involves user-selection of severai reference spots in a grid of assay capture regions, followed by automated detection of the other spots in the grid. Additionally, if is possible to automatically detect registration marks such as the blue dots (4 corners on right array of Figure 21), and then use these locations to define the iocations of the assay spots of interest.
  • the image here shows the four detected registration marks and the 12 detected assay spots (each marked with an "x"). The intensity of the spot correlates with the amount of anaiyte present in the sample.

Abstract

Disclosed are methods and devices for rapid parallel molecular affinity assays performed in a microfluidic environment. The invention exploits hydrodynamic addressing to provide simultaneous performance of multiple assays in parallel using a minimal sample volume flowing through a single channel.

Description

UW.18-WG-U1
IViETHOD AND DEVICE FOR RARD PARALLEL MiCROFLUIDiC MOLECULAR
AFFINITY ASSAYS
This application claims the benefit of United States provisional patent application number 80/828, 127, filed October 4, 2008, the entire contents of which are incorporated herein by reference.
TECHNICAL FIELD OF THE iNVENTiON
This invention relates generally to methods and devices for rapid parallel molecular affinity assays performed in a microfluidic environment. The invention exploits hydrodynamic addressing to provide simultaneous performance of multiple assays in parallel using a minima! sample volume flowing through a single channel
BACKGROUND OF THE INVENTiON
Immunoassays take advantage of the specific binding abilities of antibodies to be widely used in selective and sensitive measurement of small and large molecular anaiytes in complex samples. The driving force behind developing new immunological assays is the constant need for simpler, more rapid, and less expensive ways to analyze the components of complex sample mixtures. Current uses of immunoassays include therapeutic drug monitoring, screening for disease or infection with molecular markers, screening for toxic substances and illicit drugs, and monitoring for environmental contaminants.
Some assays have made use of laminar flow and diffusion profiles of anaiytes compiexed with binding particles (see, e.g., U.S. Patent No. 8,541213 and U.S. Patent Application 2006/0166375; published July 26, 2006). Such assays, however, are limited by their inability to provide for detection of multiple anaiytes in a single sample and in a single fiuidic channel.
There remains a need for a device that allows for simultaneous performance of dozens of immunoassays in a minimum of time using a minimum of sample volume and in a minimal space. The invention described herein meets these needs and more through the use of hydrodynamic addressing and parallel flow. SUMMARY QF THE INVENTION
The invention provides a method and assay device for detection of an anaiyte in a fiuidic sample. In one embodiment, the device comprises:
(a) a microfluidic chamber having a first inlet; (b) a first surface in communication with the first inlet, wherein the first surface comprises a plurality of capture regions: (c) a plurality of capture agents immobilized on the capture regions, wherein the capture agents specifically bind the anaiyte;
(ci) a reagent storage depot in communication via a single fiuidic channel with the first surface, wherein the storage depot comprises a plurality of reagent regions; and,
(e) a plurality of detection reagents that specifically bind the anaiyte and that become mobile upon contact with fluid, wherein the detection reagents are disposed within the reagent regions.
The first surface can comprise a porous carrier, such as a membrane or other porous structure, a fiat surface, or other structure to which the capture agents can be immobilized while retaining the ability to be brought into contact with analytes delivered via fluid passing over the first surface,
The reagent storage depot can comprise one or more cavities, and/or a polymeric compound immobilized on the device. The storage depot is provided by stabilizing the reagents in a solid state using, for example, a porous matrix (e.g., a polymer, ge! or soluble salt) that either swells on contact with the fluid and releases the reagents or completely dissolves thereby delivering the reagent. The storage depot can also be provided by locating the detection reagents, in dry form, in physical cavities, such that contact with fluid mobilizes the reagents. In each embodiment, the reagent(s) is immobile in its dry form and becomes mobilized upon contact with fluid such that the reagent is delivered, upon mobilization, to the first surface where it can react with the captured anaiyte.
In one embodiment, the storage depot comprises a porous membrane that is aligned parallel to the first surface. The device is well-suited to an embodiment having a first surface in which the plurality of capture regions are arranged linearly and perpendicular to the long axis of the single fiuidic channel thai provides communication between the storage depot and the first surface. The reagent regions are likewise arranged linearly and perpendicular to the long axis of the single fiuidic channel, such that the linear arrangement of reagent regions is parallel to the iinear arrangement of capture regions. As fluid traverses the single fluidic channel, flowing from the storage depot to the first surface, reagents are mobilized in the reagent regions and flow to the capture regions. The flow conditions of the channel are such that differing reagents disposed on the reagent regions travel in parallel to corresponding capture regions.
The device typically comprises a plurality of polymeric layers. The polymeric layers can be used to devise the configuration of inlets, channels, cavities and surfaces suitable for a particular embodiment. In some embodiments of the device, for example, a second inlet is provided in communication with the storage depot The second iniet can be used to deliver fluid to effect mobilization of the reagents stored in the storage depot. Alternatively, the same fluid stream that delivers analyte to the first surface can also serve to effect mobilization of the reagents stored in the storage depot.
In another embodiment, an outlet is provided in communication with the first surface. Such an outlet can be used, for example, to draw fluid away from the first surface if desired. Those skilled in the art can appreciate that the outlet allows one to analyze the effluent or to draw off excess fluid prior to delivery of a subsequent fluid stream, in addition to other uses.
The device can further comprise one or more channels that provide communication between the first inlet and the first surface and/or between the second inlet and the storage depot. In one embodiment, 3 channels provide communication between the first inlet and the first surface. Multiple channels from the inlet to the first surface, for example, can be used to deliver multiple analytes, or, in a typical embodiment, three channels are used to deliver one analyte sample and two control samples (e.g., positive and negative controls).
