WO2008103824A1 - Immunoessai à l'or lié à un anticorps par point amélioré en sensibilité pour la détection de virus - Google Patents

Immunoessai à l'or lié à un anticorps par point amélioré en sensibilité pour la détection de virus Download PDF

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Publication number
WO2008103824A1
WO2008103824A1 PCT/US2008/054579 US2008054579W WO2008103824A1 WO 2008103824 A1 WO2008103824 A1 WO 2008103824A1 US 2008054579 W US2008054579 W US 2008054579W WO 2008103824 A1 WO2008103824 A1 WO 2008103824A1
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WO
WIPO (PCT)
Prior art keywords
virus
insert
channel
fluid material
antibody
Prior art date
Application number
PCT/US2008/054579
Other languages
English (en)
Inventor
Mingqiang Zou
Qiang Xue
Jinfeng Li
Yong Jin
Peng Zhou
Lincoln Young
Original Assignee
Chinese Academy Of Inspection And Quarantine (Caiq)
Rheonix, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chinese Academy Of Inspection And Quarantine (Caiq), Rheonix, Inc. filed Critical Chinese Academy Of Inspection And Quarantine (Caiq)
Publication of WO2008103824A1 publication Critical patent/WO2008103824A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • the systems and methods described herein generally pertain to the field of virus detection.
  • Embodiments of the invention pertain to the field of influenza virus detection, in particular, avian influenza virus (AIV) detection.
  • the systems and methods also pertain to the use of antibody-conjugated gold nanoparticle solutions and signal amplification solutions for virus detection.
  • the systems and methods described also pertain to the field of microfluidics.
  • the systems and methods described herein pertain to the use of microfluidic diaphragm structures, microfluidic chips, and portable automated microfluidic reagent processing systems for use in the detection of pathogens such as viruses.
  • AIV avian influenza viruses
  • Antibody/antigen interaction-based immunoassays present the most straightforward methods for detection of AIV, but often lack necessary sensitivity.
  • Molecular diagnosis that is carried out, for example, using a nucleic acid amplification technique such as polymerase chain reaction (PCR), may be extremely sensitive but calls for using sophisticated equipment, trained lab personnel and can only be conducted under a tightly controlled environment. Therefore, the inventors have recognized that a simple, inexpensive, portable, and sensitive methodology is needed for the detection of viruses such as AIV.
  • PCR polymerase chain reaction
  • Microfluidics generally refers to systems, devices, and methods for processing small volumes of fluids. Because microfluidic systems can integrate a wide variety of operations to manipulating fluids, such as chemical or biological samples, these systems have many application areas, such as biological assays (for, e.g., medical diagnoses and drug delivery), biochemical sensors, or life science research in general.
  • biological assays for, e.g., medical diagnoses and drug delivery
  • biochemical sensors for, e.g., biochemical sensors, or life science research in general.
  • Microfluidic chips may include micro-scale features (or "microfeatures"), such as channels, valves, pumps, and/or reservoirs for storing fluids, for routing fluids to and from various locations on the chip, and/or for reacting fluidic reagents.
  • micro-scale features or "microfeatures”
  • microfluidic devices are restricted for one specific use and cannot be easily adapted or customized for other applications without being completely redesigned. These devices lack modularity, and therefore cannot share common device components that allow one design to perform multiple functions. This lack of flexibility leads to increased production costs as each use requires the production of a different system.
  • many existing microfluidic systems lack any means for straightforward end-point assays that are able to easily detect interactions or existence of analysts resulting from the assays. By way of example, visual detection of sample color changes after an assay is often used to evaluate the assay results, but this technique is rarely applied in a microfluidic system.
  • a system and method for detecting a virus of interest in a field or clinical sample is provided.
  • the virus of interest is an influenza virus.
  • the influenza virus is an avian influenza virus (AIV).
  • a method for detecting a virus of interest in a field or clinical sample comprising the steps of: obtaining the field or clinical sample suspected of containing the virus of interest; providing an insert for performing a dot-Antibody Linked Immunogold Assay (dot-ALIGA); applying to the insert the field or clinical sample suspected of containing the virus of interest; providing a microfluidic device comprising a channel disposed therein; inter-fitting the insert within the channel; and performing the dot-ALIGA in the microfluidic device to detect an antigen of the virus of interest, wherein the dot-ALIGA comprises: flowing a first fluid material through the channel to contact the insert therein, wherein the first fluid material comprises anti-virus antibody-labeled colloidal gold, wherein the anti-virus antibody is an antibody directed against the virus of interest, for a time sufficient to allow the anti-virus antibody-labeled colloidal gold and the antigen of the virus of interest in the field or clinical sample suspected of containing the virus of interest to
  • the system and method uses sensitivity-enhanced immunogold nanoparticles for the detection of the virus.
  • antibody-conjugated gold nanoparticles are used in the sensitivity-enhanced dot-ALIGA as virus-specific immunoassay binding agents.
  • the virus detection limitation of the sensitivity-enhanced dot- ALIGA can be comparable to a conventional microtiter plate-based ELISA and about 2 5 times more sensitive than a hemagglutination assay.
  • the dot-ALIGA is a monoclonal antibody-based dot-ALIGA and the anti-virus antibody is an anti-virus monoclonal antibody (mAb).
  • the method additionally comprises, before the step of flowing the first fluid material through the channel, the steps of: flowing a fluid comprising a blocking agent through the channel to contact the insert therein for a time sufficient to block non-specific binding sites on the insert; and drawing the fluid comprising the blocking agent away from the insert.
  • the blocking agent is bovine serum albumin (BSA), nonfat milk powder, gelatin or casein.
  • BSA bovine serum albumin
  • the method additionally comprises, before the step of detecting an interaction on the insert, the steps of: flowing a fluid comprising a washing reagent or buffer through the channel to contact the insert therein for a time sufficient to wash the insert; and drawing the washing reagent or buffer away from the insert.
  • the virus of interest is an influenza virus.
  • the influenza virus is selected from the group consisting of an avian influenza virus, an influenza A virus, an influenza B virus, an influenza C virus, a canine influenza virus, a feline influenza virus, an equine influenza virus and a swine flu virus.
  • the influenza virus is an avian influenza virus (AIV)-
  • the insert comprises a nitrocellulose membrane.
  • the sensitivity enhancement reagent comprises hydroxylamine chloride and chloroauric acid.
  • the concentration of hydroxylamine chloride can be in the range of 0.001 - 0.01 mM, 0.01 - 0.1 mM, 0.1 - 1.O mM, 1.0 - 10.O mM, 10.0 - 100.0 mM, or 100.0 mM - 1.0 M.
  • the concentration of chloroauric acid can be in the range of 0.01 - 0.1%, 0.1 - 1.0%. 1.0 - 10.0% or 10.0 - 20.0%.
  • the sensitivity enhancement reagent comprises 0.1 - 1.0 mM hydroxylamine chloride and 1 - 10% chloroauric acid.
  • the sensitivity enhancement reagent comprises 1.0 mM hydroxylamine chloride and 1-5% chloroauric acid.
  • the sensitivity enhancement reagent comprises Z,(+)-ascorbic acid and chloroauric acid.
  • the concentration of L(+)-ascorbic acid can be in the range of 0.01 - 0.1%, 0.1 - 1.0%, 1.0 - 10.0% or 10.0 - 20.0%.
  • the concentration of chloroauric acid can be in the range of 0.01 - 0.1%, 0.1 - 1.0%. 1.0 - 10.0% or 10.0 - 20.0%.
  • the sensitivity enhancement reagent comprises 0.1 - 1.0% Z,(+)-ascorbic acid and 1 - 10% chloroauric acid.
  • the sensitivity enhancement reagent comprises 0.15% /-(+)- ascorbic acid and 1 -5% chloroauric acid.
  • the detecting step detects the presence of the antigen-bound anti-virus antibody-labeled colloidal gold conjugate in the range of 0.015 - 0.02 HAU.
  • flowing the first fluid material or the second fluid material comprises actuating a distribution valve to flow a reagent from a reagent reservoir to a plurality of outlet reservoirs.
  • flowing the first fluid material or the second fluid material comprises repeatedly shuttling the first fluid material or the second fluid material in a first direction towards a first reservoir connected to the channel and in a second direction towards a second reservoir connected to the channel, wherein a distribution valve coupled to the channel substantially confines the fluid material in the channel when the distribution valve is in a closed state.
  • drawing the first fluid material or the second fluid material away from the insert comprises flowing the first fluid material or the second fluid material in at least one of a first direction towards a first reservoir connected to the channel and a second direction towards a second reservoir connected to the channel.
  • the method further comprising transporting waste from the channel to a waste reservoir connected to the channel.
  • detecting the interaction comprises visualization of color intensity, fluorescence intensity or chemiluminescence intensity.
  • detecting the interaction comprises generating an intensity value corresponding to at least one sample of the insert.
  • the intensity value is selected from the group consisting of color intensity value, fluorescence intensity value and chemiluminescence intensity value.
  • generating the color (or fluorescence or chemiluminescence) intensity value comprises: digitizing a color (or fluorescence or chemiluminescence image) corresponding to the sample to generate a plurality of pixels: providing a plurality of numerical values for respective ones of the plurality of pixels; and averaging the plurality of numerical values to provide the color (or fluorescence or chemiluminescence) intensity value.
  • the method further comprises computing a threshold value and comparing the color (or fluorescence or chemiluminescence) intensity value to the threshold value to detect the interaction.
  • the method further comprises storing at least one of the (or fluorescence or chemiluminescence)intensity value and the threshold value in a database.
  • the threshold value is computed using at least one negative control sample.
