WO2013142847A1 - Acide nucléique confiné dans une membrane pdms et éléments de capture à tube de longueur micrométrique fonctionnalisés avec des anticorps/antigènes, et systèmes les utilisant - Google Patents

Acide nucléique confiné dans une membrane pdms et éléments de capture à tube de longueur micrométrique fonctionnalisés avec des anticorps/antigènes, et systèmes les utilisant Download PDF

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Publication number
WO2013142847A1
WO2013142847A1 PCT/US2013/033610 US2013033610W WO2013142847A1 WO 2013142847 A1 WO2013142847 A1 WO 2013142847A1 US 2013033610 W US2013033610 W US 2013033610W WO 2013142847 A1 WO2013142847 A1 WO 2013142847A1
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WO
WIPO (PCT)
Prior art keywords
micro
channel
microfluidic
elements
microfluidic device
Prior art date
Application number
PCT/US2013/033610
Other languages
English (en)
Inventor
Martin A. Putnam
John H. Leamon
Jeffrey T. BRANCIFORTE
Charles O. STANWOOD
Original Assignee
Cyvek, 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
Priority claimed from US13/427,857 external-priority patent/US9216412B2/en
Application filed by Cyvek, Inc filed Critical Cyvek, Inc
Publication of WO2013142847A1 publication Critical patent/WO2013142847A1/fr
Priority to US14/479,287 priority Critical patent/US9855735B2/en
Priority to US14/479,283 priority patent/US9500645B2/en
Priority to US14/479,291 priority patent/US9546932B2/en
Priority to US14/479,285 priority patent/US10065403B2/en
Priority to US14/479,284 priority patent/US10022696B2/en
Priority to US14/479,290 priority patent/US9651568B2/en
Priority to US14/479,288 priority patent/US9759718B2/en
Priority to US14/479,286 priority patent/US9700889B2/en
Priority to US15/105,297 priority patent/US10401463B2/en
Priority to US15/340,661 priority patent/US10220385B2/en
Priority to US15/477,902 priority patent/US10786800B2/en
Priority to US15/581,526 priority patent/US10076752B2/en
Priority to US15/638,526 priority patent/US10209250B2/en
Priority to US16/118,985 priority patent/US10414143B2/en
Priority to US16/570,127 priority patent/US11292237B2/en
Priority to US17/711,601 priority patent/US11938710B2/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • 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
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/527—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for microfluidic devices, e.g. used for lab-on-a-chip

Definitions

  • the invention concerns assays in microfluidic systems, including systems that employ portable microfluidic devices.
  • Some versions of microfluidic devices are in the form of microfluidic cartridges (cassettes) that are actuated and read by an associated apparatus such as a bench-top instrument that both conducts the assay protocol within the cartridge and reads the results.
  • the invention also concerns multiplex microfluidic assays in which multiple assays performed in a microfluidic system are read or scanned with epi-fluorescence.
  • the invention in particular relates to monitoring assays performed within microfluidic systems, to detecting assay results after microfluidic assays have been run (performed), and to determining the precise relative location of a microfluidic system to a precise detection system, for conducting monitoring or detection with precision.
  • the invention has broad aspects that are applicable to microfluidic assay systems, in general, and more specific aspects that concern the assays conducted within portable microfluidic cartridges, and particularly cartridges in which the relative position of the cartridge and a precise outside scanning system is not precisely determined. Particularly important applications of the invention concern microfluidic assay cartridges that are inserted into a multi-function apparatus that both causes the assay to be performed within the cartridge and the results detected.
  • the invention also particularly concerns capture devices and systems for capturing a substance, e.g. antigen, antibody or nucleic acid, that may be used immediately in an assay or may subsequently be the subject of study, assay or further processing.
  • a substance e.g. antigen, antibody or nucleic acid
  • Examples of failure modes for microfluidic assay systems relate to flows in microfluidic channels and to valves and pistons that control the flows according to a pre-determined assay protocol.
  • the failure modes occur with any microfluidic system, but can be of particular concern when the assay is performed within a microfluidic cartridge. Blockage of a microfluidic channel and inability of a valve to open or close are examples of failure. If a valve does not open, flow is prevented; if it does not close completely, valve leakage may occur at an inopportune time. There is also the possibility of a contaminant in the microfluidic channels.
  • microfluidic channels are very tiny (e.g., 100 - 200 microns cross section width and depth, only millimeters in length) flows are difficult to visualize.
  • the channels are so small that it is difficult for the human eye to observe the fact that liquid is not flowing where it is desired.
  • the assay reagents are typically transparent, compounding the difficulty of visual or optical observation.
  • the problems are especially acute when seeking highly accurate quantification in a microfluidic assay.
  • a given amount of immobilized capture agent be exposed to a given amount of various fluids to enable reactions over defined times so that results can be compared to a standard to enable the quantification.
  • Results need to be determined with an overall coefficient of variation of less than 10% (accuracy within 10%), preferably much less.
  • assays require consistent run-to-run performance.
  • concentration and the volume of the buffer or wash liquid, of secondary reagents such as antibodies, and of fluorescent dye all need to be the same from run to run if one is to compare the result to a standard calibrating curve precisely generated from previous calibrating runs.
  • This is particularly true in blood testing in which a patient human plasma or serum sample is measured for the presences or the quantity of specific health-related analytes, for instance, antibodies such as interleukins (a class of antibodies called cytokines), e.g., IL5 or IL6.
  • cytokines a class of antibodies called cytokines
  • DNA and other nucleic acid determinations that are desired in the drug development, medical and other fields, including public health, agriculture and food processing.
  • the result of an assay is typically measured by detecting an emanation, e.g., a fluorescence intensity, from a reaction site.
  • the emanation may come from a bead, a micro particle or an immobilized spot.
  • an immobilized glass nano-reactor GNR in the form of a small hollow tube or micro- length tube, of length no more than 1000 micron, typically less than 500 micron, with capture agent, e.g., antibody, immobilized on its inside surface.
  • the fluorescence intensity from the region of the capture agent is essentially all that is measured at the completion of many assays. That fluorescence intensity is compared to a calibration curve. From the calibration curve the unknown
  • concentration of the analyte is determined.
  • concentration of the analyte is determined.
  • the invention features a microfluidic device comprising a microfluidic channel network sealed on one side by a membrane sheet, the sheet having PDMS defining at least the surface sealing the channel, the membrane sheet on its opposite side sealing one side of a pneumatic channel, the pneumatic channel arranged to enable pneumatic deflection of a deflectable portion of the membrane sheet into contact with an opposed surface to control flow in a channel of the network, the membrane sheet confining in a channel of the network at least one micro-particle functionalized with a capture agent that has been inserted into that channel.
  • Implementations of this feature may have one or more of the following features.
  • the micro-particle is functionalized with antibody, antigen or nucleic acid. At least two micro-particles are inserted and confined by the membrane sheet in a channel, one being functionalized with antibody or antigen and the other being functionalized with nucleic acid.
  • the PDMS surface of the membrane sheet is permanently bonded to a mating surface of structure defining the microfluidic channel network.
  • the PDMS surface of the membrane sheet is permanently bonded to mating surfaces of structure defining the fluidic channel network as the result of surface activation and contact for a cure period with the mating surfaces, the PDMS surface of the deflectable portion of the membrane sheet, having been surface-activated, being in a treated state as the result of a series of make and break contacts with its opposed surface during the cure period.
  • the opposed surface comprises a surface that has been surface-activated.
  • the opposed surface is comprised of PDMS, silicon-based rigid material, or a bi- functional layer adhered to an underlying material.
  • the intermediate bi-functional layer comprises organo-functional silane or an oxide layer.
  • the PDMS surface of the membrane sheet is removably bonded to a mating surface of structure defining the fluidic channel network in manner enabling access to and removal of the micro-particle following exposure of the capture agent of the micro-particle to fluid possibly containing the target of the capture agent.
  • the micro-particle is a micro-length tube within which capture agent is immobilized.
  • the micro-length tube is a glass nano reactor.
  • the micro-particle, micro-length tube or glass nano reactor is formed by dicing or cutting a running strand or tube of material, batch functionalized with respective capture agent, and inserted in the micro-fluidic channel.
  • the micro-particles, micro-length tubes or glass nano reactor is placed by a pick-and-place instrument.