The invention further provides a method of detecting the presence of an analyte in a Ruidtc sample. The method typically comprises;
(a) delivering a fiuidic sample into the first inlet of a device of claim 1 under conditions permitting contact between the sample and the capture agents immobilized on the first surface;
(b) contacting a single stream of fluid with the plurality of detection reagents under conditions effecting migration of the detection reagents to the first surface: (c) detecting the presence of detection reagent bound to anaiyte that is bound to the immobilized capture agents, whereby presence of detection reagent is indicative of the presence of the anaiyte, in a typical embodiment, the delivering of step (a) comprises pumpsng the flutdic sample into the first inlet. The method can further compnse delivering one or more control sampies via laminar flow into the first inlet. Where controls are desired, step (a) comprises delivering one stream of a test fluidic sample, one stream of a positive control fluidic sample, and one stream of a negative control fluidic sample. In one embodiment, the streams of fluidic sample are delivered via a single channel, in another embodiment, the streams of fluidic sample are delivered via separate channels For example, a 3-channei embodiment can deliver test sample, positive control sample and negative control sample, each via a separate channel Alternatively, the 3 streams can be delivered in one channel using controlled fluid pumping to avoid mixing of streams.
in one embodiment, the contacting of step φ) comprises pumping fluid into a second inlet that is tn communication with the reagent storage depot. The fluid is typically a buffer and serves to mobilize the reagent so that it can contact and bind anaiyte that has been immobilized on the first surface upon binding capture agent. Those skilled in the art understand that rinsing or washes can be used to clear out unbound reagents between steps of the method.
In some embodiments, the delivering of step (a) provides the contacting of step (b), whereby the fluidic sample, upon contact with the detection reagents, effects migration of the detection reagents. In other words, steps (a) and (b) can be accomplished with a single stream of fiuidϊc sample. Those skilled in the art can appreciate design arrangements for the device that would facilitate implementation of such an embodiment. For example, the reagent regions can be positioned between the first inlet and the capture regions.
in a typica! embodiment, the capture agents and the detection reagents comprise antibodies and/or antigens. In some embodiments, the contacting of step (b) further comprises delivering to the first surface an amplification reagent that binds to the detection reagents. The defection reagents are labeled, either directly or indirectly, and the detectable signal can be provided or amplified using known techniques and materials. Detection of signal can be achieved by a variety of means known in the art, including but not limited to, measuring an optica! property such as optica! absorbance, reflectivity, optica! transmission, chemiiuminβscence or fluorescence, in some embodiments, signal can be detected by eye. Optica! readers are preferred when a quantitative measurement is desired.
FiG. 1A; Schematic design of version 1 of the polymeric disposabie in which the secondary reagents are contained within cavities in the disposable. Two sets of fluid inlets are located at the right and left ends of the disposable as well as a single outlet path beiow the embedded membrane (center), which is Soaded with molecules.
FIG. 1B: Close-up of the centra! portion of the same image as in FigurelA.
FIG. 1C: Cut-away view of the same device (central portion) as in Figures 1A and 1 B, showing the relative locations of the different layers1 the capture membrane and the secondary reagent depots. The exit port for the device is below the membrane, and fluid exits to the right,
FiG. 2; Schematic of the minimal set of structural layers required to assemble version 1 of the immunoassay device.
FIG, 3. Schematic of assembled immunoassay device with three inlet holes on the right, one on the left, and one outlet hole invisible below the porous membrane. Secondary antibodies are printed on a membrane (left column of dots) with three cycles of the same set. Capture membrane (right column of dots) is spotted or striped with capture antibody and blocked. The relative locations of 4 valves are indicated along the bottom of the figure.
FiG. 4- Schematic illustration of how buffer is used to wet out the device from the right. Step 1 involves closing valves 2 and 3, opening valves 1 and 4, Buffer is pumped from the right (valve 4 to valve 1) to wet out both membranes. Valve at left is closed and pumping stopped.
FIG. 5: First version of sample load, in version 1, step 2 comprises pumping in sample from the right, with valves 1 and 2 closed. Sample exits below the membrane via an outlet not shown here, No flow over the secondary antibody membrane, which antibodies do not diffuse away because of high molecular weight. FIG. 8: Illustration of how, in the second version of the sample load, everything is the same as in the previous version, except that iaminar flow is used to flow 2 or 3 different solutions across the capture reagent membrane. With valves 1 and 2 closed, three solutions are pumped in: sample, positive control (all anaSytes at high levels), and negative control (no sample antigens). No flow over the secondary antibody membrane, which antibodies do not diffuse away because of high molecular weight,
FiG. 7; illustrates the rinse. Vaives 1 and 2 are closed; 3 and 4 are open. Rinse with buffer to remove excess sample from membrane.
FiG. 8: Illustrates the ioading of secondary antibodies. Close valves 1 and 4; pump buffer from vaive 2 to 3, pushing 2" antibody from left membrane through the one at the right. Continue until sufficient 2" antibody is transferred to capture zones.
FIG. 9; illustrates the rinsing of secondary antibodies. Using fluids from either vaive 1 or 2 (with valve 3 open and 4 closed), flush until ail excess secondary antibody is pushed through capture membrane Detect (if this is Au-iabeled antibody, for example) by measuring optical density of spots. Assay is complete.
FIG. 10: illustrates a detection step. This and further steps are only necessary if using BΠ amplification step. Pump secondary reagent from right at slow rate, Postsve controls and positive sample spots darken over a few seconds to minutes.
FIG. 11 : Schematic of version 2 of the device and system.
FIG. 12: Schematic of the minimal set of structural layers required to assemble version 2 of the immunoassay device as shown in Figure 11.
FIG. 13: Assay results showing the decrease in signal (from left to right) seen as the anaiyte concentration in the sample decreases. The analyte is Plasmodium falciparum Histidine-Rich Protein II, or PfHRP2. The red spots (upper 6 rows) show the results generated using an antibody-conjugated gold particle as a detection molecule; the blue spots (lower 2 rows) use an enzyme-conjugated antibody as the detection molecule, followed by an enzyme substrate that becomes a blue precipitate in the presence of the enzyme,
FiG. 14: Otagram of mini-vacuum format
FiG. 15A-B; A self-contained microfiuidic format, consisting of a laminate device in which connecting fluidic channels are formed, a membrane patterned with capture molecules, a porous pad containing dried detection reagent, and an external fluid- pumping and imaging system. The multiple fluid inlets are each fed by separate pumps in this preliminary design, sidestepping the need for valves. The device is pictured as a diagram (Figure 15A) and photograph (Figure 15B) of two revisions of the design.
FiG. 16A-B; Functional schematic (FiG. 16A) and CAD design (FIG. 16B) for assay card with single fluid inlet to the reaction chamber (the location of the assay membrane) .