  • a method for detecting an avian influenza virus (AIV) in a field or clinical sample comprising the steps of: obtaining the field or clinical sample suspected of containing AIV; providing an insert for performing a monoclonal antibody-based dot-ALIGA; applying to the insert the field or clinical sample suspected of containing the AIV; providing a microfluidic device comprising a channel disposed therein; inter-fitting the insert within the channel; and performing the monoclonal antibody-based dot-ALIGA in the microfluidic device to detect an AIV antigen, wherein the monoclonal antibody-based dot-ALIGA comprises: flowing a first fluid material through the channel to contact the insert therein, wherein the first fluid material comprises anti-AlV monoclonal antibody-labeled colloidal gold, for a time sufficient to allow the anti-AIV monoclonal antibody-labeled colloidal gold and the AIV antigen in the field or clinical sample suspected of containing the AIV to bind together to form
  • the sensitivity enhancement reagent comprises L(+)-ascorbic acid and chloroauric acid.
  • an apparatus for detecting a virus of interest in a field or clinical sample comprising: a microfluidic device, wherein the microfluidic device comprises a channel disposed therein; and an insert for performing a dot-ALIGA, wherein the insert is capable of being inter-fitted within the channel and wherein the dot-ALIGA comprises: flowing a first fluid material through the channel to contact the insert therein, wherein the first fluid material comprises anti-virus antibody-labeled colloidal gold, wherein the anti-virus antibody is an antibody directed against the virus of interest, for a time sufficient to allow the anti-virus antibody-labeled colloidal gold and the antigen of the virus of interest in the field or clinical sample suspected of containing the virus of interest to bind together to form an antigen- bound anti-virus antibody-labeled colloidal gold conjugate; drawing the first fluid material away from the insert; flowing a second fluid material through the channel to contact the insert therein, wherein the second fluid material comprises a sensitivity enhancement reagent or
  • the dot-ALIGA is a monoclonal antibody-based dot-ALIGA.
  • the anti-virus antibody is an anti-virus monoclonal antibody (mAb).
  • the virus of interest is an influenza virus.
  • the influenza virus is selected from the group consisting of an avian influenza virus, an influenza A virus, an influenza B virus, an influenza C virus, a canine influenza virus, a feline influenza virus, an equine influenza virus and a swine flu virus.
  • the influenza virus is an avian influenza virus (AIV).
  • a kit for detecting a virus of interest in a field or clinical sample is also provided.
  • the kit comprises in one or more containers: a microfluidic device, wherein the microfluidic device comprises a channel disposed therein; and an insert for performing a dot-ALIGA, wherein the insert is capable of being inter- fitted within the channel and wherein the dot-ALIGA comprises: flowing a first fluid material through the channel to contact the insert therein, wherein the first fluid material comprises anti-virus antibody-labeled colloidal gold, wherein the anti-virus antibody is an antibody directed against the virus of interest, for a time sufficient to allow the anti-virus antibody-labeled colloidal gold and the antigen of the virus of interest in the field or clinical sample suspected of containing the virus of interest to bind together to form an antigen- bound anti-virus antibody-labeled colloidal gold conjugate; drawing the first fluid material away from the insert; flowing a second fluid material through the channel to contact the insert therein, wherein the second fluid material comprises a sensitivity enhancement reagent or at least one component thereof and wherein the sensitivity enhancement reagent or at least
  • the kit additionally comprises in one or more containers the first fluid material comprising anti-virus antibody-labeled colloidal gold.
  • the kit additionally comprises in one or more containers the second fluid material comprising the sensitivity enhancement reagent or at least one component thereof.
  • the system and method for detecting the virus comprises employing a plastic microfluidic chip configured to process one or more reagents, including but not limited to buffers, washing reagents, blocking reagents, reagents comprising anti-virus antibody-labeled colloidal gold or at least one component thereof, and sensitivity enhancement reagents or at least one component thereof.
  • the chip may comprise various microfluidic features including valves, pumps, channels and reservoirs.
  • the micro-features are interconnected to allow various combinations of fluid flow patterns that can be user specified and tailored to a particular application.
  • the chip allows for the transport of one or more reagents from respective reagent reservoirs on a reagent cartridge to multiple assay channels via a transport structure.
  • the microfluidic chip includes a plastic substrate having a plurality of channels, a distribution structure for introducing a reagent into at least one of the channels, and a configurable transport system for controllably directing a flow of the reagent in the channels.
  • the channels include a plurality of inlet channels, a plurality of outlet channels and a plurality of assay channels.
  • the configurable transport system comprises a distribution valve connected to the inlet channels and outlet channels for distributing reagents to the assay channels.
  • the assay channels are configured for conducting biological assays.
  • the inlet channels, outlet channels, assay channels and distribution structure are disposed in the substrate body.
  • the porting device is a separate reagent cartridge that is detachably coupled to a top surface of the substrate and has a plurality of reagent reservoirs fiuidly communicating with the respective inlet channels.
  • the inlet channels are individually valve controlled to deliver reagents from the respective reagent reservoirs to the assay channels through the distribution valve and the outlet channels.
  • a buffer reservoir is aligned with an inlet channel to the distribution valve.
  • the buffer reservoir features a substantially larger storage volume than the individual reagent reservoirs for storing a washing buffer.
  • a diaphragm valve located beneath the buffer reservoir controllably releases the washing buffer into the assay channels through the distribution valve.
  • one or more shuttle reservoirs and outlet reservoirs store reagents and buffer that are transported during reaction incubation.
  • the shuttle reservoirs are connected to the corresponding outlet reservoirs through respective assay channels.
  • the volumes of a shuttle reservoir and an outlet reservoir are substantially larger than the volume of an assay channel so that a reaction reagent in the assay channel can be transported into the shuttle reservoir and/or the outlet reservoir during reaction incubation.
  • an on-chip waste reservoir is aligned with an outlet channel to the distribution valve.
  • the waste reservoir features a substantially larger storage volume than the buffer reservoir for storing all used reagents and washing buffer.
  • An independently actuated diaphragm valve located beneath the waste reservoir regulates fluid flow into the waste reservoir from the shuttle and/or outlet reservoirs via the distribution valve.
  • one or more bi-directional fluidic pumps are each coupled to at least three valves respectively controlling a fluid flow through an assay channel, a shuttle reservoir and an outlet channel to the distribution valve.
  • the pump-and-valves structure enables multiple fluid drawing and delivery patterns such as from a reagent reservoir to a shuttle reservoir, from a reagent reservoir to an assay channel to an outlet reservoir, from a shuttle reservoir to an outlet reservoir via an assay channel, from an outlet reservoir to a shuttle reservoir via an assay channel, from an outlet reservoir to a waste reservoir and from a shuttle reservoir to a waste reservoir.
  • the porting device comprises a separate reagent chip including the inlet channels, the distribution valve and a plurality of reagent reservoirs.
  • the reagent reservoirs are aligned with the inlet channels for introducing reagents to the distribution valve.
  • the porting device also includes a ducting chip having the outlet channels disposed therein.
  • the ducting chip is adapted to detachably couple to the reagent chip and the substrate for introducing the reagents from the reagent chip to the assay channels in the substrate.
  • an insert (or assay strip) is disposed in a void volume of an assay channel for conducting biological assays or chemical reactions, wherein the assay channel is configured to receive the insert and prevent a reaction surface of the insert from contacting the channel surface.
  • the assay channel is adapted to receive the insert (or assay strip) from an opening of the outlet reservoir connected to the assay channel.
  • the void volume of the assay channel includes an opening to the top surface of the substrate wherein the insert (assay strip) can be disposed, and a lid for removably covering the opening of the void volume.
  • the reaction surface of the insert or assay strip may include one or more samples analytes or agent for potentially interacting with reagents delivered from the reagent cartridge.
  • the samples analytes or agents are chosen for specific applications.
  • the insert includes a perforated membrane film strip and at least one membrane disk coupled to a surface of the membrane film strip and aligned with an aperture on the membrane film strip.
  • the membrane disks are each coated with an agent sample containing a biological and/or chemical material such as a target analyte or analyte-capturing antibodies.
  • the apertures include a central circular region and two rectangular regions open to the circular region. The rectangular regions are configured to trap air bubbles in a fluidic flow through the assay channel.
  • the film strip is made from a non-elastomeric plastic adhesive material.
  • the non-elastomer plastic material includes polymethyl methacrylate, polystyrene, polycarbonate and acrylic.
  • the membrane disks are made from nitrocellulose, PVDF and/or nylon.
  • a heating element is coupled to the microfluidic chip for controlling the assay temperature for enhanced assay repeatability, speed and sensitivity.
  • the insert is spotted with the field or clinical sample (or specimen) suspected of containing the virus of interest. After one or more sample-spotted inserts are disposed into the appropriate assay channels in the microfluidic chip, reagents from the reagent cartridge can be made to flow through the assay channels via the distribution structure, thereby contacting the reaction surfaces of the inserts. Washing buffer from the buffer reservoir can also flow through the assay channels to contact the inserts in the channels.
  • reaction reagents and/or washing buffer in the assay channels are pumped back and forth between a shuttle reservoir and an outlet reservoir connected to each assay channel.
  • fluidic wastes stored in the shuttle reservoirs and the outlet reservoirs are pumped into the waste reservoir via the distribution structure.
  • the microfluidic chip can be used to perform an immunoassay or other biological assay on each membrane disk in order to detect the target analytes.
  • the shuttle reservoirs are used as reagent reservoirs for creating individual assay conditions in each assay channel. Unlike a reagent delivered from the reagent reservoir that creates uniform assay conditions in all assay channels, different reagents or reagents of different concentrations in the shuttle reservoirs may be individually delivered to the assay channels for performing parallel, but non-uniform biological assays.
  • the end result of an assay is detected by color changes on the inserts using an automated image analysis procedure. The procedure involves quantitatively digitizing an array of color-spotted samples in the assay chip and quantitatively determining the color intensity corresponding to each pixel of a sample spot to generate an averaged, or pixilated, value for each sample.
  • the sample color intensity values yield information about the biological samples on corresponding membrane disks.
  • a threshold value may be computed by using negative control samples.