  • the micro-particle is a micro-length tube or glass nano reactor, the device adapted to perform an assay in which the micro-length tube or GNR has transparent walls, and a region of the capture agent to be read for fluorescence resides only on the inside surface of the micro-length tube or GNR.
  • Structure defining side walls of the pneumatic channel are defined by a thickness of double sided pressure sensitive adhesive (PSA) sheet adhered on one side to the membrane sheet.
  • PSA pressure sensitive adhesive
  • the membrane sheet consists of PDMS.
  • the membrane is a composite sheet, comprising a film or coating of PDMS and a thin flexible sheet of another material.
  • the flexible sheet of material is stiff relative to the PDMS, and has stress relief channels providing increased ability of the composite sheet to be elastically deflected.
  • the device is constructed to perform an assay on the target captured by the capture agent.
  • a micro-fluidic channel contains at least two classes of micro-particles, micro-length tubes, or GNRs, the two classes being functionalized with different capture agents specific to different targets.
  • the capture agent for one class is nucleic acid and for the other class is antigen or antibody.
  • a first class of capture agent is functionalized to capture a target in a sample and a second class is functionalized to capture a tracer added to the target for control purposes. There are more members in the first class than the second class. Capture of a target is effected by back-and forth flows of successive segments of the sample.
  • Each slug has a length of the order of 100 times the length of a micro-particle, micro-length tube element, or GNR, and a multiplicity of slugs are subjected to multiple back and forth movements to carry out the intended phase of the procedure.
  • a channel contains a series of spaced-apart micro-length tube elements or glass nano reactors immobilizing a nucleic acid capture agent to their interior surfaces for passive capture or assay (i.e. without amplification) of a native nucleic acid (or more than one) in a sample.
  • Another feature of the invention is a microfluidic device having a micro fluidic channel containing at least two micro-length tubes or glass nano reactors, one functionalized with Nucleic acid and another with antibody or antigen.
  • Another feature of the invention is a microfluidic device having a microfluidic channel containing at least one micro-length tubes or glass nano reactor functionalized to capture nucleic acid, the device constructed to enable recovery of the nucleic acid captured by the device.
  • each of the features may be implemented in the form of a portable cartridge, with a microfluidic network devoted to a single sample, or multiple microfluidic networks having respectively different sample sources.
  • the device may be constructed to conduct multiple independent assays on the same sample, with or without a tracer.
  • the invention features a microfluidicdevice that defines a micro-fluidic flow channel having a flow axis, in which a series of discrete, axially- spaced apart, hollow flow elements ) are secured in fixed position, each flow element having at least one axially-extending flow passage through its interior, assay capture agent fixed to the interior surface of the elements for capture of a a target in liquid flowing through the interior of the flow elements, the exterior axially-extending surfaces of the flow elements are free of active capture agent, while at least part of the interior surfaces carry deposits of active capture agent exposed to flow through the elements, and the channel in which the elements are secured is closed by a membrane having a PDMS surface, bonded to the material forming the side walls of the channel, other portions of the same membrane forming a pneumatically deflectable valve member for control of fluid through the channel.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following.
  • the hollow flow elements are micro-length tubes
  • At least one flow path may extend along the axially-extending exterior of each element. At least one flow path may have aggregate by-pass flow cross-section area (A 2 ) at least as great as the aggregate flow cross-section (Ai) through the interior of the element.
  • the hollow flow elements may have interior and exterior surfaces extending in parallel in the direction of the channel axis, and end surfaces may extend transversely to the axis, the surfaces of the elements may be exposed to liquid in the channel, and the device may be constructed to enable light to be transmitted into and out of the elements transversely to the flow axis for excitation and reading of fluorescence from captured analyte.
  • the end surfaces of each element may be free of active capture agent.
  • At least one by -pass flow path may extend along the axially- extending exterior of each element, in which the by -pass flow cross-section A2 is at least 1.5 times as large as the aggregate flow cross-section Ai through the hollow element.
  • a hollow flow element may comprise a micro-length tube element having a length L less than about 700 ⁇ . Length L may be about 250 ⁇ .
  • a hollow flow element may be of glass or glass-like substance and may defines an internal volume of the order of 1 nano liter, the element may constitute a glass nano reactor for assay reactions.
  • a hollow flow element may have interior flow cross section width between 75 + 50 ⁇ .
  • a hollow flow element may have interior and exterior concentric cylindrical surfaces, the interior surface may have diameter between about 75 + 50 ⁇ .
  • the ratio of exterior width (respecting claims 1-10) or diameter (respecting claim 1 1) of the hollow element to respectively the interior width or diameter of the element may be between about 1 and 4.
  • a hollow element may be of fused silica and of straight, cylindrical form having interior diameter of about 70 um, exterior diameter of about 125 um, and axial length of about 250 um, the end surfaces may be planar, lying at a substantial angle to the element axis.
  • the hollow element may be a segment of drawn tubing.
  • the assay device may have end surfaces lying at a substantial angle to the element axis as a result of cutting drawn tubing.
  • the flow channel may be defined by spaced apart, opposed sidewalls, the channel may have greater width than the width or diameter of a flow element fixed in it, the element may be in contact with one of the sidewalls such that by-pass flow cross-section area on the side of the element opposite the wall with which it makes contact is greater than on the side on which the element makes contact.
  • At least the side of the flow channel in contact with the flow element may be is defined by a material that has electrostatic attractive properties relative to the exterior surface of the element.
  • the flow channel may be defined at its bottom by a rigid base surface, preferably low fluorescent glass, and at its sides by opposed, cut surfaces of an elastomeric sheet.
  • Channel side walls may be comprised of polydimethylsiloxane (PDMS).
  • a flow channel may be defined by an open channel of depth slightly less than the corresponding dimension of the hollow element, the hollow element may reside in this open channel, and a transparent elastic closing member may be disposed over and may close the open side of the open channel, the closing member elastically bearing against the portion of the element lying outside the open channel to form the flow channel and simultaneously secure the element in its fixed position within the flow channel.
  • the elastic closing member may be a portion of an elastomeric sheet lying over multiple flow elements in the flow channel, securing them in their respective positions in the flow channel.
  • the assay device may comprise a rigid base plate having a planar surface, upon which may be secured one side of a parallel first sheet of elastomer in which a through, open-slot has been cut, the side surfaces bounding the slot and the corresponding exposed surface of the base plate forming the open channel, and the closing member may comprise a transparent second elastomeric sheet lying parallel against and attracted to the opposite side of the first sheet and the element protruding from the open channel in manner that locally deforms the second sheet against the element and may causes it to elastically press the element against the base plate, fixing it in position. Respective portions of the same sheet may lie against and secure each of a plurality of the elements in the flow channel.
  • the assay device may be associated with a positive-displacement pump arranged to introduce a segment of liquid sample, and may cause the sample to move back and forth to produce capture of analyte only in the interior surface of the element, and to repeat this action for successive segments of liquid sample.
  • At least end margins of the interior surface of an element may be free of active capture agent.
  • a section of the interior surface of an element, lying inwardly from the ends of the element, may be free of active capture agent.
  • Active capture agent on the interior surface of the element may be configured to define a code.
  • the code may be a bar code.
  • the active capture agent may be an antibody.
  • the active capture agent may be an antigen.
  • the active capture agent may be an oligomer.
  • the invention features for use in a micro-fluidic flow channel (44) having a flow axis, a transparent hollow flow element (32) adapted to be secured in fixed position, the flow element having at least one axially-extending flow passage through its interior, assay capture agent fixed to the interior surface of the element for capture of an analyte in liquid flowing through the interior of the flow element, the element constructed to enable light to be transmitted out of the element for reading of fluorescence from captured analyte, wherein the exterior axially- extending surface of the flow element is free of active capture agent, while at least part of the interior surface carries a deposit of active capture agent for exposure to flow through the element.
  • the invention features a micro-length tube element for use in an assay device comprising a hollow body with a through-passage for liquid flow, wherein active capture agent for a given analyte resides only on a portion of the interior surface of the hollow body for interaction with fluid sample; the exterior surfaces of the micro-length tube element being free of active capture agent, and un- reactive with the analyte in the sample.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • Margins of the interior surface of the element may be free of active capture agent and un-reactive with the analyte in the sample.
  • a length L may be less than about 700 ⁇ .
  • length L may be about 250 ⁇ .
  • the element may have interior flow cross section width between about 75 + 50 ⁇ .
  • the element may have interior and exterior concentric cylindrical surfaces, the interior surface may have diameter between about 75 + 50 ⁇ .