F!G. 17A: Functional schematic of assay card shown in FIG. 17B.
FiG. 17B: CAD design of assay card with multiple inlets to reaction chamber,
FiG. 18: Two capture reagents patterned in two 4x4 arrays on a membrane. On the left, a PfHR P2 capture molecule is patterned; on the right, an aldolase capture molecule.
FiG. 19: Five sequential frames from a video of a dry-reagent pad being rehydrated.
FiG. 20: Three frames from a video of an assay showing (1) sample introduction to membrane; (2) rehydrated conjugate introduced to membrane; and (3) capture spot labeled by conjugate.
FiG. 21: images indicating steps of automated optical measurement. On the left, four separate registration marks are identified in an image; on the right, the analyzed image (captured by a flatbed scanner, 48-bit RGB, 3200 dpi) with simulated blue registration marks and red assay spots, the location of each marked with an "X."
OVERVI EVV OF THE INVENTION
The invention relates to a method and device for performing rapid molecular binding assays, including immunoassays, and in particular, sandwich immunoassays. The method invoives binding a plurality of primary capture reagents to a plurality of locations on a porous membrane, placing a matched set of secondary (or detection) binding molecuies in a line of cavities or on a porous membrane aligned parallel to the reagent storage locations, but separated by a gap, and a method for sandwiching the anaiyte in question between them using laminar flow in a microfiuidic device. The sample is loaded onto the first membrane by pumping it through said first membrane, where sample anaiyte molecuies become bound to the capture molecules immobilized on that membrane. Fluid is then pushed past the storage depot line or through the second membrane to release the secondary capture molecules and transport them to the first membrane to "sandwich" the anaiyte molecules. Detection is then possible by either directly (if the secondary capture molecule is directly observable (such as a fiuorescently- or Au-!abβied secondary antibody) or indirectly (using for example, secondary antibodies labeled with enzymes such as horseradish peroxidase (HRP) followed by flow over the first membrane of a solution producing an observable signal, such as precipitatable tetramethylbenzidine (TMB).
The device allows the simultaneous performance of dozens of immunoassays (as well as positive and negative control reactions) in a minimum of time using a minimum of sample volume and in a minima! space. Reading the results of the immunoassays may either be made directly (by eye), or with the aid or a quantitative optical reader. Conventional off-the-shelf reagents can be used to minimize cost. It is particularly well adapted for performance of multiple immunoassays on an inexpensive polymeric disposable device that may be read out directly or using an optical reader.
Appjjc^tion.s.oi.the.jnvention
The invention disclosed herein is a design for a molecular binding assay (and a method of using that design). This assay system is well suited to use as the basis of immunoassays such as "sandwich immunoassays". Although the reagents and assays are referred to herein as immunoassay reagents and immunoassays, respectively, it is understood by those skilled in the art that a device that could perform any other assay (based on proteins, aptamers, nucleic acids, or other molecules) that involves molecules capable of binding to each other would fall under the scope of this invention.
In a typical embodiment of this assay, the device is fabricated from inexpensive polymeric components combined with porous membranes capable of binding to and immobilizing capture reagents such as capture immunoassays or target antigens, depending on the format of the immunoassay. The arrangement allows for storage of both capture reagents and secondary reagents in dry form on the polymeric rnicroflutdic device, thereby creating a self-contained disposable that can be used with or without a reader technology. By allowing the storage of multiple reagents in parallel, the disposable can be made to perform multiple immunoassays in parallel, as well as perform measurements of multiple anaiyte concentrations in samples, positive control solutions, and negative control solutions simultaneously. The assay assumes laminar flow conditions in all components, and microfluidic dimensions. The immunoassay format can be manufactured very inexpensively, such that a polymeric disposable is suitable for use in point-of-care assays. Optical detection methods (optica! absorption, diffuse reflectance absorption, or fluorescence) are typically utiiized, although other methods are not excluded. The assays can operate in a simple qualitative yes/no fashion, or in a quantitative manner (using, for example, a quantitative optica! reader). Detection of the optical signal indicating the binding of the anaiyte can be performed in either of two well-understood ways; One version involves the use of an optically detectable secondary antibody, such as an antibody bound (covaientiy or noncovaientiy) to colored microspheres, fluorescent molecules or nanoparticies, or strongiy absorbing dyes of nanoparticles (such as gold nanoparticies). In a more sensitive version, the assay is an ELiSA assay, in which the secondary antibody is labeled with an enzyme, and the final step after binding of the secondary antibody to the anaiyte is exposure of the enzyme-loaded capture membrane with a "developing solution"; examples are to be taken from the list of a!l known ELISA systems, including any of several commercially available horseradish peroxidase/ precipitating tetramethyibenzidine systems.
A likeiy appiication for such a disposabie (with or without use of a quantitative reader) is a point-of-care immunoassay system for use in the developing world, although use as an inexpensive point-of-care diagnostic system is also possible. The disposable polymeric immunoassay system can be coupled to other types of assays in a single integrated device.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS QF THE JNVENTIQN
Exemplary versions of the device are described. The first is shown in Figure 1A.
The device can be fabricated from seven polymeric layers. A representative example of a multiple polymeric layered device is shown in Figure 2,
A schematic of the minima! set of structural layers required to assemble version 1 of the immunoassay device (of Figure 1A) is shown in Figure 2, The layers are numbered in order of assembly. Layers 1, 4 and 8 are just C (carrier) layers (plain sheets of an appropriate polymer such as Mylar, PMMA, or others), whereas layers 2 and 7 are ACA (adhesive, carrier, adhesive) layers. Layers 3 and 6 are AC layers, with adhesive on one side that serve to seal layer 5« the membrane, in place. Layer 1 is the top cover of the device, and must consist of a clear (optically transparent) material to allow optica! observation of the !ayer 5, Layer 2 is the main fluid cavity. Layer 3 is the "floor" of the main fluid cavity, which contains a (here large and rectangular) hole for fluid outflow, as we!! as a multiplicity of storage depots for storage of secondary reagents. These secondary reagents can be placed into the storage depots as one of the last steps of assembly of the polymeric device. Layer 4 is the cavity that localizes the permeable membrane. Layer 5 consists of a permeable membrane onto which capture molecules are immobilized prior to final assembly of the device, and which is placed within the rectangular cavity in layer 4, The deposition of the different capture molecules onto layer 5 can be in any form, but are shown here as circular spots. Layer 6 supports the permeable membrane. Layer 7 collects all flow through the membrane to a single port. Layer 8 is the floor of the device and couples to inlets and outlets for the device. Note that, in this schematic, the right side of all layers {but 5) are shown with two holes. In the schematic below either one hole or three are used, as explained below. Note that further embodiments of the device can be assembled in part using injection molded parts to reduce the part count and reduce fabrication costs.