  • the threshold value, the color intensity values, and the various images corresponding to the sample array may be stored and archived for future reference.
  • the end result of an assay is detected by changes in fluorescence or chemiluminescence on the inserts using the automated image analysis procedure described above.
  • a microfluidic chip can be ported to a controller capable of driving the pump and valve structures on the chip.
  • the controller may be electronically or wirelessly connected to a computer or a Personal Digital Assistant (PDA), such as BLACKBERRY® or PALM PILOT®, providing an interface for a user to programmably control the assay reactions on the chip.
  • PDA Personal Digital Assistant
  • the microfluidic chips are made entirely from plastic materials.
  • an entire microfluidic chip suitable for portable immunoassay is made from polystyrene, which results in extremely low fabrication costs.
  • An enabler for the use of polystyrene in such an application while preserving the integrity and reliability of the microfeatures disposed therein is the use of weak solvent bonding.
  • FIG. 1 illustrates one embodiment of a microfluidic chip for carrying out the assays of the invention.
  • FIG. 2 illustrates an alternative view of the microfluidic chip of FIG. 1.
  • FIGS. 3a-b illustrate a microfluidic valve used in the embodiment shown in FIG. 1.
  • FIGS. 4a-4f illustrate a microfluidic pump used in the embodiment shown in FIG. 1.
  • FIGS. 5a-c illustrate an inlet valve used in the embodiment shown in FIG. I.
  • FIGS. 6a-b illustrate a cartridge and a reservoir used in the embodiment shown in
  • FIG. 7 shows an assay chip having ducts that connect to a separate reagent chip.
  • FIGS. 8-10 illustrate steps for manufacturing the device of FIG. 7.
  • FIGS. 1 la-c illustrate an exemplary insert sized and shaped to inter-fit within the embodiment shown in FIG. 1.
  • FIG. 12 illustrates an embodiment of a chip in which a single driving force distributes a reagent to a plurality of outlet reservoirs.
  • FIG. 13 illustrates an embodiment of a chip in which multiple driving forces distribute a reagent to a plurality of outlet reservoirs.
  • FIG. 14 illustrates an embodiment of a chip having multiple driving forces distributing a plurality of reagents to a plurality of outlet reservoirs.
  • FIGS. 15a-c illustrate a method of inter-fitting the exemplary insert of FIGS. 1 la-c within a channel of the embodiment shown in FIG. 1.
  • FIGS. 16a-b show the results of a microfluidic-based on-chip immunoassay process.
  • FIG. 17 illustrates steps in identifying samples containing a target analyte.
  • FIG. 18 shows a complete and self-contained microfluidic system including a computer, a controller and a chip.
  • FIG. 19 illustrates an alternate embodiment of a chip coupled to a controller.
  • FIG. 20 shows the results of a sensitivity-enhanced dot- ALIGA. Two-fold serial dilution of reference virus was detected using gold-conjugated AIV antibody. Top row: treated with sensitivity enhancement reagent. Bottom row: not treated with sensitivity enhancement reagent. See Section 6.1 for details.
  • FIG. 21 shows the results of another sensitivity-enhanced dot- ALIGA.
  • Two vertical test columns (“SE” and “WO SE”) were spotted with the AIV reference strain and two vertical test columns (“SEC” and “WO SEC”) were spotted with negative controls. See Section 6.2 for details.
  • FIG. 22 shows an insert exhibiting the results of performing a sensitivity enhanced dot-ALIGA for AIV using a microfluidic device. See Section 6.3 for details.
  • the invention in various embodiments, provides immunoassays and methods for detecting pathogens including, but not limited to, avian influenza virus.
  • the immunoassays and methods described herein are preferably conducted on microfluidic chips or systems.
  • Microfluidic systems that are suitable for use in carrying out the assays and methods described herein are known in the art.
  • U.S. Patent Application Publication No. 2007/0166199 Al application Serial No. 1 1/594,444, entitled “Microfluidic systems and control methods" by Zhou et al. discloses a microfluidic system that is suitable for use in carrying out the assay.
  • the microfluidic system comprises a pneumatic manifold having a plurality of apertures, and a chip manifold having channels disposed therein for routing pneumatic signals from respective ones of the apertures to a plurality of valves in a microfluidic chip, wherein the channels route the pneumatic signals in accordance with a configuration of the plurality of valves in the microfluidic chip.
  • Microfluidic chips that are suitable for use in carrying out the assay are known in the art.
  • a microfluidic chip and inserts such as disclosed in U.S. Patent Application Publication No. 2007/0166200 Al (application Serial No. 1 1/650,006, entitled "Microfluidic chips and assay systems" by Zhou et al.) are used in carrying out the assay.
  • Such a microfluidic chip and inserts can be used to provide an efficient and accurate approach for conducting parallel assays for detecting a pathogen such as avian influenza virus.
  • the microfluidic chip has a plurality of micro features interconnected to provide a configurable fluid transport system for processing at least one reagent. Inserts are provided to removably interfit into one or more of the microfeatures of the chip, wherein the inserts include sites for interactions with the reagent.
  • FIG. 1 illustrates a microfluidic system 1 that includes an assay chip 5 and a cartridge 10 disposed on the chip 5 along a width of the chip 5.
  • the cartridge 10 includes a plurality of reagent reservoirs 12 having side walls that define chambers to hold fluid reagents.
  • the chip 5 includes a buffer reservoir 16 having a cylindrical sidewall to hold a washing buffer, a plurality of shuttle reservoirs 17 adapted to hold reagents during an assay operation, and a waste reservoir 18 adapted to hold used reagents and used buffer after the assay operation.
  • the chip 5 also includes a plurality of inlet valves 14 positioned to align with the various reservoirs. The inlet valves 14 serve to control fluid flows between the reservoirs and respective microchannels in the chip 5.
  • the chip 5 includes a plurality of inlet channels 20, a distribution valve 25, an inlet 30, a waste channel 38, a plurality of reagent and or buffer outlet channels 35, assay channels 40, fluid pumps 44, and outlet reservoirs 48.
  • the distribution valve 25 controls the release of fluid from the inlet channels 20 to the inlet 30.
  • the distribution valve 25 controls the release of fluid from the inlet 30 to the waste channel 38.
  • the inlet 30 serves as an inlet to outlet channels 35 which are in fluidic communication with the assay channels 40.
  • the pumps 44 pump fluid in a direction 60 towards the outlet reservoirs 48, but can also be programmed to pump fluid generally in the direction 62 towards the shuttle reservoirs 17 and the inlet 30.
  • the chip 5 is generally constructed from a first substrate 6, a second substrate 7, and a membrane 8 (not shown) disposed in between the two substrates 6 and 7.
  • the membrane 8 has a thickness of between about lO ⁇ m and about 150 ⁇ m, or between about 15 ⁇ m and about 75 ⁇ m.
  • the depicted first substrate 6 and second substrate 7 each has a thickness substantially larger than the thickness of the membrane 8, but in other implementations, has a thickness similar to or less than the thickness of the membrane 8.
  • the microfluidic channels 20, 25, 38, and 40 may be of any suitable dimension, but in certain embodiments have cross-sectional dimensions of between about 1 ⁇ m and about 500 ⁇ m, or between about 1 ⁇ m and about 50 ⁇ m.
  • the first substrate 6, the second substrate 7, and the membrane 8 are all made of plastic.
  • Exemplary materials include non-elastomeric polymers, such as polymethyl methacrylate, polystyrene, polycarbonate, and acrylic. These materials are beneficial at least in part because they are reasonably rigid, which is suitable for the first substrate 6 and the second substrate 7. Moreover, these materials can be deformable when used in thin layers, which is suitable for the membrane 8 which may deflect towards and away from the first 6 and second 7 substrates.
  • the system 1 provides automated "many-to-many" reagent dispensing and processing.
  • inlet valves 14, distribution valve 25 and fluid pumps 44 By selectively operating inlet valves 14, distribution valve 25 and fluid pumps 44, various combinations of fluid flow patterns among reagent reservoirs 12, buffer reservoir 16, waste reservoir 18, shuttle reservoirs 17 and outlet reservoirs 48 can be achieved.
  • the distribution valve 25 may be constructed in accordance with the valve structure described with respect to FIGS. 3a-b.
  • FIGS. 3a-b show a three-layer active planar valve structure 399, which may be formed using acetonitrile assisted bonding.
  • the valve structure 399 includes a first substrate 300 having interdisposed microchannels 301 and 303.
  • a membrane layer 304 is selectively bonded to the first substrate 300 in areas 306, thus creating a diaphragm structure 308.
  • a second substrate 302 is bonded to the membrane 304.
  • the second substrate includes a drive chamber 310.
  • the channel pumps 44 of FIG. 1 may be constructed in accordance with the pump structure described with respect to FIG. 4a-f or as described in U.S. Patent Application Publication No. 2006/0076068 Al (application Serial No. 11/242,694, entitled “Microfluidic pump and valve structures and fabrication methods" by Young et al.).
  • U.S. Patent Application Publication No. 2006/0076068 Al discloses plastic microfluidic structures having a substantially rigid diaphragm that actuates between a relaxed state wherein the diaphragm sits against the surface of a substrate and an actuated state wherein the diaphragm is moved away from the substrate.
  • the microfluidic structures formed with this diaphragm can be employed as valves and pumps.
  • a microfluidic pump generally refers to any structure or group of structures capable of applying positive and/or negative pressure to a fluid and/or facilitating the flow of fluid in one or more desired directions.
  • the depicted micro-diaphragm pump 400 generally includes three valves: an inlet valve 402, a drive valve 404 and an outlet valve406, interconnected by portions 418b and 418c of microchannel 418.
  • the pump 400 pumps fluid through the microfluidic channel 418 by cycling through six states that are activated sequentially to produce a peristaltic-like pumping effect.
  • FlG. 4 depicts three valve structures 402, 404 and 406 that make up the pump 400, other pump embodiments may contain four or more valve structures.