  • the ratio of exterior width or diameter of the hollow element to the interior width or diameter may be between about 1 and 4.
  • the element may be of fused silica and of straight cylindrical form having interior diameter of about 70 ⁇ , exterior diameter of about 125 ⁇ , and axial length about 250 ⁇ , the end surfaces may be planar, lying at a substantial angle to the tubular axis.
  • the element may be a segment of drawn tubing.
  • the element may have end surfaces lying at a substantial angle to the element axis as a result of cutting drawn tubing.
  • the capture agent on the interior surface of the element may be configured to define a code.
  • the code may be a bar code.
  • the active capture agent may be an antibody.
  • the active capture agent may be an antigen.
  • the active capture agent may be an oligomer.
  • the channel may be wider than the corresponding dimension of the hollow element, and a lateral motion of the placing instrument toward the side wall of the channel may be employed to bring the element into proximity of the wall of the channel to enable the close-field electrostatic attraction to attract the element from the placement instrument.
  • the microfluidic channel into which the element is placed may have at least one side wall formed of elastomeric material, the channel may have a width less than the corresponding dimension of the element, and the placing action may forces the element into the channel by force-fit until compression of the elastomeric material grips the element sufficiently to detach the element from the placing instrument and enable withdrawal of the instrument.
  • the element may be inserted into its microfluidic channel by pick-and-place motion effected by automated tweezer fingers engaging oppositely directed portions of the element.
  • the oppositely directed portions may be parallel planar surfaces.
  • the element may be inserted into its microfluidic channel by pick-and-place motion effected by automated vacuum pick up.
  • the vacuum pickup may be effected by a device which engages an outer cylindrical surface of the element.
  • a flexible sheet in places of substantial area may be joined by bonding to an opposed surface effectively to secure the position of a hollow flow element in its channel, and another portion or portions of the area of the sheet may perform a further function within the microfluidic device, including flow channel closure, providing flexible diaphragm for fluid-actuated valve or providing on-board pump diaphragm, preferably portions performing all three.
  • the flexible sheet may comprise elastomer, preferably PDMS.
  • the device may be constructed to conduct multiplex assays within a single portable assay cartridge (chip). At least some parts of the device may be joined by co-valent bonding of activated surfaces of bondable material, a contiguous portion of the same sheet fixing the position of a said detection element in its flow channel.
  • At least some parts of the device may be joined by co-valent bonding of activated surfaces of bondable material, a contiguous portion of the same sheet forming a flexible pump diaphragm. At least some parts of the device may be joined by co-valent bonding of activated surfaces of bondable material, a contiguous portion of the same sheet forming a flexible valve diaphragm.
  • the flexible valve diaphragm portion may engage a valve seat originally formed of surface-activated bondable material that has been subjected to a series of make-and break contacts that interrupt covalent bonding of the valve diaphragm portion with its opposed seat.
  • At least some parts of the device may be joined by co-valent bonding of activated surfaces of bondable material, and respective contiguous portions of the same sheet may seal an open side of a flow channel, may fix the position of a said detection element in its flow channel, may form a flexible pump diaphragm or form a flexible valve diaphragm, preferably respective portions of the sheet performing all of these functions.
  • the assay device may be formed by preparation of two
  • each may have a backing of relatively rigid material and an oppositely directed face suitable for bonding to a mating face of the other subassembly, followed by bonding the assemblies face-to-face that has the effect of capturing the hollow flow elements and closing the channels in which they have been placed.
  • the capture agent may be antibody for conducting ELISA.
  • the device may contain a means of providing a fluorophor label to captured analyte, and the detection elements may be exposed to a window transparent to outwardly proceeding fluorescent emission for detection.
  • the window may be transparent to exterior-generated stimulating light emission to enable epi-fluorescent detection.
  • the invention features a method of preparing hollow flow detection elements for an assay according to any of the preceding claims comprising batch coating hollow flow elements in solution, and drying, and thereafter picking and placing the elements in flow channels of a micro fluidic device, and preferably capturing the flow elements by bonding two opposed layers that capture the elements while sealing the flow channels.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • a method of preparing hollow flow elements for an assay may comprise batch coating the detection elements to coat the elements with capture agent, including eliminating or preventing the occurrence of active capture agent on outside surfaces of the hollow elements.
  • a suspension of the hollow elements in fluid with the capture agent may be aggressively agitated (preferably by vortexing) to impart disrupting shear forces to the exterior surface of the elements, the shear force may act to prevent binding of the capture agent to outside surface of the elements.
  • the exterior surface of the hollow elements may be in treated condition that prevents formation of a coating on the exterior surface.
  • a continuous tube may be formed by a drawing or extruding process, during which the exterior surface of the tube may be treated to prevent functionalization of its outer surface, followed by dicing the tube to produce micro-length hollow tubes, and functionalizing the hollow tubes by batch process.
  • Coated capture agent may be removed or rendered inactive by selective exposure to a laser elimination process that removes or de-activates capture agent from a surface of a coated element.
  • the elements may be suspended in a sugar-based stabilizing solution, and may be flowed upon a pick-up plate from which they are to be picked, including the step of wicking excess fluid from the plate with an absorbent wicking substance, without physical contact of the wicking substance with the elements.
  • the pick-up plate on which the elements are flowed may be a plate having grooved which the elements become lodged in alignment, and the wicking may be done by positioning the wicking material to communicate with the grooves at a location spaced from the elements. There may be a series of pockets formed along the grooves in which the elements discretely lodge.
  • the pickup plate may define a flat element-receiving surface, and the wicking is accomplished by contacting the puddle of solution at locations on opposite sides of the puddle.
  • the pickup plate may be contacted with a ring of absorbent material having an inside diameter larger that the perimeter of the collection of elements to be drained.
  • the elements may be stored on a pick-up plate coated with a sugar-based stabilizing solution, under controlled humidity conditions, of relative humidity of at least 50%.
  • the invention features a method of making an assay device comprising providing micro-elements in the form of micro-particles or micro-length tube detection elements and thereafter with an automated tool, picking and placing the micro-elements into open-sided microfluidic channels in a body.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: including bonding a layer to the body to close the open sides of the channels, enclosing the micro-elements and completing the respective portions of the microfluidic channels.
  • the layer comprises a PDMS membrane which is surface-activated
  • the body defines an opposing surface that is bondable by covalent bonds to the membrane layer, and the act of closing is performed by so bonding the membrane and the body.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the body has an opposed surface defined by PDMS that is surface- activated.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the picking and placing is performed by an automated vision-based picking-placing control system.
  • the open microfluidic channels may have widths greater than the micro-elements and an automated placement tool is caused to move laterally after entry of the elements into the respective channels, to enable the micro-elements to lodge against predetermined side- walls of the channels.
  • the open microfluidic channels may have widths greater than the micro-elements and the placing into the channels includes use of electrostatic attraction to disengage the micro-elements from the automated placement tool and attract them to side walls of the channels.
  • the microfluidic channels may be defined in an elastomeric channel- defining member, including immobilizing the micro-elements within the microfluidic - channels by applying a force to the micro-elements, wherein the force causes the elastomeric channel-defining member to resiliently deform and exert an immobilizing force on the micro-elements.
  • the micro-elements may be force- fit into microfluidic channels of narrower width.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: an automated placement tool, picking up the elements from grooves or pockets in a surface, and placing the elements into the microfluidic channels.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the predetermined channels may be located on a platform, wherein the position of the platform is computer-controlled in the X and Y directions and the placement tool does not move in those directions.
  • the placement tool comprises grippers, wherein the movement of the grippers is computer-controlled.
  • the placement tool comprises a vacuum pickup head and the movement of the pickup head is computer-controlled.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: picking up the micro-elements from a smooth surface; placing the micro-elements into the predetermined channel, wherein the predetermined channel comprises an elastomeric channel-defining layer; immobilizing the micro- elements within the micro-channel by applying a force to the micro-elements, wherein the force causes the elastomeric channel-defining layer to resiliently deform and exert an immobilizing force on the micro-elements.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: picking up the micro-elements from a smooth surface; placing the micro-elements into the predetermined channel, wherein the predetermined channel comprises an elastomeric channel-defining layer; and in conjuction with electrostatic attraction, immobilizing the micro-elements within the micro-channel against a side- wall of the predetermined channel by moving laterally within the channel to a point at which the micro-elements may be drawn to and attach to the sidewall.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: picking up the micro-elements from a smooth surface; placing the micro-elements into the predetermined channel, in which the predetermined channels may be located on a platform, wherein the position of the platform is computer- controlled in the X, Y and the placement tool moves in theta direction about a vertical axis to align with the orientation of an automated, visually identified micro-particle.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following forming: the micro-particles or micro-detection elements for an assay includes the step of coating the detection elements in bulk with capture agent before picking and placing the elements into microfluidic channels.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: batch coating the micro-detection elements with capture agent by mixing in solution, and drying before picking and placing the detection elements into the microfluidic channels.