Shown in Figure 3 is an operation sequence for version 1 of the device as shown in Figures 1A and 2, in this schematic, the assembled device has three inlet holes on the right, one on the left, and one outlet hole invisible below the porous membrane, The cavity is designed In such a way that fluid entering the main cavity is "fully developed ", and, therefore, flowing almost exclusively horizontally and at the same horizontal velocity top to bottom (as shown in this figure) by the time it reached either the membrane from the right, or the secondary reagent storage depots from the ieft
As illustrated in Figure 4, buffer is used to wet out the device from the right. Such a process proceeds with the exit below the device closed, so that almost all fluid flows from right to left. This wets the secondary reagent storage depots1 necessitating that they begin to hydrate and dissolve. The high molecular weight of the secondary reagents prevents them from diffusing appreciably in the vertical direction (as shown in this figure) during the complete operation of the device. If it necessary to minimize vertical diffusion, the capping layer (layer 1 in Figure 2) can be manufactured with ftns that fit between the secondary reagent storage depots. The wet-out pushes minimum fluid through the membrane.
Figure 5 illustrates a first version of sample load, In the simplest case, the valves "below" the left side of the device are closed and the sample is pumped in through a single inlet from the right forcing the sample to flow through the semi-permeabie membrane. in the second version of the sample load (Figure 6) everything is the same as in the previous version, except that laminar flow is used to flow 2 or 3 different solutions across the capture reagent membrane. One of these is the sample, but the other two are positive and negative controls (meaning solutions, presumably buffer, containing a high concentration of each anaiyte to be measured, and no analyies, respectively.) Under laminar fiow conditions, only a controlled amount of interdiffusion between the streams occurs before they arrived at the capture membrane, and since fiow then goes through the membrane, three distinct zones are maintained with respect to capture of anaiytes. This allows "reai time calibration" of the immunoassay with a very simple format,
As shown in Figure 7, in either case; buffer is flushed from the right (with the vaive under the ieft side closed) to clear excess (free) anaiyte from the device and flush the capture membrane.
The secondary reagent (2° Ab, for example) is then loaded onto the anaiyte moiecuSes that are bound to the capture membrane (via the capture molecules) by pumping buffer from the ieft iniet (with ail the right inlet valves closed; see Figure 8). This continues until aii of the 2° Abs are transferred. Laminar flow (or channels or fins, if necessary) wili ensure that the appropriate T Abs are transported to the appropriate capture molecule regions on the membrane.
The remaining 2" Ab is rinsed from the system to ensure that ail capture zones receive equivalent doses of that reagent (Figure 9). If a directly observable secondary reagent (such as a goScMabeied or fluorescently-labeied 2s* Ab) is used, it is possible to observe and quantify the intensity of the observable signal on the appropriate locations of the capture membrane to measure anaiyte concentrations. If not, the defection method shown in Figure 10 is used.
Assuming that an enzyme~iabeied2':' Ab is used as the secondary reagent, a separate detection step is empioyed (Figure 10). In this case the left-most vaive is closed and a solution of a detection reagent is pumped from the right and through the membrane at a controlled rate. Spots then become observable as product built up. An example of a system that has proven useful in this regard is the horseradish peroxidase/precipitating tetramethySbenzidine system, although many other ELISA detection schemes have been demonstrated and couid be used here. Those that produce a precipitated product are preferred because of the buiid-up of signal possible on the membrane over time and pushing of reagents through the membrane, but non-precipitating systems can aiso be used. Alternatively, other detection reagents can be stacked on top of the 2a Ab layer to produce strong signals using fluorescence or optical absorbance.
The above-mentioned scheme relies on the deposition of the secondary reagents onto an impermeabie surface to form depots for subsequent movement to the capture membrane. An alternative that allows the use of technology demonstrated tn other types of assays is to use a second permeable membrane as the depot for the 2C Abs, allowing these reagents to be preloaded into a membrane before assembly of the card, and washed out of this membrane by flowing buffer up through the membrane. The preliminary design is shown in Figure 11. This design allows all the reagents to be printed onto large sheets of membrane using commercial printing mechanisms for great simplification of manufacturing and. thereby, cost savings. Furthermore, the secondary reagent membrane can be prepared in the same way as the secondary reagents are in lateral flow immunoassay devices (immunochxomatographic test strips),
Shown in Figure 1 1 is a schematic of version 2 of the device and system; tt is very similar to that shown in Figure 1A, except that the 2n Ab storage is now on a permeable membrane that sits in a cavity like that for the capture membrane, there is a second channel below the second membrane (which is an inlet, not an outlet) and the 2° Ab spots are deposited (in a matrix of preserving chemicals) on the second membrane (at left). The second membrane is of a type with no or very low protein retention.
Schematic of the minimal set of structural layers required to assemble version 2 of the immunoassay device as shown In Figure 11. The layers are numbered in order of assembiy and have the same characteristics as those mentioned in version 1 above. Layer 3 is the "floor" of the main fluid cavity, which contains 2 (here large and rectangular) holes for fluid passage. Layer 4 contains the cavities that localize the permeable membranes. Layer 5 consists of a two separate (and different) permeable membranes. The one onto which capture molecules are immobilized prior to final assembiy of the device is identical to that described in version 1 (Figures 1A and 2), The one at the left is for storage of the 2s reagents (e.g., Abs). Both sets of reagents am "spotted" or "striped" onto the membranes and dried prior to insertion into their respective cavities in layer 4. Layer 6 supports the permeable membranes. Layer 7 now has two separate cavities for controiling flow in the vicinity of the membranes. The one at right is identical to that in version 1 , and collects all flow through the membrane to a single port. The new cavity at left delivers fluid flow to the 2° reagent storage membrane at left, as described befow. Layer 8 is the floor of the device and couples to inlets and outlets for the device.