  • the inlet valve 402 opens and draws fluid from an inlet portion 418a of the microfluidic channel 418 into volume 425 between the membrane 408 and the second substrate 432.
  • the drive valve 404 opens and draws more fluid into the pump system.
  • the inlet valve 402 closes.
  • the outlet valve 406 opens.
  • the drive valve 404 closes, and thereby forces fluid through the outlet valve 406 and into an outlet portion 418d of the microfluidic channel.
  • the outlet valve 406 then closes.
  • the pump 400 is bidirectional. If the cycle is reversed, portion 418d is an inlet portion of the microfluidic channel 418, portion 418a is an outlet portion of the microfluidic channel 418, and fluid flows from portion 418d to portion 418a.
  • valve structures 402, 404, and 408 are independently actuatable, in that any one of the valve structures can be actuated with little or substantially no effect on the state of the other valve structures.
  • Those skilled in the art will recognize that alternate sequences of states may produce a pumping effect, and that other pumps can also be used in practicing the invention.
  • FIGS. 5a-b illustrate an exemplary inlet valve structure 14 of FIG. 1.
  • the valve 14 includes a first substrate 508 with a drive chamber 510 fabricated therein, a second substrate 515 and a membrane 520.
  • a reservoir may be disposed above the second substrate 515 and aligned with reservoir port 540 to provide a source of fluid for porting into channel 545.
  • the reservoirs will be discussed in detail with respect to FIGS. 6a-b.
  • FIG. 5c illustrates an exemplary structure including a plurality of inlet valves 14 of FIG. 1 connected in series.
  • Various embodiments and alternatives may be applied to the pump and valve structures. In particular, three or more valves similar to the valve structure 565 in FIG.
  • FIG. 5c may be connected in series by microchannels to form a pump that operates with a peristaltic-like mechanism, such as the pumps 44 of FIG. 1.
  • Other arrangements of valve structures interconnected by microchannels can also form generic pumping configurations.
  • a reservoir may be disposed above the second substrate 515 and aligned with reservoir port 540 to provide a source of fluid for porting into channel 545.
  • FIGS. 6a-b show a cartridge 610 with a top side 602 and a bottom side 604 having a reagent reservoir 612 formed thereon.
  • the cartridge 610 is provided with its top side 602 and bottom side 604 both sealed by suitable adhesive materials.
  • the top adhesive material 605 is a sealing tape
  • the bottom sealing material (not shown) may also be a sealing tape.
  • Other suitable adhesive materials may also be used.
  • FIG. 6a depicts the cartridge 610 having only the reagent reservoirs 612 disposed thereon, although various other cartridge configurations are possible.
  • a cartridge includes a buffer reservoir 616, a waste reservoir 618 and a plurality of shuttle reservoirs 617 in addition to the reagent reservoirs 612.
  • a cartridge includes the reagent 612 and buffer 616 reservoirs.
  • the shuttle 617 and waste 618 reservoirs may be integrally constructed onto the chip 615 or provided on a separate cartridge.
  • three separate cartridges are provided respectively including the shuttle reservoirs 617, the reagent reservoirs 612, and the buffer 616 and waste 618 reservoirs.
  • a cartridge has only the shuttle reservoirs 617 for distributing different reagents to assay channels 630-635.
  • FIGS. 7-10 illustrate an alternate method for coupling multiple reservoirs to an assay chip.
  • FIG. 7 shows an assay chip 705, a reagent chip 710, and a ducting chip 715.
  • the reagent chip 710 includes a reagent cartridge 720 and a reagent loading chip 725.
  • the ducting chip 715 serves to provide bi-directional fluid flows between the reagent chip 710 and the assay chip 705.
  • the reagent chip 710 allows several reagent reservoirs 735-739 to dispense reagents into reservoir 740 before being ported to the assay chip 705 through the ducting chip 715.
  • one of the reagent reservoirs 735-739 may be a buffer reservoir for storing a buffer solution.
  • one of the reservoirs 735-739 may be a waste reservoir for storing used reagents after an assay.
  • the ducting chip 715 is rigid enough to provide the necessary structural support to duct the assay chip 705 to the reagent chip 710. However, the ducting chip 715 is deformable such that reagent chip 710 and assay chip 705 need not be exactly aligned along a vertical axis
  • the ducting chip 800 includes a cover layer
  • the ducting chip further includes a first support layer 810, a channel layer 815, and a second support layer 820. Layers 805 and 810 are provided with apertures 825 that are aligned to allow fluid to flow from channels 830 in a downward 832 direction.
  • the channel layer includes a plurality of inter-disposed channels 830.
  • the first support layer 810, the channel layer 815, and the second support layer 820 include apertures 845 that are substantially aligned to allow fluid to flow in a downward 832 direction from a reservoir 840.
  • An adhesive O-ring 835 adheres the reservoir
  • FIG. 8b shows the ducting chip 800 of FIG. 8a after assembly.
  • the reagent loading chip 925 includes a bottom substrate layer 905 with drive chambers 907, a membrane layer 910, and a top substrate layer 915 with microchannels etched therein.
  • the layers may be attached with suitable lamination methods described herein.
  • FIG. 9b shows a top view of the reagent loading chip 925.
  • FIG. 10 illustrates an exploded view of the full structure including the ducting chip
  • FIG. 10 shows the reagent cartridge 1020 being laminated to the reagent loading chip
  • the ducting chip 1015 being coupled to the reagent loading chip 1025, and the assay chip
  • void regions 1060-1065 may be disposed in the respective assay channels. These void regions 1060-1065 may be open to a top surface of the chip 1005. A cover adhesive layer may be disposed over each channel void region 1060-
  • a temperature-modulating device such as a heater or a cooler, may be coupled to the microfluidic systems 1 and 1000 to regulate the temperature of the fluids in the systems for providing an optimal environment wherein on-chip biological and/or chemical reactions may occur.
  • a temperature-modulating device such as a heater or a cooler
  • FIG. 1 there are six reagent reservoirs 12, six shuttle reservoirs 17, six outlet reservoirs 48, one waste reservoir 18 and one buffer reservoir 16.
  • the assay channels may be provided with biological or chemical materials that react with reagents introduced into the microfluidic system.
  • inserts are provided with chemical and/or biological agents for insertion into the microchannels for the purpose of reacting with the reagents.
  • Exemplary inserts are shown in FIGS, l la-b.
  • the insert is a flexible plastic strip with an adhesive coating on one side.
  • the insert is a thin polystyrene strip.
  • the insert has a thickness of between about 50 microns to about 500 microns in thickness, a width of between about 1 mm to about 5 mm, and a length of between about 5mm to about 100mm.
  • the assay channels are configured accordingly in order to accommodate the inserts disposed therein.
  • an insert may be provided with chemical and/or biological agents.
  • an insert in one exemplary implementation, includes a membrane 1 104 having adhesive disposed on its surface and membrane disks 1 1 10 adhered to the membrane 1 104, wherein the membrane disks 1 1 10 are provided with chemical and/or biological agents.
  • the membrane 1 104 is further provided with apertures 1 1 15 over which the membrane disks 1 1 10 lie.
  • the apertures 1 1 15 may be included in a perforated cover strip 1105 adhering to the membrane 1 104.
  • the apertures serve to allow fluid contact between the bottom side of the membrane disks 1 110 and a fluid flow through channel 1 130 wherein the insert 1 107 is disposed.
  • the apertures 1 115 are circular. In one example as shown in FlG.
  • the apertures 1 1 15 each includes a central circular region 1120 with two opposing rectangular regions 1122 open to the circular region 1120.
  • the rectangular regions 122 are oriented on the insert 1107 in a direction 1132 aligned with a direction of fluid flow when the insert 1 107 is disposed in the assay channel 1 130. This feature enables the insert 1 107 to trap air bubbles in the fluid.
  • the membrane disks 11 10 are preferred to be circular, although other shapes are possible.
  • the apertures 11 15 are shaped and sized to provide structural support for the membrane disks 1 1 10. For the case of circular disks and circular apertures as illustrated in FIG.
  • the disks 1 1 10 are preferred to have a diameter of between about lmm and about 5 mm, and the apertures 1 1 15 are preferred to have a diameter that is between about 5% and about 10% less than the diameter of the disks 11 10.
  • a diameter of the central circular regions 1120 of the apertures 11 15 may be between about 5% and about 10% less than a major diameter of the membrane disks 1 1 10.
  • a width 1 124 of the rectangular regions 1122 may be between about 5% to about 10% less than the diameter of the central circular regions 1120.
  • the membrane disks 1 1 10 may be made of a porous material such as nitrocellulose.
  • the porosity of the membrane disks 1110 may be sufficiently large to allow fluid and salt passing through but small enough to interact with macromolecules, viruses or bacteria in the fluid.
  • the membrane disks 1 110 may be made of nitrocellulose, PVDF and/or nylon, which are suitable materials for use in a microfluidic-based dot-chip process as will be described below.
  • the membrane disks 1 1 10 and the apertures 1 1 15 may be formed by, for example, a die cut or laser cut. The operations of various components of the microfluidic system I of FIG. 1 will be described below. By selectively operating the inlet valves 14, distribution valve 25, and channel pumps 44, various combinations of fluid flow patterns might be achieved.
  • FIGS. 3a-b illustrate one method for operating the distribution valve 25 of FIG. 1.
  • a positive upward pressure is applied to the diaphragm 308 via the drive chamber 310, the membrane 308 is pushed away against the valve seat 312 between the two microfeatures 301 and 303, effectively preventing any transfer of fluid between them.
  • FIGS. 4a-f illustrate one method for pumping fluid through the pump structure 44 of FIG. 1. The method comprises cycling the pump structure though six states that are activated sequentially to produce a pumping effect.
  • the inlet valve 402 is opened and fluid is drawn from inlet microchannel 412 into the volume 402a between the membrane 408 and the first substrate 410.