  • micro-elements for an assay comprise micro-length hollow flow elements.
  • micro-length hollow flow elements comprise glass nano reactors.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: preparing micro-length hollow flow elements for an assay comprising batch coating the elements to coat the elements with capture agent including eliminating or preventing the occurrence of active capture agent on an outside surface of the hollow flow elements.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: a suspension of the hollow flow elements in fluid with the capture agent is aggressively agitated to impart disrupting shear forces to an exterior surface of the micro-length elements, the shear force acting to prevent binding of the capture agent to the outside surface of the elements.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the hollow elements may be aggressively agitated by vortexing.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: functionalization of the elements (i.e. coating the elements with capture agent), the elements may be suspended in a sugar-based stabilizing solution, and flowed upon a pick-up plate from which they may be to be picked for placement in the channels.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the step of wicking excess fluid from the pick-up plate with an absorbent wicking substance, without physical contact of the wicking substance with the elements.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: pick-up plate on which the elements may be flowed is a plate having grooves in which the elements become lodged in alignment, and the wicking is done by positioning the wicking material to communicate with the grooves at a location spaced from the elements.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: a series of pockets formed along the grooves in which the elements discretely lodge.
  • the pickup plate defines a flat element-receiving surface
  • the wicking is accomplished by contacting the solution at locations on opposite sides of a collection or clump of the micro-elements in the solution.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the pickup plate is contacted with a ring of absorbent material having an inside diameter larger that the perimeter of the collection of micro-elements to be drained.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: preparing the micro-elements, the elements may be stored on a pickup plate while coated with a sugar-based stabilizing solution, under controlled humidity conditions, of relative humidity of at least 50%.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following wherein the micro-element comprising micro-length hollow flow elements having a length L less than about 700 ⁇ . Preferred implementations of this aspect of the invention may incorporate one or more of the following the length L is about 250 ⁇ .
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the element is of glass or glass-like substance.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the element is hollow and defines an internal volume of the order of 1 nano liter, the element constituting a glass nano-reactor ("GNR") for assay reactions.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the element is a hollow micro-element of fused silica and of straight, cylindrical form having interior diameter of about 70 um, exterior diameter of about 125 um, and axial length of about 250 um, the end surfaces being planar, lying at a substantial angle to the element axis.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the element is a segment of a drawn material.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the drawn material comprises tubing.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the element has end surfaces lying at a substantial angle to the element axis as a result of cutting drawn substance or tubing.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the micro-fluidic channel is defined by spaced apart, opposed sidewalls, the channel has greater width than the width or diameter of a detection element to be fixed in it, during placing motion the element being brought into contact with one of the sidewalls.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the element is positioned relative to the channel such that by -pass flow cross-section area on the side of the element opposite the wall with which it makes contact is formed greater than on the side on which the element makes contact.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following a side of the flow channel is defined by a material that has electrostatic attractive properties relative to the exterior surface of the element, to which the element is drawn during placing of the element in the channel.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following channel side walls may be comprised of polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following a flow channel is defined by an open channel of depth slightly less than the corresponding dimension of the element, the element residing in this open channel, and a closing member is disposed over and closes the open side of the open channel, the closing member elastically bearing against the portion of the element lying outside the open channel to form the flow channel and simultaneously secure the element in its fixed position within the flow channel.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the layer is an elastic membrane.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the elastic closing member is a portion of an elastomeric sheet lying over multiple flow elements in one or more flow channels, securing each element in position in its respective flow channel.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the channel is formed by a rigid base plate having a planar surface, upon which is secured one side of a parallel first sheet of elastomer in which a through, open-slot has been cut, the side surfaces bounding the slot and the corresponding exposed surface of the base plate forming the open channel that receives the element, and a closing member comprises a second elastomeric sheet lying parallel against and attracted to the opposite side of the first sheet and the element protruding from the open channel in manner that locally deforms the second sheet against the element and causes it to elastically press the element against the base plate, fixing it in position.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following respective portions of the same elastic or elastomeric sheet may be caused to lie against and secure each of a plurality of the elements in the flow channel.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following close- field electrostatic attraction is employed to define the position of the element and enable withdrawal of the placement tool.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the micro fluidic channel into which the micro-element is placed has at least one side wall formed of elastomeric material, the channel having a width less than the corresponding dimension of the element, and the placing action forces the element into the channel by force- fit until compression of the elastomeric material grips the element sufficiently to detach the element from the placing instrument and enable withdrawal of the instrument.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following the element is inserted into its microfluidic channel by pick-and- place motion effected by automated tweezer fingers engaging oppositely directed portions of the element.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following gripping elements of the tweezer may have width in the transverse direction to the axis of the channel that may be less than the separation of side walls of the channel, enabling insertion and withdrawal of the tweezer elements without interference with the channel walls.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the oppositely directed portions may be parallel planar surfaces orthogonal to the axis of the element.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the element is inserted into its microfluidic channel by pick-and- place motion effected by an automated vacuum pick up.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the vacuum pickup is effected by a device which engages an outer cylindrical surface of the element.
  • a microfluidic assay device in forming a microfluidic assay device, the steps of formation comprising: forming micro-particles in bulk, wherein the micro-particles may be sized for insertion in a predetermined open microfluidic channel; placing the micro- particles into the predetermined channel by means of an automated placement tool; and covering the channel to form an enclosed channel containing the micro-particles.
  • covering the channel further comprises applying a continuous membrane having a portion that forms a deflectable valve membrane, using surface activated flexible membrane to adhere to surface activated adjoining surface, wherein adhering the flexible membrane includes making and breaking contact with the valve seat during cure to inhibit valve sticking.
  • the enclosed channel is formed by joining a rigid-backed fluidic layer and a rigid backed pneumatic layer on which a membrane sheet is bonded, the membrane sheet closing the open channel.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the membrane layer and the body in which the open channels may be formed comprise PDMS.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: the PDMS surfaces may be bonded by covalent bonding.
  • micro fluidic assay device formed by the methods of any of the foregoing claims.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following: in the form of a portable micro fluidic cartridge.
  • the invention features in assembling a portion of a microfluidic device by conducting bonding action by contacting faces of opposed bondable materials, one comprising a flexible sheet, the method, while maintaining continual contact of the faces in a region Rl until bonding is completed, of employing repeated make-and-break-contact manufacturing protocol on a second region R2 of the contacted faces of the bondable materials, thereby over time neutralizing the tendency for permanent bonds to form in that region R2, thus to enable making and breaking actuated movements of the second region R2 of the flexible sheet relative to the portion of the other material that it opposes.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • the faces of opposed bondable materials may have the capability of forming molecular bonds, and molecular bonds between the faces may be formed in the region Rl of the continual contact.
  • the materials may have the capability of forming covalent bonds and covalent bonds between the faces may be formed in the region Rl of the continual contact.
  • At least one of the materials may be a surface-activated bondable material.
  • At least one of the materials may be surface-activated PDMS. Both materials may be PDMS.
  • the flexible membrane portion may define a valve diaphragm and the opposed portion of the other material may define a valve seat which the flexible membrane portion engages.
  • the bonding of the flexible sheet material to the opposed material simultaneously may act to complete and seal a flow channel, may fix the position of an inserted element in a flow channel, or may form the diaphragm of the piston of a membrane pump. All actions may be simultaneously performed.
  • the repeated make-and break-contact manufacturing protocol may be performed by applying, respectively, positive and negative air pressure to the back of the flexible membrane portion.
  • the negative air pressure may be of larger magnitude than the positive air pressure.
  • the method may include providing a chamber defining a deflection cavity into which the flexible membrane portion may be drawn away from contact with the opposite material while other regions of the faces of the two materials are in contact for bonding; bringing the regions of the faces of the two materials into continuing contact for bonding and applying vacuum to the deflection cavity to draw the flexible membrane portion into the cavity, away from the opposite material; thereafter, in cyclical manner, repeatedly applying a positive pressure pulse followed by a negative pressure pulse to cause the flexible membrane portion to repeatedly make and break contact with a surface portion of the opposite material during cure of the bond between adjacent surface portions of the two materials that are in continuing contact.