Reference is made to Figures 3-10 for a usage sequence for version 2 that is similar to that described above for version 1. Note that in step 4 (2" Ab ioading) of version 2 (Figure 8), the flow of fluid is up through valve 1 and the 2" Ab storage membrane and over to and down through the capture membrane. Using fluids from either valve 1 or 2, (with valve 3 open and 4 closed) flush until ail excess secondary Ab is pushed through capture membrane is shown in Figure 9. The next step is to detect (if this is Au- labeied Ab, for example) by measuring optical density of spots.
The 6th and further steps are necessary only if using an ampiification step (Figure 10).
Representative formats used for assay development
A. 96-weii plate vacuum manifold - BioDot
The BioDot vacuum manifold is suitable for testing of the flow-through immunoassays of the invention, it consists of 96 individual open-bottom wells and a vacuum plenum that applies a low pressure beiow each well. Between the welis and the plenum is placed a porous membrane, patterned with capture molecules against analytes of interest. Reagents such as the sample, washing buffers, and detection molecule are added sequentially to the wells and drawn through by the applied vacuum. Pictured is an example of the assay results. Each circle in the grid lies underneath a single well and represents a unique set of assay conditions.
The assay results presented in Figure 13 show the decrease in signal (from left to right) seen as the analyte concentration in the sample decreases. The analyte is Plasmodium falciparum Histidine-Rich Protein II, or PfHRP2. The red spots (first 6 rows) show the results generated using an antibody-conjugated gold particle as a detection molecule; the blue spots (last 2 rows) use an enzyme-conjugated antibody as the detection molecule, followed by an enzyme substrate that becomes a biue precipitate in the presence of the enzyme.
B. Mini-vacuum
A similar format to the 96-weii plate is the mini-vacuum or "minivac" format, it also uses an applied vacuum to draw fluid from a reservoir through a membrane. The reservoir in this case addresses a larger area of membrane, and the membrane is supported by a metal mesh. Pictured in Figure 14 is a diagram of the format The mesh is depicted in the inset
C, On~card assay ~ dry reagent
The assay can be run in a self-contained microfiuidic format, consisting of a iaminate device in which connecting fluidic channels are formed, a membrane patterned with capture moiecuies, a porous pad containing dried detection reagent, and an external fluid-pumping and imaging system. The muitipie fluid inlets are each fed by separate pumps in this design, sidestepping the need for valves. The device is pictured in Figure 15A-B as a diagram (15A) and photograph (15B) of the design.
With respect to FIG. 15A, the self-contained microfiuidic format consists of a laminate device 150 in which connecting flυidic channels are formed by a sample ioop 152 that ts met by a second channel 155 delivering mobilized reagents. Their contents combine into a single channel 130 through the membrane 153. The device 150 aiso includes air vents 180, a membrane 153 patterned with capture molecules, a porous pad 156 containing dried detection reagent, and an external fluid-pumping and imaging system (not shown; representative example is microFlow™ System available from Micronics, Redmond, WA). The muitipie fluid inlets include a sample inlet 151 and a second iniet 154, each fed by separate pumps in this design, sidestepping the need for valves. The second inlet 154 is used to introduce fluid that is directed to the conjugate pad 156 via second channel 155 that feeds into the sample loop 152 before it enters reaction chamber 169 and contacts the membrane 153. A bubble vent 157 can withdraw bubbles from the sample ioop 152 and an outlet 158 exits the reaction chamber 169 via waste line 159.
D. On-card assay ~ wet reagent
More sophisticated vaived devices have been developed for controlling fluid motion from a single pump. Pictured in Figures 16B and 17B are two alternate designs for the assay cards. They include reagent reservoirs for liquid reagents instead of the dried reagent pads described in part C above.
Figure 16A- B depicts a functional schematic (18A) and CAD design (16B) for assay card with single fluid iniet to the reaction chamber (the location of the assay membrane). With respect to FIG. 16B, air vents 160 are positioned in waste reservoirs 161, 162, and a bubble vent 163 is provided for priming. Valves 170 disposed throughout provide control points, such as between pipette loading vents 184 and reagent reservoirs 165-188, between pipette loading points 172 and reagent reservoirs 165-168, and between reagent reservoirs 185-168 and reaction chamber 169, as well as between pumps 174, 176 and reaction chamber 169.
Figure 17B depicts a CAD design of assay card with multiple inlets to the reaction chamber.
Representative results
A. Piuraiity of capture reagents patterned on porous substrate
Pictured in Figure 18 is an example of two capture reagents patterned in two 4x4 arrays on a membrane. On the left, a PIHRP2 capture molecule is patterned; on the right, an aldolase capture molecule. Both PfH RP2 and aldolase were introduced to the system, followed by a gold-conjugated antibody against PfHRP2, an enzyme- conjugated antibody against aldolase, and an enzyme substrate. The PIHRP2 capture regions thus can be seen in red (left array) whiie the aldolase capture regions appear blue (right array). This assay was run in a simplified wet-reagent on-card assay.
B. Rehydration of secondary reagent stored in dry form
Pictured in Figure 19 are five frames from a video of a dry-reagent pad being rehydrated. Fluid moves from left to right. Apparent is the lightening of the pad to its original white color as red fluid - the dried gold-antibody conjugate - passes out the channel. The reagent's functionality is seen in the following section C.
C. Storage depot in communication with assay substrate
Following from section B above, the rehydrated gold-antibody conjugate is used in an on-card assay, using the card design pictured in Figure 15B. In this assay, the following steps are performed:
1 , Analyte-containing sample is injected into the sample loop. 2. Buffer fluid pushes the sample from the sample loop through the membrane.
3. Buffer washes unbound sample components from the membrane.
4. Buffer rehydrates the gold-antibody conjugate stored in the conjugate pad, and the air ejected is pulled into a bubble vent line.
5. Gold-antibody conjugate is passed through the membrane, binding to the captured analyte.
6. Buffer washes unbound conjugate from the membrane. Frames from a video of the assay are pictured in Figure 20. In the first frame, sample is introduced to membrane, in the second frame, rehydrated conjugate is introduced to membrane. In the third frame, the capture spot is labeled by conjugate.