  • FIG. 4a the inlet valve 402 is opened and fluid is drawn from inlet microchannel 412 into the volume 402a between the membrane 408 and the first substrate 410.
  • the drive valve 404 is opened, drawing more fluid into the pump system.
  • the inlet valve 402 is closed.
  • the outlet valve 406 is opened.
  • the drive valve 404 is closed, forcing fluid out through the outlet valve 406 into outlet microchannel 418.
  • the outlet valve 406 is then closed.
  • FIGS. 5a-b illustrate one method for operating the inlet valves 14 of FIG. 1.
  • a positive pneumatic force 525 is applied through drive chamber 510, forcing the valve 500 to be in a closed position wherein there is no fluidic communication between inlet channel 545 and reservoir port 540.
  • a negative pneumatic force 530 is applied through drive chamber 510, forcing the valve 500 to be in an open position wherein reservoir port 540 is in fluidic communication with inlet channel 545.
  • FIG. 5c illustrates the operation of a plurality of inlet valves being connected in series.
  • communication between inlet valves 550 and 557 may be controlled by actuating a valve structure 565 connected to the inlet valves.
  • a positive pneumatic force 570 may be applied through the drive chamber 586 disposed in the bottom substrate 593. This force will push the membrane 588 into conformal contact with a region 590 of the top substrate 592.
  • the valve is in a closed position with substantially no fluidic communication between adjoining microchannels 572 and 573.
  • a negative pneumatic force 575 applied through the drive chamber 586 will pull the membrane 588 away from the top substrate 592, such that the membrane 588 forms a cavity towards the drive chamber 586 into the region 587.
  • the valve is in an open position in which adjoining microchannels 572 and 573 are in fluidic communication.
  • FIGS. 12-14 illustrate various embodiments for distributing fluids through the chip 1 of FIG. 1 by actuating the pump and valve structures described above.
  • FIG. 12-14 illustrate various embodiments for distributing fluids through the chip 1 of FIG. 1 by actuating the pump and valve structures described above.
  • FIG. 12 illustrates a single driving force for distributing a reagent from a reagent reservoir 1205a among a plurality of microchannels 1220-1223 on a chip 1200.
  • the single driving force is produced by an inlet valve 1215a and a drive diaphragm 1224 located in between the area of an inlet valve 1215a and an outlet valve 1225.
  • These three valves may operate according to the peristaltic-like pumping mechanism described above with respect to FIG. 4 to transport fluid contents of reservoir 1205a among the outlet channels 1210-1213.
  • reagent contents of reservoirs 1205b-d may be delivered to outlet channels 1210-1213 via pumping action produced by respective ones of inlet valves 1215b-d, drive diaphragm 1224 and outlet valve 1225.
  • the tlow resistances of outlet channels 1210-1213 impact the fluid flow rate on assay channels 1220-1223.
  • the flow rate in each channel of an assay chip is inversely proportional to the flow resistance of that channel.
  • the outlet channels 1210-1213 may be fabricated to have different flow resistances if an application calls for different channels to have different respective flow rates.
  • FIG. 13 illustrates an embodiment of the chip 1 in FIG. 1 that overcomes the variation in flow rates resulting from varying channel flow resistances.
  • Each assay channel 1310-1315 and each outlet channel 1360-1365 are associated with a respective fluid pump 1320- 1325.
  • FlG. 13 illustrates a reagent from reagent reservoir 1350 being distributed (see arrows) among outlet channels 1360- 1365 via distribution valve 1352.
  • a plurality of reagents from their respective reagent reservoirs 1350-1355 are delivered to the distribution valve 1352 wherein the reagents may be mixed to create a reagent mixture.
  • the reagent or reagent mixture may be further distributed to selected assay channels 1310-1315, outlet reservoirs 1330-1335, and/or shuttle reservoirs 1340-1345.
  • FIG. 14 illustrates additional fluid distribution patterns of the microfluidic system shown in FIG. 1.
  • each shuttle reservoir 1440-1445, assay channel 1410-1415 and outlet channel 1460-1462 are connected in series to form a fluid pump 1420-1425, wherein each fluid pump 1420-1425 provides bi-directional fluid flow to and from the respective micro- features.
  • fluid pumps 1420-1425 provides bi-directional fluid flow between shuttle reservoirs 1440-1445 and outlet reservoirs 1430-1435 interconnected by the respective assay channels 1410-1415.
  • a reagent in outlet reservoir 1432 is delivered through outlet channel 1461 and distribution valve 1462 to waste reservoir 1464.
  • a reagent in shuttle reservoir 1443 is delivered to waste reservoir 1464 via outlet channel 1461 and distribution valve 1462.
  • different reagents or reagents of different concentrations may be introduced to the assay channels 1410-1415 from the corresponding shuttle reservoirs 1440-1445. Introducing reagents from shuttle reservoirs permits variability in assay channel conditions through tailored reagent delivery.
  • the pumps and valves of FIG. 1 may be selectively and programmably actuated. In particular, by selectively actuating certain inlet valves 14, a user may release selected reagents stored in selected reagent reservoirs 12 and/or washing buffer stored in buffer reservoir 16.
  • the microfluidic system 1000 of FIG. 10 separates the assay functionality from the reagent delivery functionality. In situations where a particular assay needs to be performed repeatedly, it may be more inconvenient to use a larger cartridge repeatedly than several smaller ones. In one example, the microfluidic system 1000 may be used to run a number of identical assays in parallel.
  • reagent reservoirs 1035-1039 are provided with enough-reagents to run several assays, and the reagent chip 1010 supplies reagent to several chips as their respective assays are being performed.
  • ducting chip 1015 may be used to duct used reagents from assay chip 1005 into reservoir 1040 on reagent chip 1005. The used reagent in reservoir 1040 is then ported to waste reservoir 1035 for disposal. Waste reservoir 1035 may be utilized to store used reagents from one or more assay chips.
  • the microfluidic system 1000 operates by flowing fluids from reagent reservoirs 1035-1039 into reservoir 1040.
  • a fluid may be delivered from reservoir 1037 to reservoir 1040 via valve 1041 much like the process shown in FIG. 5c according to which a fluid from valve 550 is delivered toward valve 555 via valve 565.
  • actuating valve 1050 delivers fluid into channel 1072
  • actuating valve 1041 delivers fluid into channel 1073
  • actuating valve 1055 delivers fluid into reservoir 1040.
  • a fluid flows from reservoir 1040 into a reagent reservoir 1036 by a similar mechanism as that illustrated in FIG. 5c.
  • valves 1055, 1041 and 1062 all in open states, actuating valve 1055 pushes fluid into channel 1073, actuating valve 1041 pushes fluid into channel 1064, and actuating valve 1062 pushes fluid into reservoir 1035.
  • the insert 1 107 is first deposited into an assay channel 1 130 through an opening of the outlet reservoir 1 134 that is located at the end of the assay channel 1 130 and has a width substantially the same as the width of the assay channel 1 130.
  • the insert 1 107 is slid into the channel 1 130 until it spans a length 1 136 of the channel.
  • the insert is inserted into the assay channel 760 through channel void 730.
  • the channel void 730 is provided with an open top in which the insert is disposed.
  • FIG. 15a illustrates the insertion of an insert 1507, and in particular, shows an exemplary channel structure that facilitates the use of the insert 1507.
  • the channel 1520 is a stepped channel including a wide bottom portion 1522 and a narrow top portion 1524.
  • the insert 2017 is inserted into the stepped channel 1520 such that it generally overlies membrane 1510, as shown in FIG. 15c. More specifically, FIG.
  • the insert 1507 having an aperture 1515 and a membrane disk 1525.
  • the insert 1507 is situated in the channel 1520 such that the top surface of the membrane disk 1525 does not contact a top surface 1517 of the channel 1520, allowing for fluid in channel 1520 to flow around and contact the membrane disk 1525.
  • the insert is used to perform an assay similar in operation to a dot- ELISA method.
  • the dot-ELISA is a method, known in the art, for detecting the presence of a target analyte within samples.
  • Drawbacks of the conventional dot-ELISA process include difficulties with standardization. Many of the steps are often performed by hand in Petri dishes and the specification of these procedures is vague. Additionally, sample locations are hardly controllable. When sample is spotted on a membrane surface, the hydrophilicity of the material may lead to rapid sample spreading and diffusion. Larger sample amounts result in larger spotted areas. Moreover, since detection sensitivity is related to analyte density per unit area, this diffusion means that larger sample amounts do not necessarily result in lower detection limitation.
  • the present invention employs a similar assay processing, but allows for standardized and more efficient handling, treatment, and analysis.
  • samples are applied to a membrane disk 1 1 10 as shown in FIGS. 1 la-b.
  • the samples are air dried, and then the insert 1 105 is disposed in an assay channel of a microfluidic chip, similar to that of FIG. 1.
  • FIG. 1 the operation of the microfluidic chip 1 in performing assays will be discussed.
  • Various reagents are stored in reagent reservoirs 12 for conducting on- chip immunoassay.
  • the reagents include fluids that will be employed in a dot-ALIGA.
  • various reservoirs may include one or more washing buffers, antibody-conjugated immunogold or at least one component thereof, or sensitivity enhancing agents or at least one component thereof.
  • a buffer reservoir 16 may be used to store a washing buffer.
  • the buffer reservoir 16 may feature a substantially larger void volume than the individual reagent reservoir 12.
  • the reagents are released from their respective reservoirs 12 by activating respective inlet valves 14 and then distributing the reagents throughout the assay channels 40 using the activation of distribution valve 25 and channel pumps 44.
  • the washing buffer in buffer reservoir 16 may also be released into the assay channels 40 in a similar manner. The order and timing of release of the reagents and buffer from their respective reservoirs will correspond to the steps of the assay method used.
  • the reagents may correspond to the reagents described above with respect to the immunoassay process, and are released in accordance with the order and timing of the steps mentioned above.