  • the vacuum may be applied to the deflection cavity prior to and during initial contacting of the regions of the faces of the two materials for bonding, so that the membrane portion does not bond at the time of initial contact of the regions of the faces of the materials.
  • the material may comprise the flexible sheet has its face comprised of PDMS, the face having been surface- activated.
  • the material may be a layer comprising PDMS throughout its thickness.
  • the flexible sheet may be comprised substantially of a material other than PDMS, but may have its face opposing the other material comprised of an activatable adhesive coating that has been surface-activated for bonding.
  • the activatable adhesive may be PDMS.
  • the material opposite the flexible sheet may have its face for bonding comprised of PDMS, the face may be surface-activated.
  • the material opposite the flexible sheet may have its face for bonding comprised of glass, presenting OH groups to the activated PDMS surface.
  • the material opposite the flexible sheet may have a main body formed of a material, on the face of which may be a linker layer bonded to that material, which may present OH groups to the activated surface of the PDMS membrane portion.
  • the material opposite the flexible sheet may comprise synthetic resin.
  • the synthetic resin may be one of the group comprising polymethacrylate, polystyrene, or polysulfane, which has been surface-activated and bonded to the linker layer.
  • the material opposite the membrane portion may not deflect during the bonding process.
  • the material opposite the flexible sheet having a flexible membrane portion may also have a flexible sheet the flexible membrane portion opposite the first a flexible membrane portion; including, providing, at the face of the second membrane, a second chamber defining a deflection cavity into which the second flexible membrane portion may be drawn away from contact with the opposite material while other regions of the faces of the two materials are in contact for bonding; bringing the faces of the two materials into continuing contact for bonding and applying vacuum to the deflection cavities to draw each of the flexible membrane portions into its respective cavity, away from the opposite material; thereafter, in cyclical manner, repeatedly, in synchronized manner, applying a positive pressure pulse followed by a vacuum pulse to each of the chambers to cause the membrane portions to repeatedly make and break contact with the opposite membrane during cure of the bond between adjacent face portions of the two materials that are in contact.
  • the flexible membrane portion may be an elastomer subject to thinning under tension (as described by Poisson), and the membrane portion may be deflected initially with a negative pressure of a first magnitude, and later at a magnitude of a second magnitude of substantially larger value, the initial value assuring bonding of contacting may face up to the perimeter of the flexible membrane portion.
  • a micro fluidic structure may be produced. 27.
  • the micro fluidic structure may be in the form of a pneumatically operable, hand held bio-assay cartridge containing microfluidic channels, reservoirs for sample and reagents and membrane pumps and valves formed of flexible portions of the flexible sheet.
  • the invention features a method of forming a pneumatically controlled microfluidic device containing a micro-fluidic network that includes micro-elements in the form of micro-particles or micro-length hollow flow elements, and a pneumatic network that includes pneumatic micro-channels and micro-valve features enabling membrane valves to operate to control fluid conditions in the microfluidic network, including the steps (a) forming two portions, denominated fluidic layer, in which microfluidic channels are formed, and pneumatic layer, in which pneumatic micro-channels and micro-valve features are formed, each having a backing that is rigid in the plane of extent of the layers, (b) providing an intervening elastic membrane, and (c) permanently bonding both layers to opposite sides of the membrane, the permanent bonding of the membrane to the fluidic layer being effective to permanently enclose a set of inserted micro-elements in the fluidic network and relate the two layers to enable pneumatic control of fluid conditions in the microfluidic network.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • micro-elements are functionalized to serve as assay capture elements.
  • micro-elements are glass nano reactors.
  • the method including piston chamber features in the pneumatic network.
  • the pneumatic layer defines a well or reservoir communicating with the fluidic layer by vias formed through the membrane.
  • sample wells and reservoirs for buffer liquid, reagent and waste are defined by the rigid backing of the pneumatic layer.
  • the backing for the pneumatic layer is a machined or injection molded component.
  • the rigid backing of the fluidic layer is a transparent member of rigid material through which results of the assay may be read.
  • the in which the transparent member is glass or glass-like material of silicon.
  • microfluidic channels are formed in a sheet of material permanently bonded to the rigid backing of the fluidic layer and to the membrane.
  • layer is comprised of PDMS.
  • a sheet forming channels of the fluidic layer is provided with a surface activated surface that permanently bonds with molecular bonding to the fluidic layer, the rigid backing of the fluidic layer or both.
  • the membrane layer and the sheet are comprised of PDMS having opposed surfaces that are permanently bonded together.
  • the layer of material is a composite formed of a central relatively stiff sheet and sheet- form pressure-sensitive adhesive on each side of the sheet.
  • the method in which the layer in which pneumatic micro-channels or channel segment are formed comprises low fluorescent material.
  • micro-elements have dimensions such that they have portions that protrude from the open microfluidic channel, and the membrane is elastically distorted about them, producing a compressive load that secures the microelements in position.
  • a unitary membrane sheet seals the microfluidic channels and performs at least one further function selected from the group consisting of securing the micro-elements in position, defining vias, defining movable vale membrane portions, defining movable membrane piston portions an , with an extension beyond the fluidic layer, defining a compliant surface about pneumatic ports for leak tight sealing to a source of pneumatic pressure.
  • the membrane simultaneously forms all features defined in claim 26.
  • the invention features a microfluidic device for conducting a fluid assay formed with the method of any of the foregoing claims, for example a biological assay, having a microfluidic flow channel in which is inserted at least one discrete detection micro-element (preferably a micro-length tube with length less than about 700 micron, preferably less than about 500 micron, and internal diameter of between about 75 +/- 50 micron, preferably in many instances, 50 +/- 25 micron, in fixed position), that is provided with capture agent, the detection micro-element being positioned for exposure to fluid flows within the device for conducting an assay.
  • a discrete detection micro-element preferably a micro-length tube with length less than about 700 micron, preferably less than about 500 micron, and internal diameter of between about 75 +/- 50 micron, preferably in many instances, 50 +/- 25 micron, in fixed position
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • the detection element comprises a short hollow flow element of length less than 700 micron, preferably less than approximately 500 micron, having oppositely directed planar end surfaces and a cylindrical outer surface extending between those end surfaces, and preferably so located in the flow channel to permit flow through the element, and by -pass flow of at least equal volume along the outside of the fixed element.
  • the device in which the pick and place motion is effected by automated tweezer fingers engaging oppositely directed portions of the flow element, preferably oppositely directed parallel planar surfaces.
  • the device in which the pick and place motion is effected by automated vacuum pick up.
  • the device in which the vacuum pickup device engages an outer cylindrical surface of the flow element engages an outer cylindrical surface of the flow element.
  • the device may be adapted to be read by a detector of epi-fluorescence type.
  • the device may be a portable cartridge constructed to conduct multiplex assays on a sample.
  • the device may be a portable cartridge constructed to conduct multiplex assays on multiple samples.
  • the device may have at least some parts of the device are joined by co-valent bonding of activated surfaces of bondable material, a contiguous portion of a covalently bound sheet fixing the position of a detection micro-element in its microfluidic channel.
  • the device may have at least some parts of the device are joined by co-valent bonding of activated surfaces of bondable material, a contiguous portion of a covalently bound sheet forming a flexible pump diaphragm.
  • the device may have at least some parts of the device are joined by co-valent bonding of activated surfaces of bondable material, a contiguous portion of a covalently bound sheet forming a flexible valve diaphragm.
  • the device may have a portion of the membrane of flexible sheet forming a flexible valve diaphragm portion that engages a valve seat originally formed of surface- activated bondable material that has been subjected to a series of make-and break contacts that interrupt covalent bonding of the valve diaphragm portion with its opposed seat.
  • the device may have parts that may be permanently secured by co-valent bonding of selected regions of faces of surface-activated bondable materials.
  • the device in which the form of activation is oxidation.
  • the device in which at least one of the parts comprises surface-activatable elastomer.
  • the device in which the elastomer is PDMS.
  • the device in which the bond is formed by opposed surfaces of surface-activated PDMS.