D, Optical detection of assay results
Optica! measurement of assay results has been performed using several methods, images have been captured by both a flatbed scanner (48-bit RGB, 3200 dpi) and a USB "webcam." The assay results from captured images can be quantified by measuring the pixel count in one or more of the color channels. This measurement has been assisted by a semi-automated measurement process that involves user-selection of severai reference spots in a grid of assay capture regions, followed by automated detection of the other spots in the grid. Additionally, if is possible to automatically detect registration marks such as the blue dots (4 corners on right array of Figure 21), and then use these locations to define the iocations of the assay spots of interest. The image here shows the four detected registration marks and the 12 detected assay spots (each marked with an "x"). The intensity of the spot correlates with the amount of anaiyte present in the sample.
Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention ts not limited except as by the appended ciaims.

Claims

What is claimed is:
1 , An assay device for detection of an aπalyte in a fluidic sample, the device comprising:
(a) a microfluidic chamber having a first inlet;
(b) a first surface in communication with the first inlet, wherein the first surface comprises a plurality of capture regions;
(c) a plurality of capture agents immobilized on the capture regions, wherein the capture agents specifically bind the analyte, (d) a reagent storage depot in communication via a single fluidic channel with the first surface, wherein the storage depot comprises a plurality of reagent regions; and,
(e) a plurality of detection reagents that specifically bind the analyte and that become mobiie upon contact with fluid, wherein the detection reagents ar& disposed within the reagent regions.
2. The device of claim 1. wherein the first surface comprises a porous carrier,
3, The device of claim 1 , wherein the storage depot comprises one or more cavities,
4. The device of ciaim 1 , wherein the storage depot comprises a polymeric compound immobilized on the device.
5, The device of ciaim 1 , wherein the storage depot comprises a porous membrane.
6. The device of ciaim 1 , further comprising a second iniet in communication with the storage depot.
7. The device of ciaim 1 , wherein the capture agents and the detection reagents are in dry form.
8. The device of ciaim 1 , which comprises a plurality of polymeric layers.
9. The device of c!aim 1 , further comprising one or more channels that provide communication between the first inlet and the first surface and/or between the second inlet and the storage depot,
S 10. The device of claim 9, comprising 3 channels that provide communication between the first Wet and the first surface.
11. The device of claim 1 : further comprising an outlet in communication with the first surface. 0
12. A method of detecting the presence of an analyte in a flυidic sample, the method comprising:
(a) delivering a fluidic sampie into the first inlet of a device of claim 1 under conditions permitting contact between the sample and the capture agents 5 immobilized on the first surface;
(b) contacting a single stream of fluid with the plurality of detection reagents under conditions effecting migration of the detection reagents to the first surface;
(c) detecting the presence of detection reagent bound to anaiyte thai is bound0 to the immobilized capture agents, whereby presence of detection reagent is indicative of the presence of the anaSyte.
13. The method of claim 12, wherein the delivering of step (a) comprises pumping the fluidic sample into the first inlet. 5
14. The method of claim 12, further comprising delivering one or more control sampies via laminar flow into the first inlet.
15. The method of claim 14, wherein step (a) comprises delivering one stream of a0 test fluidic sarnpie, one stream of a positive control fluidic sample, and one stream of a negative control fluidic sample.
16. The method of claim 14, wherein the streams of fluidic sample are delivered via a single channel. 5
17. The method of claim 14, wherein the streams of fluidic sample are delivered via separate channels.
18. The method of claim 12, wherein the contacting of step (b) comprises pumping fluid into a second inlet that is in communication with the reagent storage depot.
S 19. The method of claim 12, wherein the delivering of step (a) provides the contacting of step (b), whereby the fluidic sample, upon contact with the detection reagents, effects migration of the detection reagents.
20. The method of claim 12, wherein the capture agents and the detection reagents 0 comprise antibodies and/or antigens.
21. The method of claim 12, wherein the contacting of step (b) further comprises delivering to the first surface an amplification reagent that binds to the detection reagents. 5
22. The method of claim 12, wherein the detecting comprises measuring an optical property selected from optica! absorbance, reflectivity, optica! transmission, chemϋumtnescence or fluorescence.
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102272600A (en) * 2008-12-31 2011-12-07 雅培医护站股份有限公司 Method and device for immunoassay using nucleotide conjugates
CN103649759A (en) * 2011-03-22 2014-03-19 西维克公司 Microfluidic devices and methods of manufacture and use
US9415392B2 (en) 2009-03-24 2016-08-16 The University Of Chicago Slip chip device and methods
US9447461B2 (en) 2009-03-24 2016-09-20 California Institute Of Technology Analysis devices, kits, and related methods for digital quantification of nucleic acids and other analytes
US9464319B2 (en) 2009-03-24 2016-10-11 California Institute Of Technology Multivolume devices, kits and related methods for quantification of nucleic acids and other analytes
US9500645B2 (en) 2009-11-23 2016-11-22 Cyvek, Inc. Micro-tube particles for microfluidic assays and methods of manufacture
US9546932B2 (en) 2009-11-23 2017-01-17 Cyvek, Inc. Microfluidic assay operating system and methods of use
US9651568B2 (en) 2009-11-23 2017-05-16 Cyvek, Inc. Methods and systems for epi-fluorescent monitoring and scanning for microfluidic assays