  • the released reagents flow through the assay channels 40 and contact the inserts 70 therein.
  • a fluid flowing through the narrow portion 1524 of the stepped channel 1520 contacts and reacts with agents on the membrane disks 1525.
  • Apertures 1515 provide for the possibility of additional fluid contact along a bottom side of the membrane disks 1525.
  • the channels may be provided with materials with which the fluid reagents react, i.e., reagents may flow through assay channels with membrane disks disposed therein, thereby causing the occurrence of interactions between the reagents and the analysts on the membrane disks. It may be desirable to allow dynamic flow conditions or longer incubation times for the reactions via multiple passes of the reaction reagent through channels. This is achieved in part by the bidirectional pumping functionality.
  • the bidirectional channel pumps 44 are used to repeatedly shuttle a reagent back and forth between the shuttle reservoirs 17 and outlet reservoirs 48 along respective assay channels 40. This cycling action provides multiple passes for much greater efficiency at longer reaction time.
  • the outlet reservoirs 48 and shuttle reservoirs 17 are directly vented to the atmosphere, thereby allowing release of air from the channels 40 during the pumping cycles.
  • the void volume of each shuttle reservoir 17 and each outlet reservoir 48 are substantially larger than the void volume of each assay channel 40 so that reagents in the channels 40 may be stored in the reservoirs during the back and forth pumping action. After the assay operation, used reagents are then transported to the waste reservoir 18 for disposal.
  • the void volume of waste reservoir 18 is substantially larger than the void volume of the buffer reservoir 16 for storing all used reagents and washing buffer after an assay operation.
  • the hydrophobic nature of the insert 1 107 along with the inherent surface tension of the liquid sample allows a user to apply a larger amount of sample to a membrane disk 1 1 10 without diffusion or spreading of the sample to other disks 1110 nearby.
  • sample spotting onto the insert 1 107 is accomplished by placing the insert 1107 on an absorbent backing material such as a chromatograph paper with-membrane disk surface touching the paper.
  • the combination of the water-absorbent ability of the backing material and the sample-retaining ability of the insert 1 107 give rise to rapid sample absorption and concentration effects during spotting. Furthermore, the sample droplet diffusion area is substantially defined by the area of the membrane disk 1 110. This results in several advantages, such as after a larger amount of sample has dried on the membrane disk, a higher density of sample within the area defined by the membrane disk 1110 is achieved. In addition, since there is less risk of diffusion and contamination of sample material between different membrane disks 11 10, the membrane disks 11 10 may be placed closer together than the sample spots 2210 would be placed on the monolithic membrane 2205 as shown in FIG. 22, thus resulting in improved space efficiency for on-chip processing and potential reagent savings.
  • FIG. 16a illustrates a plurality of inserts 1705 in channels after an assay has been performed. As shown, certain membrane disks 1710a have been colored as positive results by an enzyme-substrate reaction, indicating the presence of a target analyte in a sample disposed on the corresponding membrane disk. Other membrane disks 1710b are substantially not colored, indicating no target analyte in a sample disposed on the corresponding membrane disk.
  • each insert 1705 includes eight membrane disks 1710. Each chip may include six or more assay channels, and therefore at least 48 samples may be assayed simultaneously.
  • an image analysis method for the automated processing of on-chip immunoassay results.
  • a microfluidic chip may be scanned utilizing, for example, a photo scanner or a digital camera to capture one or more colored images of the inserts after an assay operation.
  • FlG. 16a provides an exemplary image of an 8x6 sample- spotted array.
  • the scanned images may be stored in a handheld device for further off-line manipulation or sent to a remote computer for off-line image analysis.
  • Image analysis software may then be used to analyze the color intensities of the membrane disks from the captured color images. The intensity of each membrane disk 1710 is subsequently digitized into pixels with a numerical value assigned to each pixel.
  • each membrane disk 1710 in a sample array is uniquely identifiable by a combination of a barcode embedded in the chip and a set of coordinates specifying the channel and insert positions at which a membrane disk is located. For example, as shown in FIG.
  • a membrane disk 1710c on the upper-left corner of a chip that is bar- coded as CHIP-0001 may be labeled as CHIP-0001-Al, where Al indicates a combination of the column 1712 and row 1714 positions where the disk 1710c lies.
  • a protocol is provided for interpreting a color intensity value 1716 for identifying the presence of a target analyte in a sample disposed on the corresponding membrane disk 1710.
  • a threshold value is computed using negative control disks such that a color intensity value 1716 is interpreted as having a positive result for target analyte if the color intensity value is above the threshold value.
  • FIG. 17 provides an illustration for determining the presence of a target analyte in eight exemplary samples. These samples are disposed on membrane disks 1814 and correspond to computed color intensity values 1812.
  • the threshold value 1810 in this particular embodiment is 26.8 by arithmetically averaging Cl, Fl, B2, E2, H2, C3, F3, B5, E5 and H5 as shown in FIG. 16b .
  • the membrane disks 1814 in positions A, B, D, E, G, H are identified as having coated with the target analyte-containing solution.
  • the samples and target analytes for the assay may be any samples and targets suitable for use with immunoassay processes.
  • the samples may include control samples and experimental samples. Experimental samples are generally taken from a subject with a condition of interest, and control samples generally mimic the subject but exclude the analyst of interest. Typically, experimental samples are taken from a potentially diseased patient.
  • a subject may be, for example, a human, animal or plant.
  • samples can include bodily fluids, discharges or effluents such as blood, urine, saliva, fecal matter, sputum, nasal discharges, tears, etc.
  • FIG. 18 shows a complete system including an assay chip 1905, a cartridge 1910, a controller 1915, and a computer 1920.
  • the controller 1915 allows for automated control of the various pump and valve structures of the chip 1905.
  • the chip 1905 includes pneumatic drivers 1920 (not shown) positioned to be substantially aligned with the pump and valve structures of the chip 1905. Positive or negative pneumatic pressure is applied via the drivers 1920 in accordance with input signals provided through input wires 1925.
  • the computer 1920 may provide a user interface for controlling the controller 1915. A user may provide inputs specifying requirements on a particular assay run using a graphical user input provided by the computer 1920.
  • the computer is electrically connected to the controller 1915 and provides signals to the controller 1915 so it acts in accordance with the user inputs.
  • FIG. 19 illustrates an embodiment with an assay chip 2005 ducted to a separate reagent chip 2010 on a programmable controller 2015.
  • the controller 2015 includes a group of pneumatic solenoid valves. Each of the pneumatic signals from the solenoid valves is routed through the chip to one or a series of microfluidic valves on a specific chip layout. For example, in one embodiment there is an individual solenoid valve connected to each of the corresponding reagent reservoirs 12 of FIG. 1, but all six of the channel pumps 44 are connected in parallel to a set of four solenoid valves so they may act together. There is a solenoid drive board in the controller 2015 that takes the signals from the computer and turns on the appropriate solenoid valve to actuate the required microfluidic valve.
  • the microprocessor on the control board includes a memory which may store the sequence and thus an assay may be run independently of external computer control.
  • the microfluidic chip generally includes a top substrate 7, a bottom substrate 6, and a membrane 8 disposed therebetween.
  • the microfeatures e.g., pumps, valves, or reservoirs
  • the top substrate 7 and the membrane 8 are laminated together, and similarly the membrane 8 and the bottom substrate 6 are laminated together.
  • U.S. Patent Application Publication No. 2006/0078470 Al application Serial No. 10/964,216, entitled “Laminated microfluidic structures and method for making” by Zhou et al. discloses laminated, polymeric microfluidic structures and methods for making such structures. This publication is incorporated herein by reference in its entirety.
  • these layers are laminated by: 1) using a weak solvent bonding agent, and 2) laminating the layers under mild conditions, such as under low heat or low pressure. This is beneficial at least in part because this lamination method reduces or eliminates damage to the microfeatures during the lamination process. More particularly, in an exemplary use, the weak solvent bonding agent is applied to one or both surfaces to be adhered, and then mild pressure (e.g., from moderate heat or moderate physical pressure pressing the surfaces together) adheres the surfaces. [0156] According to an aspect, the weak solvent bonding agent may be chemically defined as:
  • the weak solvent may have a chemical formula of:
  • the weak solvent may have a chemical formula of:
  • the weak solvent bonding agent is acetonitrile.
  • Acetonitrile is a versatile solvent that is widely used in analytical chemistry and other applications. It is 100% miscible with water and exhibits excellent optical properties.
  • the ability of acetonitrile to have little or no effect on polymeric surfaces under ambient conditions but adhere to surfaces under moderate pressure makes it highly suitable for laminating polymeric materials such as polystyrene, polycarbonate, acrylic and other linear polymers. For example, microstructures disposed on a polystyrene substrate that was treated with acetonitrile at room temperature for at least several minutes did not exhibit any noticeable feature damage.
  • acetonitrile-based lamination allows substrate alignment for structures containing multi-component layers or fluid networks constructed utilizing both a cover plate and a base plate.
  • acetonitrile at room temperature may gently soften the surface.
  • an operator may slide the two surfaces against each other to adjust their alignment. After aligning the surfaces, the operator may then apply pressure to the surfaces to laminate them together.
  • the virus of interest is an influenza virus.
  • the influenza virus is an avian influenza virus (AlV).
  • a method for detecting a virus of interest in a field or clinical sample comprising the steps of: obtaining the field or clinical sample suspected of containing the virus of interest; providing an insert for performing a dot-ALIGA; applying to the insert the field or clinical sample suspected of containing the virus of interest; providing a microfiuidic device comprising a channel disposed therein; inter-fitting the insert within the channel; and performing the dot-ALIGA in the microfiuidic device to detect an antigen of the virus of interest, wherein the dot-ALIGA comprises: flowing a first fluid material through the channel to contact the insert therein, wherein the first fluid material comprises anti-virus antibody-labeled colloidal gold, wherein the anti-virus antibody is an antibody directed against the virus of interest, for a time sufficient to allow the anti-virus antibody-labeled colloidal gold and the antigen of the virus of interest in the field or clinical sample suspected of containing the virus of interest to bind together to form an antigen- bound anti
  • antibody-conjugated gold nanoparticles are used in the sensitivity-enhanced dot-ALIGA as virus-specific immunoassay binding agents.