  • the device in which the bond is formed by one opposed surface of surface-activated PDMS and the other surface is surface-activated glass or polymer other than PDMS may be formed by preparation of two subassemblies, each having a backing of relatively rigid material and an oppositely directed face suitable for bonding to a mating face of the other subassembly, followed by permanently bonding the assemblies face-to-face.
  • the device in which the bonding creates a permanent bond with material such as of PDMS, in which a bond of like surface-activated surfaces in which the original structure of mating surfaces is substantially eliminated by molecular diffusion.
  • the detection micro-element comprises a cylindrical, hollow flow element of length no greater than 700 micron, preferably less than about 500 micron, most preferably about 200 micron and internal diameter of approximately 50 micron +/- 25 micron, the element being substantially uniformly coated on its inner surface with capture agent for a selected fluid assay.
  • the device in which the capture agent is antibody for conducting ELISA.
  • the device in which capture agent is substantially absent from the outer cylindrical surface or all outer surfaces of the element, and the detection element is sized, relative to the channel in which it is inserted, to define a substantial flow path through the element and a substantial by -pass flow path along the exterior of the element.
  • the device in which the detection element is of depth greater than the depth of an open channel in which it is inserted, and a capturing layer closes and seals the channel, the capturing layer being elastically deformed by its contact with the flow element thereby applying forces thereto that fix the location of the element in the channel.
  • the device in which the capturing layer is co-valently bonded to the substance defining the open channel.
  • the device constructed to perform ELISA biological assay.
  • the device in which a series of between about 3 and 10 spaced-apart discrete flowmicro-elements of less than 700 micron length, preferably less than about 500 micron, are fixed in a given channel.
  • the device in which the device contains a means of providing a fluorophor label to captured analyte, and the flow elements are exposed to a window transparent to outwardly proceeding fluorescent emission for detection.
  • the device in which the window is transparent to exterior-generated stimulating light emission to enable epi-fluorescent detection.
  • the invention features a microfluidic device for conducting a fluid assay, for example a biological assay, having a flow channel in which is inserted at least one discrete detection micro-element that is provided with capture agent, microelement being positioned for exposure to fluid flows within the device for conducting an assay, the device formed by preparation of two subassemblies, each having a backing of relatively rigid material and an oppositely directed face suitable for bonding to a mating face of the other subassembly, followed by permanently bonding the assemblies face-to-face.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • the device in which the bond is permanent formed by bonding together two surface- activated surfaces.
  • the device in which the member defining one of the surfaces has portions that fix the position of a said detection micro-element in its flow channel, form a flexible pump diaphragm or form a flexible valve diaphragm, preferably respective portions of the sheet performing all of these functions.
  • a flexible valve diaphragm portion engages a valve seat originally formed of surface-activated bondable material that has been subjected to a series of make-and break contacts that interrupt covalent bonding of the valve diaphragm portion with its opposed seat.
  • the device in which the surfaces are both of PDMS.
  • the invention features a microfluidic device for conducting a fluid assay, for example a biological assay, having a flow channel in which is inserted at least one discrete hollow flow element (preferably a micro-length element with length less than about 700 micron, preferably less than about 500 micron, and internal diameter of between about 75 +/- 50 micron, preferably in many instances 50 +/- 25 micron, in fixed position), that is provided with capture agent only on its interior, the element being positioned for exposure to fluid flows within the device for conducting an assay, the flow channel being of rectangular cross-section, the exterior of the element being of cylindrical cross-section, and by -pass flow paths are defined along the exterior of the element.
  • a discrete hollow flow element preferably a micro-length element with length less than about 700 micron, preferably less than about 500 micron, and internal diameter of between about 75 +/- 50 micron, preferably in many instances 50 +/- 25 micron, in fixed position
  • the invention features a discrete detection element in the form of a micro-length hollow flow element with length less than about 700 micron, preferably less than about 500 micron, and internal diameter of between about 50 +/- 25 micron, the flow element provided with capture agent, the flow element being constructed to be fixed in position for exposure to fluid flows within a device for conducting an assay.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • the hollow flow element in which capture agent resides only on the interior surface of the element.
  • the invention features a hollow flow element carrying on its interior surface, but not its exterior surface, an assay capture agent, the element fixed in position in a fluid channel in manner that provides at least about 50 % by-pass flow capacity relative to the flow capacity through the element.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • the invention features a method of manufacturing the device or element of any of the foregoing device claims.
  • the invention features a method of use of the device or element of any of the foregoing device claims.
  • the invention features a method of preparing detection elements for an assay comprising batch coating the detection elements, preferably hollow flow elements by mixing in solution, and drying, and thereafter picking and placing the elements in flow channels of a micro fluidic device formed according to any of the method claims herein, and preferably capturing the flow elements by bonding two opposed layers that capture the elements while sealing the flow channels.
  • the invention features a method of flowing a fluid with a tracer in a microfluidic channel of an assay device and detecting the tracer for determining the channel location or condition of the channel.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • An assay in which an assay fluid having a desired property may be present within a micro fluidic channel at a given phase of the assay protocol, including the step of providing the assay fluid with a detectable tracer that is benign (e.g. inert) to the respective phase of the assay, and, during conduct of that phase of the assay, under required assay conditions, monitoring a selected region of the microfluidic channel with a detection system to detect the tracer, and comparing the detected results with a standard of acceptable results.
  • a detectable tracer that is benign (e.g. inert)
  • a method may be conducted to determine the precise location of a portion of a microfluidic channel relative to a detection system, including the step of providing a fluid with a detectable tracer, and performing a detection operation that locates the microfluidic channel or a portion of it by a detection system that detects the tracer.
  • this method may be conducted during a step of an assay in which the tracer is inert with respect to an assay fluid in which it is carried.
  • a detectable property of the tracer may be used to verify the assay phase. Different values of the detectable property of the tracer may be used to confirm the phase of an assay, e.g. different and unique concentrations of the a tracer are used for each phase of an assay.
  • the property of the tracer may be fluorescence intensity.
  • the property of the tracer may be optical density.
  • the detection steps may be performed by an epi-fluorescent system employing a light to excite fluorescence within a microfluidic channel, and to translate the beam with relation to the channel over a set of channels or along the channel for detecting position, monitoring, or assay-reading purposes.
  • the light source may be a laser.
  • the laser beam may have an aspect ratio of at least 2: 1. Relative movements along a channel may be used to index between monitoring locations, and to progressively read assay results, e.g. from one or a set of immobilized detection elements, such as micro- length tubes (or glass nano-reaction vessels).
  • the microfluidic flow channel may have a flow axis, along which a series of discrete, axially-spaced apart, transparent hollow flow elements may be secured in fixed position, each hollow flow element may have at least one axially-extending flow passage through its interior, the elements may have interior and exterior surfaces extending in parallel in the direction of the channel axis, and end surfaces may extend transversely to the axis, the surfaces of the elements may be exposed to liquid in the channel, and assay capture agent may be fixed to the interior surface of the elements for capture of an analyte in liquid flowing through the interior of the hollow flow elements, the device may be constructed to enable light to be transmitted into and out of the elements transversely to the flow axis for excitation and reading of fluorescence from captured analyte.
  • the invention features a device for performing any method recited herein.
  • the assay method or device associated with a positive-displacement pump may be arranged to introduce a segment of liquid sample, and may cause the sample to move back and forth with respect to a hollow flow element to produce capture of analyte only in the interior surface of the element and to repeat this action for successive segments of liquid sample.
  • Capture agent on the interior surface of a hollow element in the channel may be configured to define a code.
  • the code may be a bar code.
  • the invention may feature an assay method or a device for performing a method in which the active capture agent is an antibody, antigen, or oligomer.
  • the invention features a micro-length tube element for use in an assay device comprising a hollow body with a through passage for liquid flow, wherein active capture agent for a given analyte resides on at least a portion of the interior surface of the hollow body for interaction with fluid sample, the capture agent on the interior surface of the element being configured to define a code.
  • the invention features a micro-length tube element for use in an assay method or device comprising a hollow body with a through-passage for liquid flow, wherein active capture agent for a given analyte resides only on a portion of the interior surface of the hollow body for interaction with fluid sample; the exterior surfaces of the micro-tube element being free of active capture agent, and un-reactive with the analyte in the sample and in which the capture agent on the interior surface of the element is configured to define a code.
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • the code may be a bar code.
  • the active capture agent is an antibody.
  • the active capture agent may be an antigen.
  • the active capture agent may be an oligomer.