US9700889B2 (en) 2009-11-23 2017-07-11 Cyvek, Inc. Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results
US9759718B2 (en) 2009-11-23 2017-09-12 Cyvek, Inc. PDMS membrane-confined nucleic acid and antibody/antigen-functionalized microlength tube capture elements, and systems employing them, and methods of their use
US9855735B2 (en) 2009-11-23 2018-01-02 Cyvek, Inc. Portable microfluidic assay devices and methods of manufacture and use
US10065403B2 (en) 2009-11-23 2018-09-04 Cyvek, Inc. Microfluidic assay assemblies and methods of manufacture
US10196700B2 (en) 2009-03-24 2019-02-05 University Of Chicago Multivolume devices, kits and related methods for quantification and detection of nucleic acids and other analytes
US10228367B2 (en) 2015-12-01 2019-03-12 ProteinSimple Segmented multi-use automated assay cartridge

Families Citing this family (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8101403B2 (en) 2006-10-04 2012-01-24 University Of Washington Method and device for rapid parallel microfluidic molecular affinity assays
WO2009034563A2 (en) * 2007-09-14 2009-03-19 Nanocomms Patents Limited An analysis system
US20110151479A1 (en) * 2008-08-25 2011-06-23 University Of Washington Microfluidic systems incorporating flow-through membranes
WO2011063408A1 (en) 2009-11-23 2011-05-26 Cyvek, Inc. Method and apparatus for performing assays
EP2555871B1 (en) 2010-04-07 2021-01-13 Biosensia Patents Limited Flow control device for assays
EP2912432B1 (en) 2012-10-24 2018-07-04 Genmark Diagnostics Inc. Integrated multiplex target analysis
US20140322706A1 (en) 2012-10-24 2014-10-30 Jon Faiz Kayyem Integrated multipelx target analysis
WO2014150905A2 (en) 2013-03-15 2014-09-25 Genmark Diagnostics, Inc. Systems, methods, and apparatus for manipulating deformable fluid vessels
DE102013105491A1 (en) * 2013-05-28 2014-12-04 Endress + Hauser Conducta Gesellschaft für Mess- und Regeltechnik mbH + Co. KG Method and apparatus for primary signal acquisition for an affinity assay
US9498778B2 (en) 2014-11-11 2016-11-22 Genmark Diagnostics, Inc. Instrument for processing cartridge for performing assays in a closed sample preparation and reaction system
USD881409S1 (en) 2013-10-24 2020-04-14 Genmark Diagnostics, Inc. Biochip cartridge
BR112016009958B1 (en) * 2013-11-06 2021-08-03 Becton, Dickinson And Company MICROFLUIDIC DEVICE, METHOD, SYSTEM AND KIT
US9598722B2 (en) 2014-11-11 2017-03-21 Genmark Diagnostics, Inc. Cartridge for performing assays in a closed sample preparation and reaction system
US10005080B2 (en) 2014-11-11 2018-06-26 Genmark Diagnostics, Inc. Instrument and cartridge for performing assays in a closed sample preparation and reaction system employing electrowetting fluid manipulation
US10775380B2 (en) 2015-04-10 2020-09-15 Tumorgen, Inc. Rare cell isolation device and method of use thereof
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US11648200B2 (en) 2017-01-12 2023-05-16 Duke University Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature triggered hierarchical self-assembly
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WO2019079301A2 (en) 2017-10-18 2019-04-25 Group K Diagnostics, Inc. Single-layer microfluidic device and methods of manufacture and use thereof
US11649275B2 (en) 2018-08-02 2023-05-16 Duke University Dual agonist fusion proteins
USD879999S1 (en) 2018-11-02 2020-03-31 Group K Diagnostics, Inc. Microfluidic device
US11512314B2 (en) 2019-07-12 2022-11-29 Duke University Amphiphilic polynucleotides
EP4121208A2 (en) * 2020-03-17 2023-01-25 Nordetect APS A microfluidic device, production of a microfluidic device and method and system for performing inorganic determinations
CN116134315A (en) * 2020-05-29 2023-05-16 雅培快速诊断国际无限责任公司 Machine-readable diagnostic test and method and apparatus for manufacturing and/or processing same

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR19990036069A (en) * 1995-08-03 1999-05-25 이.에이치. 리링크 Diagnostic device
KR20000036176A (en) * 1996-09-17 2000-06-26 쟈콥 월스태더 Multi-array, multi-specific electrochemiluminescence testing
WO2003038436A2 (en) * 2001-11-02 2003-05-08 University Of Strathclyde Microfluidic ser(r)s detection
US6664104B2 (en) * 1999-06-25 2003-12-16 Cepheid Device incorporating a microfluidic chip for separating analyte from a sample

Family Cites Families (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL7610608A (en) 1976-09-24 1978-03-29 Akzo Nv PROCESS FOR STABILIZING PEROXIDASE-CONTAINING COMPOSITIONS.
US4632901A (en) 1984-05-11 1986-12-30 Hybritech Incorporated Method and apparatus for immunoassays
DE3445816C1 (en) 1984-12-15 1986-06-12 Behringwerke Ag, 3550 Marburg Flat diagnostic agent
US5079142A (en) 1987-01-23 1992-01-07 Synbiotics Corporation Orthogonal flow immunoassays and devices
US5112739A (en) 1988-08-02 1992-05-12 Polaroid Corporation Enzyme controlled release system
US5183740A (en) 1990-02-23 1993-02-02 The United States Of America As Represented By The Secretary Of The Navy Flow immunosensor method and apparatus
US5200321A (en) 1990-09-07 1993-04-06 The United States Of America As Represented By The Secretary Of The Navy Microassay on a card
US6767510B1 (en) * 1992-05-21 2004-07-27 Biosite, Inc. Diagnostic devices and apparatus for the controlled movement of reagents without membranes
US5354654A (en) 1993-07-16 1994-10-11 The United States Of America As Represented By The Secretary Of The Navy Lyophilized ligand-receptor complexes for assays and sensors
US5409664A (en) 1993-09-28 1995-04-25 Chemtrak, Inc. Laminated assay device
GB9502112D0 (en) 1995-02-03 1995-03-22 British Biocell Int Assay device and method
US6454945B1 (en) 1995-06-16 2002-09-24 University Of Washington Microfabricated devices and methods
US5716852A (en) 1996-03-29 1998-02-10 University Of Washington Microfabricated diffusion-based chemical sensor
US6750031B1 (en) 1996-01-11 2004-06-15 The United States Of America As Represented By The Secretary Of The Navy Displacement assay on a porous membrane
US6541213B1 (en) 1996-03-29 2003-04-01 University Of Washington Microscale diffusion immunoassay