  • Immunogold reagents comprising antibody-conjugated (antibody-labeled) gold nanoparticles for use in dot-ALIGA can be made using standard methods known in the art.
  • the virus detection limitation of the sensitivity-enhanced dot-ALIGA can be comparable to a conventional microtiter plate-based ELISA and about 2 s times more sensitive than a hemagglutination assay.
  • the dot-ALIGA is a monoclonal antibody-based dot-ALIGA and wherein the anti-virus antibody is an anti-virus monoclonal antibody (mAb).
  • mAb anti-virus monoclonal antibody
  • Any polyclonal or monoclonal anti-virus antibody known in the art can be used, including commercially available antibodies and antibodies produced by methods well-known in the art.
  • the components of the fluid materials can be introduced separately or made to flow through the channel in separate steps, and/or drawn away from the insert in separate steps.
  • separate components of the first fluid material comprising anti-vims antibody-labeled colloidal gold can be introduced separately or made to flow through the channel in separate steps, and/or drawn away from the insert in separate steps.
  • separate components of the second fluid material comprising the sensitivity enhancement reagent can be introduced separately or made to flow through the channel in separate steps, and/or drawn away from the insert in separate steps.
  • a solution of hydroxylamine chloride (or Z,(+)-ascorbic acid) and a solution of chloroauric acid can be introduced separately or made to flow through the channel in separate steps, and/or drawn away from the insert in separate steps.
  • the components of fluid materials can be stored in separate reservoirs or mixed and stored in one reservoir.
  • the method additionally comprises, before the step of flowing the first fluid material through the channel, the steps of: flowing a fluid comprising a blocking agent through the channel to contact the insert therein for a time sufficient to block non-specific binding sites on the insert; and drawing the fluid comprising the blocking agent away from the insert.
  • Any blocking agent known in the art can be use, e.g., bovine serum albumin (BSA), nonfat milk powder, gelatin or casein.
  • the method additionally comprises, before the step of detecting an interaction on the insert, the steps of: flowing a fluid comprising a washing reagent or buffer through the channel to contact the insert therein for a time sufficient to wash the insert; and drawing the washing reagent or buffer away from the insert.
  • the washing reagent or buffer can be any suitable standard buffer known in the art, e.g., PBS or Tris buffer.
  • the virus of interest is an influenza virus.
  • influenza virus is selected from the group consisting of avian influenza virus, influenza A virus, influenza B virus, influenza C virus, canine influenza virus, feline influenza virus, equine influenza virus and swine flu virus.
  • influenza virus is avian influenza virus (AIV).
  • the insert can comprise any suitable membrane known in the art such as a nitrocellulose (NC) membrane.
  • the sensitivity enhancement reagent can comprise hydroxylamine chloride and chloroauric acid.
  • the concentration of hydroxylamine chloride can be in the range of 0.001 - 0.01 mM, 0.01 - 0.1 mM, 0.1 - 1.0 mM, 1.0 - 10.0 mM, 10.0 - 100.0 mM, or 100.0 mM - 1.0 M.
  • the concentration of chloroauric acid can be in the range of 0.01 - 0.1 %, 0.1 - 1.0%. 1.0 - 10.0% or 10.0 - 20.0%.
  • the sensitivity enhancement reagent comprises 0.1 - 1.0 mM hydroxylamine chloride and 1 - 10% chloroauric acid.
  • the sensitivity enhancement reagent comprises 1.0 mM hydroxylamine chloride and 1-5% chloroauric acid.
  • the sensitivity enhancement reagent comprises L(+)-ascorbic acid and chloroauric acid.
  • the concentration of L(+)-ascorbic acid can be in the range of 0.01 - 0.1%, 0.1 - 1.0%, 1.0 - 10.0% or 10.0 - 20.0%.
  • the concentration of chloroauric acid can be in the range of 0.01 - 0.1%, 0.1 - 1.0%. 1.0 - 10.0% or 10.0 - 20.0%.
  • the sensitivity enhancement reagent comprises 0.1 - 1.0%
  • the sensitivity enhancement reagent comprises 0.15% £(+)- ascorbic acid and 1 -5% chloroauric acid.
  • a sensitivity enhancement reagent can be used as a single (mixed) reagent or mixture, or at least one component of the enhancement reagent can be applied or used separately in the assay.
  • the sensitivity enhancement reagent comprises hydroxylamine chloride and chloroauric acid
  • separate solutions of hydroxylamine chloride and of chloroauric acid can be used.
  • the detecting step detects the presence of the antigen-bound anti-virus antibody-labeled colloidal gold conjugate in the range of 0.015 - 0.02 HAU.
  • flowing the first fluid material or the second fluid material comprises actuating a distribution valve to flow a reagent from a reagent reservoir to a plurality of outlet reservoirs.
  • flowing the first fluid material or the second fluid material comprises repeatedly shuttling the first fluid material or the second fluid material in a first direction towards a first reservoir connected to the channel and in a second direction towards a second reservoir connected to the channel, wherein a distribution valve coupled to the channel substantially confines the fluid material in the channel when the distribution valve is in a closed state.
  • drawing the first fluid material or the second fluid material away from the insert comprises flowing the first fluid material or the second fluid material in at least one of a first direction towards a first reservoir connected to the channel and a second direction towards a second reservoir connected to the channel.
  • the method further comprising transporting waste from the channel to a waste reservoir connected to the channel.
  • detecting the interaction comprises visualization of color intensity, fluorescence intensity or chemiluminescence intensity.
  • detecting the interaction comprises generating an intensity value corresponding to at least one sample of the insert.
  • the intensity value is selected from the group consisting of color intensity value, fluorescence intensity value and chemiluminescence intensity value.
  • generating the color intensity value comprises: digitizing a color (or fluorescence or chemiluminescence image) corresponding to the sample to generate a plurality of pixels: providing a plurality of numerical values for respective ones of the plurality of pixels; and averaging the plurality of numerical values to provide the color (or fluorescence or chemiluminescence) intensity value.
  • the method further comprises computing a threshold value and comparing the color (or fluorescence or chemiluminescence) intensity value to the threshold value to detect the interaction.
  • the method further comprises storing at least one of the color (or fluorescence or chemiluminescence) intensity value and the threshold value in a database.
  • the threshold value is computed using at least one negative control sample.
  • a method for detecting an avian influenza virus (AIV) in a field or clinical sample comprising the steps of: obtaining the field or clinical sample suspected of containing AIV; providing an insert for performing a monoclonal antibody-based dot-ALIGA; applying to the insert the field or clinical sample suspected of containing the AIV; providing a microfluidic device comprising a channel disposed therein; inter-fitting the insert within the channel; and performing the monoclonal antibody-based dot-ALIGA in the microfluidic device to detect an AIV antigen, wherein the monoclonal antibody-based dot-ALIGA comprises: flowing a first fluid material through the channel to contact the insert therein, wherein the first fluid material comprises anti-AIV monoclonal antibody-labeled colloidal gold, for a time sufficient to allow the anti-AIV monoclonal antibody-labeled colloidal gold and the AIV antigen in the field or clinical sample suspected of containing the AIV to bind together to form an AIV antigen
  • An apparatus for detecting a virus of interest in a field or clinical sample.
  • the apparatus can comprise a microfluidic device such as the one described hereinabove.
  • the microfluidic device comprises a channel disposed therein.
  • the apparatus further comprises an insert for performing a dot- ALIGA, wherein the insert is capable of being inter-fitted within the channel and wherein the dot-ALIGA comprises: flowing a first fluid material through the channel to contact the insert therein, wherein the first fluid material comprises anti-virus antibody-labeled colloidal gold, wherein the anti-virus antibody is an antibody directed against the virus of interest, for a time sufficient to allow the anti-virus antibody-labeled colloidal gold and the antigen of the virus of interest in the field or clinical sample suspected of containing the virus of interest to bind together to form an antigen- bound anti-virus antibody-labeled colloidal gold conjugate; drawing the first fluid material away from the insert; flowing a second fluid material through the channel to contact the insert therein, wherein the second fluid material
  • the dot-ALIGA is a monoclonal antibody-based dot-ALIGA.
  • the anti-virus antibody is an anti-virus monoclonal antibody
  • the virus of interest is an influenza virus.
  • influenza virus is selected from the group consisting of an avian influenza virus, an influenza A virus, an influenza B virus, an influenza C virus, a canine influenza virus, a feline influenza virus, an equine influenza virus and a swine flu virus.
  • influenza virus is an avian influenza virus (AIV).
  • kits for detecting a virus of interest in a field or clinical sample comprises in one or more containers: a microfluidic device, wherein the microfluidic device comprises a channel disposed therein; and an insert for performing a dot-ALIGA, wherein the insert is capable of being inter- fitted within the channel and wherein the dot-ALIGA comprises: flowing a first fluid material through the channel to contact the insert therein, wherein the first fluid material comprises anti-virus antibody-labeled colloidal gold, wherein the anti-virus antibody is an antibody directed against the virus of interest, for a time sufficient to allow the anti-virus antibody-labeled colloidal gold and the antigen of the virus of interest in the field or clinical sample suspected of containing the virus of interest to bind together to form an antigen- bound anti-virus antibody-labeled colloidal gold conjugate; drawing the first fluid material away from the insert; flowing a second fluid material through the channel to contact the insert therein, wherein the second fluid material comprises a
  • the kit additionally comprises in one or more containers the second fluid material comprising the sensitivity enhancement reagent or at least one component thereof.