  • the invention features reading code on a discrete hollow flow element, according to the following steps: (A) provide hollow flow element with code pattern written in capture agent on inside surface of the element; preferably provide as a micro-length tube element; preferably the code represents the identity and/or concentration of capture agent on the element; (B) Pick and place the hollow element into the flow channel; (C) Conduct analyte capture step by sample flow through channel, attaching analyte molecules to capture agent in the code pattern on inside surface of hollow element; (D) Attach fluorophore tag to captured analyte molecules by flow through the channel; complete the fluid assay; (E) Stimulate and detect pattern of fluorescent emission from tagged analyte through the wall of the hollow element; (F) Conduct pattern analysis, identify pattern of light emission from captured analyte and match the read pattern to stored code table; (G) Use the coded information associated with the specific code pattern of the hollow element; present e.g. by print-out or associate the data with stored or transmitted as
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • Step A may be provided according to the steps of (A) Provide micro- length tube element uniformly coated with capture agent on inside tubular surface with no active capture agent on outside cylindrical surface; (B) With laser beam, deactivate or remove the capture agent according to the blank spaces of a predetermined code pattern, preferably a bar-code pattern; (C) Place thus-coded micro- length tube element in flow channel constructed to enable sample flow both through the interior and past the exterior of the element, capturing analyte.
  • a predetermined code pattern preferably a bar-code pattern
  • the invention features reading code on a discrete hollow flow element, and quantifying assay data, according to the following steps: (A) Provide hollow carrier element with code pattern written in capture agent on inside surface of the element; preferably provide as a micro-tube element; preferably the code represents the identity and/or concentration of capture agent on the element;
  • step B Pick and place hollow element into flow channel;
  • step C Conduct analyte capture step by sample flow through channel, attaching analyte molecules to capture agent in the code pattern on inside surface of hollow element;
  • D Attach fluorophore tag to captured analyte molecules by flow through the channel; complete the fluid assay;
  • E Stimulate and detect pattern of fluorescent emission from tagged analyte;
  • step E Substantially simultaneously with step E, read intensity of emission from discrete hollow carrier element (preferably, a micro-length tube element), quantify the magnitude of detected emission; determine the concentration of analyte in the sample as a function of this quantification;
  • G Conduct pattern analysis, identify pattern of light emission from captured analyte and match the read pattern to stored code table;
  • H Use the coded information associated with the specific code pattern of the hollow element; present e.g. by print-out or associate the data with stored or transmitted assay data;
  • I And, or, use the detected information in data form in computer logic as part
  • Preferred implementations of this aspect of the invention may incorporate one or more of the following:
  • the tracer may comprise a substance not employed in the conduct of the assay.
  • the tracer may be a substance employed in the assay.
  • the tracer may be a fluorescent tag employed to be captured at the site of analyte to enable reading of the assay.
  • the fluorescent tag may be capable of fluorescent emission under stimulation, the method may include exciting the fluorescence.
  • the fluorescent tag may be capable of fluorescent emission by light stimulation, and the step may include reading the fluorescence by an epi-fluorescence detection system. Any methods discussed herein may include an apparatus for performing the method of any of the foregoing claims.
  • Fig. 1 is an illustration of assembly steps for an assay cassette having flow channels in which discrete micro-length tube flow elements are fixed between a Fluidic Layer subassembly and a PDMS sheet of a Control/Reservoir Layer subassembly;
  • Figs. 2A and 2B are diagrammatic plan views, on enlarged scale, depicting 4 micro-length tube elements fixed in series in a flow channel, Fig. 2A illustrating substantial liquid flow both through the flow elements and as by-pass flow through by -pass passages on the outside of the elements, while Fig. 2B illustrates the continued flow condition in the case in which one micro-length tube becomes blocked and Fig. 2C is a cross-section, on enlarged scale, depicting flow conditions in which a micro-length tube element becomes plugged (i.e. blocked);
  • Fig. 3 is a much enlarged perspective view of a portion of the cassette, denoting 4 parallel channels, in each of which are fixed six micro-length tube flow elements;
  • Fig. 4 is a flow diagram of steps A through K in the manufacture and use of the cassette (i.e. "flow chip") constructed according to the foregoing Figures;
  • Fig. 6 depicts a flow element and a micro-length tube element example, illustrating the percentage reduction of active capture agent under differing conditions of the surfaces of the element;
  • Fig. 7 depicts a device employed to aggressively agitate a suspension of micro-length tube elements in a capture agent, e.g. antibody, antigen, or oligomer- containing liquid;
  • a capture agent e.g. antibody, antigen, or oligomer- containing liquid
  • Fig. 8 illustrates diagrammatically, by vectors, sheer forces T to which the outside and inside surfaces of the micro-tubular element are exposed;
  • Fig. 9 illustrates a step in the manufacture of micro-tubular elements from micro-bore drawn filament
  • Fig. 10 is a plane view of portion of an alignment plate for micro-tubular elements
  • Fig. 10A under the heading "pick and place", is a plan (top) view of alignment pocket, GNRs (micro-length tubes) in the pockets, wicking channels, and tweezers;
  • Fig. 10B is a cross-sectional view of GNRs covered with stabilizing reagent and an absorbent pad approaching the alignment plate, while Fig. IOC, similar to Fig. 10B, shows the absorbent pad positioned on the alignment plate and stabilizing reagent greatly reduced, and Fig. IOC, similar to Fig. IOC, shows the absorbent pad removed and tweezers positioned at opposite ends of a GNR.
  • Figs. 10D and E are plan (top) and vertical cross-sectional views, respectively, of a circular absorbent pad surrounding GNRs on a flat plate; there is no Fig. 10F.
  • Fig. 10G shows a micro-length tube being fit into a narrower channel with interference fit while Fig. 10H shows the channel with the membrane enclosing the tube;
  • Fig. 11 illustrates a centrifugal dryer cooperating with a drying and alignment plate to dry the micro-length tube elements
  • Fig. 12 is, on enlarged scale, a diagrammatic representation of laser beams ablating (removing or rendering in-active) selected regions of capture agent on end and inside surfaces of a micro-length tube element;
  • Fig. 13 is a view similar to Fig 12, depicting the formation of a code of capture agent on the inside surface of a micro-length tube flow element;
  • Fig. 14 depicts a photo mask exposure scheme for forming a large laser beam into beamlets that perform the steps of Fig. 12 or 13;
  • Fig. 15 is a diagrammatic top view of a system for placing discrete micro- length tube elements into open channels of a microfluidic assay device (see also Fig.
  • Fig. 16 is a side view of a pick and place apparatus employing a tweezer instrument for engaging end surfaces of the micro-length tube elements (see also Fig.