JPH1016228A (en) 1996-07-02 1998-01-20 Canon Inc Ink jet printer and method for heat-insulating control of printing head therefor
IL119389A (en) * 1996-10-09 2001-10-31 Cargill Inc Process for the recovery of lactic acid by liquid-liquid extraction using a cation exchanger
GB9623820D0 (en) 1996-11-16 1997-01-08 Secr Defence Surface plasma resonance sensor
GB9700759D0 (en) 1997-01-15 1997-03-05 Carbury Herne Limited Assay device
US6159739A (en) 1997-03-26 2000-12-12 University Of Washington Device and method for 3-dimensional alignment of particles in microfabricated flow channels
US6020209A (en) 1997-04-28 2000-02-01 The United States Of America As Represented By The Secretary Of The Navy Microcapillary-based flow-through immunosensor and displacement immunoassay using the same
WO1999000655A2 (en) 1997-06-27 1999-01-07 Immunetics, Inc. Rapid flow-through binding assay apparatus and method
US6007775A (en) * 1997-09-26 1999-12-28 University Of Washington Multiple analyte diffusion based chemical sensor
WO1999017119A1 (en) 1997-09-26 1999-04-08 University Of Washington Simultaneous particle separation and chemical reaction
US5994150A (en) 1997-11-19 1999-11-30 Imation Corp. Optical assaying method and system having rotatable sensor disk with multiple sensing regions
US6663833B1 (en) 1998-03-10 2003-12-16 Strategic Diagnostics Inc. Integrated assay device and methods of production and use
AU3372800A (en) * 1999-02-23 2000-09-14 Caliper Technologies Corporation Manipulation of microparticles in microfluidic systems
JP2003501639A (en) 1999-06-03 2003-01-14 ユニバーシティ オブ ワシントン Microfluidic devices for transverse and isoelectric focusing
US7351376B1 (en) * 2000-06-05 2008-04-01 California Institute Of Technology Integrated active flux microfluidic devices and methods
JP4191608B2 (en) 2001-12-05 2008-12-03 ユニヴァーシティ オブ ワシントン Microfluidic devices and surface modification processes for solid phase affinity binding assays
US7279134B2 (en) 2002-09-17 2007-10-09 Intel Corporation Microfluidic devices with porous membranes for molecular sieving, metering, and separations
CA2455669A1 (en) 2003-02-04 2004-08-04 Bayer Healthcare, Llc Method and test strip for determining glucose in blood
US7300802B2 (en) 2003-04-25 2007-11-27 Biodigit Laboratories Corp. Membrane strip biosensor system for point-of-care testing
US20050136550A1 (en) 2003-12-19 2005-06-23 Kimberly-Clark Worldwide, Inc. Flow control of electrochemical-based assay devices
US7550267B2 (en) 2004-09-23 2009-06-23 University Of Washington Microscale diffusion immunoassay utilizing multivalent reactants
WO2006047591A2 (en) 2004-10-25 2006-05-04 University Of Washington Rapid microfluidic assay for analyte interactions
US7189522B2 (en) 2005-03-11 2007-03-13 Chembio Diagnostic Systems, Inc. Dual path immunoassay device
WO2006130299A2 (en) 2005-05-03 2006-12-07 Micronics, Inc. Microfluidic laminar flow detection strip
US8101403B2 (en) 2006-10-04 2012-01-24 University Of Washington Method and device for rapid parallel microfluidic molecular affinity assays
US7736891B2 (en) 2007-09-11 2010-06-15 University Of Washington Microfluidic assay system with dispersion monitoring

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR19990036069A (en) * 1995-08-03 1999-05-25 이.에이치. 리링크 Diagnostic device
KR20000036176A (en) * 1996-09-17 2000-06-26 쟈콥 월스태더 Multi-array, multi-specific electrochemiluminescence testing
US6664104B2 (en) * 1999-06-25 2003-12-16 Cepheid Device incorporating a microfluidic chip for separating analyte from a sample
WO2003038436A2 (en) * 2001-11-02 2003-05-08 University Of Strathclyde Microfluidic ser(r)s detection

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102272600A (en) * 2008-12-31 2011-12-07 雅培医护站股份有限公司 Method and device for immunoassay using nucleotide conjugates
US10543485B2 (en) 2009-03-24 2020-01-28 University Of Chicago Slip chip device and methods
US9415392B2 (en) 2009-03-24 2016-08-16 The University Of Chicago Slip chip device and methods
US9447461B2 (en) 2009-03-24 2016-09-20 California Institute Of Technology Analysis devices, kits, and related methods for digital quantification of nucleic acids and other analytes
US9464319B2 (en) 2009-03-24 2016-10-11 California Institute Of Technology Multivolume devices, kits and related methods for quantification of nucleic acids and other analytes
US9493826B2 (en) 2009-03-24 2016-11-15 California Institute Of Technology Multivolume devices, kits and related methods for quantification and detection of nucleic acids and other analytes
US10370705B2 (en) 2009-03-24 2019-08-06 University Of Chicago Analysis devices, kits, and related methods for digital quantification of nucleic acids and other analytes
US10196700B2 (en) 2009-03-24 2019-02-05 University Of Chicago Multivolume devices, kits and related methods for quantification and detection of nucleic acids and other analytes
US9651568B2 (en) 2009-11-23 2017-05-16 Cyvek, Inc. Methods and systems for epi-fluorescent monitoring and scanning for microfluidic assays
US9700889B2 (en) 2009-11-23 2017-07-11 Cyvek, Inc. Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results
US9759718B2 (en) 2009-11-23 2017-09-12 Cyvek, Inc. PDMS membrane-confined nucleic acid and antibody/antigen-functionalized microlength tube capture elements, and systems employing them, and methods of their use
US9855735B2 (en) 2009-11-23 2018-01-02 Cyvek, Inc. Portable microfluidic assay devices and methods of manufacture and use
US10022696B2 (en) 2009-11-23 2018-07-17 Cyvek, Inc. Microfluidic assay systems employing micro-particles and methods of manufacture
US10065403B2 (en) 2009-11-23 2018-09-04 Cyvek, Inc. Microfluidic assay assemblies and methods of manufacture
US9546932B2 (en) 2009-11-23 2017-01-17 Cyvek, Inc. Microfluidic assay operating system and methods of use
US9500645B2 (en) 2009-11-23 2016-11-22 Cyvek, Inc. Micro-tube particles for microfluidic assays and methods of manufacture
CN103649759A (en) * 2011-03-22 2014-03-19 西维克公司 Microfluidic devices and methods of manufacture and use
US10228367B2 (en) 2015-12-01 2019-03-12 ProteinSimple Segmented multi-use automated assay cartridge

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