  • This example demonstrates the enhanced detection of AIV by employing a sensitivity enhancement reagent, a solution of 0.15% Z,(+)-ascorbic acid and 1% chloroauric acid, in a dot-
  • the reaction product in the supernatant was precipitated by centrifugation at 13000 rpm, 4 0 C for 30 min and was re-suspended in 0.01 M TBS (Tris buffered saline, pH 8.2 with 1% BSA). This process was repeated once to completely remove free proteins in solution.
  • the gold-conjugated mAb particle was re-suspended in 10 ml TBS (pH 8.2, 0.02% Na 3 N, 1% Sucralose, 1% BSA). and stored at 4°C until use.
  • each sampling spot was treated with a sensitivity enhancement reagent, a solution of 0.15% Z.(+)-ascorbic acid and 1% chloroauric acid, at room temperature for a few minutes and stopped by washing the strip in distilled water.
  • FIG. 20 shows that the two-fold serial dilution of reference virus was detected using the gold-conjugated anti- AIV antibody.
  • the top row was treated with the sensitivity enhancement reagent and a spot can clearly be seen as low as 2 '16 HA titer.
  • the bottom row was not treated with sensitivity enhancement reagent.
  • Example 2 Sensitivity enhanced dot-ALIGA for detection of AIV
  • This example demonstrates the enhanced detection of AIV by employing a solution of 1.0 mM hydroxylamine chloride and 5% chloroauric acid as a sensitivity enhancement reagent in an indirect dot-ALIGA.
  • Colloidal gold preparation and monoclonal antibody labeling Colloidal gold was prepared as described above in Example 1. The pH value of the colloidal gold was adjusted to 8.5-9.2 by dropwise addition of 1% K 2 CO 3 . Monoclonal antibody against avian influenza H9N2 subtype virus was centrifuged at 10,00Og for Ih at 4 0 C, the supernatant was removed and the rnAb concentration was adjusted to 1 mg/ml with phosphate buffer saline (PBS, 8.0 g of NaCl, 0.2 g KH 2 PO 4 , 1.28 g Na 2 HPO 4 -H 2 O, 0.2g KCl, 1000 ml distilled H 2 O, pH 7.4). The final anti-AIV mAb-labeled colloidal gold solution contained 10 ⁇ g/ml mAb and 1% BSA.
  • PBS phosphate buffer saline
  • the shuttle reservoirs 617 and outlet reservoirs 61 1 were then attached to the six channel assay chip 615.
  • the on-chip dot-ALIGA assay was carried by sequentially pumping the following solutions through each processing channel:
  • reagent or washing buffer were shuttled between the shuttle reservoir 617 and outlet reservoir 611. All waste agents and washing buffer were stored in the waste reservoir 618.
  • FIG. 22 The results of the on-chip sensitivity enhanced dot-ALIGA are shown in FIG. 22.
  • the numbers in the top row (HAU/SE) at membrane disk positions 1, 3, 5 and 8 indicate that these disks are spotted with 1.0 ⁇ l of HA unit of two-fold diluted reference strain of avian influenza virus subtype H9N2 while the numbers in the bottom row (SEC) at the membrane disk positions 2, 4, 6, 7 indicate that these disks are spotted with 1.0 ⁇ l of two-fold diluted negative controls.
  • the results show that AIV is detectable in samples at a level of 0.016 HA unit. This is about ten times more sensitive than the conventional, widely used colloidal gold-based lateral flow assay.
  • step 4 sensitivity enhancement agent, rather than 1.0 mM hydroxylamine chloride. All steps were performed as described above except that in step 4, a solution of 0.15% £(+)-ascorbic acid and
  • AIV is detectable in samples at a level of about 0.016 HA unit.

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Abstract

L'invention concerne des systèmes et des procédés pour la détection améliorée d'un virus d'intérêt utilisant un immunoessai à l'or lié à un anticorps par point amélioré en sensibilité (ALIGA par point) effectué sur une puce microfluidique. Le virus peut être un virus de la grippe, par exemple, un virus de la grippe aviaire (AIV). La puce microfluidique peut avoir une pluralité de microparticularités interconnectées pour fournir un système de transport de fluide pouvant être configuré pour traiter les réactifs pour l'ALIGA par point amélioré en sensibilité, fournissant un format d'essai qui a une sensibilité améliorée et une détection améliorée de AIV au moins aussi faible que 0,02 HAU. La limite de détection de virus de l'ALIGA par point amélioré en sensibilité est comparable à un ELISA à base de plaques de micro-titration classique et est d'environ 25 fois plus sensible qu'un ELISA à point classique, rivalisant ainsi avec les limites de procédés de type ELISA colorimétrique à base de plaques de micro-titration.
PCT/US2008/054579 2007-02-23 2008-02-21 Immunoessai à l'or lié à un anticorps par point amélioré en sensibilité pour la détection de virus WO2008103824A1 (fr)

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CN102520185A (zh) * 2011-11-18 2012-06-27 河南省农业科学院 甘薯上马铃薯y病毒属病毒的血清学检测试剂盒及其检测方法
CN104198713A (zh) * 2014-09-05 2014-12-10 云南省农业科学院生物技术与种质资源研究所 一种快速检测蓟马体内番茄斑萎病毒的方法
US9163279B2 (en) 2009-11-02 2015-10-20 The Secretary Of State For Environment, Food & Rural Affairs, Acting Through The Animal Health And Veterinary Laboratories Agency Device and apparatus
CN105445459A (zh) * 2015-12-01 2016-03-30 浙江普康生物技术股份有限公司 一种检测h7n9亚型禽流感病毒神经氨酸酶特异性抗体的多肽-酶联免疫吸附试剂盒
CN105458288A (zh) * 2015-12-02 2016-04-06 青岛大学 一种纳米金颗粒的制备方法
CN105911277A (zh) * 2016-04-25 2016-08-31 成都盛泰尔生物医药科技有限公司 一种动物疫病抗体类病毒胶体金定量检测系统及制备方法
CN106153888A (zh) * 2015-03-11 2016-11-23 宁波大学 流体驱动用构件能够迅速脱除的亚型猪流感检测用装置
CN106153898A (zh) * 2015-03-11 2016-11-23 宁波大学 附加的驱动液流用构件易于卸除的亚型猪流感检测装置
US9777305B2 (en) 2010-06-23 2017-10-03 Iti Scotland Limited Method for the assembly of a polynucleic acid sequence
EP3325158A4 (fr) * 2015-07-24 2018-12-12 HJ Science & Technology, Inc. Systèmes microfluidiques reconfigurables et dosages immunologiques multiplexés échelonnables
CN111686826A (zh) * 2019-03-15 2020-09-22 国家纳米科学中心 分层结构的微流控芯片及其应用
US11319567B2 (en) 2014-05-27 2022-05-03 Academia Sinica Fucosidase from bacteroides and methods using the same
US11376589B2 (en) 2018-04-30 2022-07-05 Protein Fluidics, Inc. Valveless fluidic switching flowchip and uses thereof
US11884739B2 (en) 2014-05-27 2024-01-30 Academia Sinica Anti-CD20 glycoantibodies and uses thereof
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US9163279B2 (en) 2009-11-02 2015-10-20 The Secretary Of State For Environment, Food & Rural Affairs, Acting Through The Animal Health And Veterinary Laboratories Agency Device and apparatus
US9777305B2 (en) 2010-06-23 2017-10-03 Iti Scotland Limited Method for the assembly of a polynucleic acid sequence
CN102520185A (zh) * 2011-11-18 2012-06-27 河南省农业科学院 甘薯上马铃薯y病毒属病毒的血清学检测试剂盒及其检测方法
US11884739B2 (en) 2014-05-27 2024-01-30 Academia Sinica Anti-CD20 glycoantibodies and uses thereof
US11319567B2 (en) 2014-05-27 2022-05-03 Academia Sinica Fucosidase from bacteroides and methods using the same
CN104198713A (zh) * 2014-09-05 2014-12-10 云南省农业科学院生物技术与种质资源研究所 一种快速检测蓟马体内番茄斑萎病毒的方法
CN104198713B (zh) * 2014-09-05 2016-02-10 云南省农业科学院生物技术与种质资源研究所 一种快速检测蓟马体内番茄斑萎病毒的方法
CN106153888A (zh) * 2015-03-11 2016-11-23 宁波大学 流体驱动用构件能够迅速脱除的亚型猪流感检测用装置
CN106153898A (zh) * 2015-03-11 2016-11-23 宁波大学 附加的驱动液流用构件易于卸除的亚型猪流感检测装置
EP3325158A4 (fr) * 2015-07-24 2018-12-12 HJ Science & Technology, Inc. Systèmes microfluidiques reconfigurables et dosages immunologiques multiplexés échelonnables
CN105445459A (zh) * 2015-12-01 2016-03-30 浙江普康生物技术股份有限公司 一种检测h7n9亚型禽流感病毒神经氨酸酶特异性抗体的多肽-酶联免疫吸附试剂盒
CN105458288B (zh) * 2015-12-02 2018-06-12 青岛大学 一种纳米金颗粒的制备方法
CN105458288A (zh) * 2015-12-02 2016-04-06 青岛大学 一种纳米金颗粒的制备方法
CN105911277A (zh) * 2016-04-25 2016-08-31 成都盛泰尔生物医药科技有限公司 一种动物疫病抗体类病毒胶体金定量检测系统及制备方法
US11376589B2 (en) 2018-04-30 2022-07-05 Protein Fluidics, Inc. Valveless fluidic switching flowchip and uses thereof
US11839873B2 (en) 2018-04-30 2023-12-12 Protein Fluidics, Inc. Valveless fluidic switching flowchip and uses thereof
CN111686826A (zh) * 2019-03-15 2020-09-22 国家纳米科学中心 分层结构的微流控芯片及其应用
CN111686826B (zh) * 2019-03-15 2023-05-23 国家纳米科学中心 分层结构的微流控芯片及其应用
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