  • Fig. 17 depicts the ends of tweezer tines approaching the oppositely directed end surfaces of a micro-length tube element positioned in the alignment plate;
  • Fig. 19 "PLACE with tweezer", depicts the ends of tweezer tines leaving the oppositely directed end surfaces of a micro-length tube element positioned in a flow channel of the microfluidic device;
  • Figs. 21 A to 21 E represent, in large scale end cross-section views, a sequence of steps involved in placing and fixing the position of micro-length tube elements in an assay device and completing the enclosure of a flow channel in the device;
  • Figs. 22 A and B illustrate the flow cross-sections of a flow channel with micro-length tube element in place in a channel
  • Fig. 23A is a diagram of a laser and computer controlled steering mirror cutting a pattern in double sided PSA film with peelable liners;
  • Fig. 23B is a plan view of a laser-cut pattern in the material of Fig. 23 A defining pneumatic control channels and features for integrated valves and pumps while Fig. 23C is a magnified view of a portion of Fig. 23B;
  • Fig. 23 D is a vertical cross section, with parts broken away, on a magnified scale, of a microfluidic device comprising a reservoir layer, a pneumatic channel-PSA layer, a membrane, a fluidic layer containing a GNR in a fluidic channel and a glass layer, with a channel shunt formed in the membrane layer at the location of a stability bridge in the pneumatic channel-PSA layer, the membrane also closing off a fluidic channel and containing a GNR placed in the channel;
  • Fig. 23 E is a vertical cross section, with parts broken away, on a magnified scale, similar to Fig. 23D, except that the channel shunt is formed in the reservoir layer, the membrane also closing off a fluidic channel and containing a GNR placed in the channel;
  • Fig. 23F is a cross-section similar to Figs. 23D and E, depicting a valve portion of the membrane deflected into a recess cut in the pneumatic channel-PSA layer, the membrane also closing off a fluidic channel and containing a GNR placed in the channel;
  • FIG.24 A top view of the fluidic sub-assembly on an enlarged scale
  • Fig. 25 - A perspective view of parts of the pneumatic sub-assembly as the PDMS sheet comes together with the reservoir/pneumatic layer;
  • Fig. 26 A plan view, looking up at the underside of the reservoir/ pneumatic sub-assembly through its transparent PDMS membrane sheet;
  • Fig. 27 - A pian view, again of the underside of the reservoir/pneumatic subassembly and the mating upper surface of the Fluidic Layer sub-assembly;
  • Fig. 28 - A perspective view diagrammatically illustrating the mating action of the two sub-assemblies with the micro-length tubes in the Fluidic Layer;
  • Fig. 28A - A side view illustrating the PDMS layer and the mating surface respectively of the two subassemblies (Reservoir/Pneumatic Layer and Fluidic Layer) being pressed together with slight pressure;
  • FIG. 28B A magnified view of a portion of Fig. 28a denoted by a circle in Fig. 28A;
  • Fig. 28C A perspective view of the completed assembly viewed from above (as assembled with the glass layer facing up);
  • Fig. 28D A perspective view of the completed assembly, viewed, from above (after inversion, so that the reservoir layer faces up, the glass layer faces down);
  • Fig. 29 - A top view of the completed assembly
  • Fig. 30 A schematic diagram in perspective of assembly steps for another microfluidic assay device, a Figure very similar to Fig. 1 except for numeral references instead of legends;
  • FIG. 30A An exploded perspective view of the device of Fig. 30;
  • Fig. 31A A perspective view on an enlarged scale of a fluidic channel of Figs. 30 and 30A;
  • FIG..31C An even more greatly magnified view of sets of extremely small hollow flow elements disposed in channels of Figures 31 A and B(see Figs. 6 and 8 for a representation of a single flow elment;
  • Fig. 32 - A greatly magnified plan view of a portion of the channel structure, showing two channels, with four hollow flow elements disposed in each and indicating scanning;
  • Fig. 33 A plan view of a single channel, with schematic illustration of onboard pump and valve, and showing flow paths through and alongside hollow flow elements;
  • Fig. 33A Similar to Fig 33, a plan view, but in greater detail, of a microfluidic channel having a hollow flow element and a micro-piston located between two micro-valves;
  • Fig. 33A' is a cross section of the assembly of Fig. 33A taken on line 33A'- 33A' of Fig 33A;
  • Figs. 33B, 33C and 33D are magnified views of portions of Fig. 33A' as respectively designated by circles in that figure, Figs. 33B and 33D showing the membrane engaged upon a valve seat;
  • Figs. 33E is a view like the magnified cross-sections of Figs. 33B and 33D, except with the membrane deflected away from the valve seat;
  • Fig. 33F is like the magnified cross-section of Fig. 33C, except with the membrane deflected;
  • Fig. 34 A magnified diagrammatic cross section, with parts broken away of micro-fluidic channels of a device, and depicting the membrane capturing a hollow flow elment in the channel, lines of flow being indicated through and outside the flow element;
  • Fig. 34A - A view similar to Fig. 34 in which twolayers (membrane and the layer defining the side wall of the channels)m both of PDMS, have been fused by covalent bonding to close the channels and secure the hollow flow elements;
  • Fig. 41 - A diagram of steps in the assembly process for the device of preceding figures
  • Fig. 41 A, B, C, and D— Are cross-sectional views of a micro fluidic device through a hollow flow element, illustrating, diagrammatically steps in employing PDMS surface activation and covalent bonding to form the liquid-tight channels and secure the extremely small hollow flow elements in place in the channels;
  • Fig. 42 - A diagram in plan of a pick-and-place instrument positioned above an X,Y translation table, a delivery plate for discrete, extremely small hollow flow elements and a receiving channel of multiplex micro-fluidic assay devices of the preceding figures;
  • Fig, 43 a diagrammatic front view of a tweezer type pick and place device, and its support tower;
  • Fig. 44 a diagrammatic front view, similar to that of Fig. 43 of a vacuum type pick and place device, and its support tower;
  • Figs. 46 and 47 Respectively, picking, and placing side views of a vacuum pick up device;
  • Figs. 48, 49 and 49A A sequence of positions during placing of a flow element with a pick-and-place device, the + and - signs diagrammatically illustrating the use of close-space electrostatic attraction between the channel wall and the element being delivered that facilitaties placement of the element and withdrawal of the tool;
  • Fig. 49B and Fig. 50 - illustrate element-securing and channel-sealing actions occurring during assembly of the device of preceding figures
  • Fig. 50A in plan view, illustrates hollow micro-particles (here, micro-length tubes) distributed in random fashion onto a flat surface, and a pick up head is shown;
  • Fig. 50B is a three dimensional diagram showing a picking head with video system and computer controller for effecting relative movements X, Y, Z and angular orientation theta between a surface element and the picking head;
  • Figs. 50C and 50D are side cross-sectional and plan views of a placement tool in form of a vacuum tip
  • Figs. 50 E and F are side and horizontal cross section views of a tweezerr pick up tool
  • Fig. 50 G shows an element being introduced with interference fit into a channel, the width of which is less than the diameter of the element, while Fig. 50H shows the element in a channel closed by a top layer;
  • Fig. 51 is a diagrammatic view, showing the repeated cycling of a diaphragm valve formed by an overlying portion of a PDMS layer, which is bonded to the opposed structure at each side, the diaphragm valve, repeatedly closed with 3 psi positive pressure and opened with negative 8 psi pressure (vacuum), is found to overcome the molecular bonds being formed between diaphragm and valve seat, thus over time, neutralizing the tendency for permanent co-valent bonds to form between contacting surface-activated surfaces, thus enabling the thus-formed valve to properly operate;
  • Fig. 51 A- 1 is a diagrammatic showing of two opposed layers of PDMS, showing them in the natural state of the PDMS which is a hydrophobic state with methyl group endings exposed;
  • Fig. 51 A-2 is a similar Figure following plasma oxygen plasma treatment showing the separated layers are terminated in OH groups;
  • Fig. 51 A-3 is a Figure illustrating permanent bonds between the hydroxyl groups producing oxygen covalent bridging
  • Fig. 51 A-4A similar to parts of Fig. 51, illustrates, in diagrammatic cross- section, a valve as initially assembled, comprising two opposed layers of plasma- treated PDMS with a valve membrane portion of one layer deflected, the opposed PDMS sheet forming an opposed valve seat ;
  • Fig. 51 A-4B is similar to Fig. 51-A-4A following make and break process after assembly;
  • Fig. 51 A-4' is similar to Fig. 51 A-4B, but shows each PDMS layer deflected outwardly from the other in the central region.
  • Fig. 51 A-4' illustrates two regions of PDMS following plasma activation steady contact in region R2 and cyclical contact and activation or separation in what is referred to as the valve region, Ri, illustrating the initiation of permanent bonding through the hydroxyls and the condensation reaction resulting in bridging oxygen in the region R2 1, and in region Ri, where contact had occurred only temporarily and then removed, the surface having a number of methyl groups or non-bonding or lower energy state species;
  • Fig. 51 A-4 similar to Fig. A-4'. is a figure illustrating a single surface deflected opposing a planar surface.
  • Figs. 51 A-5, A- 6 and A- 7 show deflection chambers useful with, respectively, the devices of Figs. 51 -A-4', 51-4A and B, and 51 A-4";
  • Figs. 51-A-9 through 51-A-l lc illustrate composite membranes useful with the Make and Break process:
  • Fig. 51 A-9 illustrates, in cross-section, a composite membrane comprising a thin flexible sheet (PET) and a PDMS film of greater thickness;
  • Fig. 51 A-10 illustrates, in cross-section, a thin flexible sheet (PET) and a PDMS coating of lesser thickness
  • Fig. 51 A-l 1 illustrates, in plan view, a PET/PDMS lamination having circular stress relief channels formed in PET film
  • Fig. 51 A-l la illustrates, in cross-section, a composite membrane similar to that of Fig. A-9, but having stress relief slots formed in the thin flexible sheet (PET);
  • Figs 51 A- l ib and 51 A-l l-c are similar to Fig. 51 amd Fig. 51 A-6, illustrate in cross-section a composite, corresponding to that of Fig. 15-A-l la, in respectively un-deflected and deflected states;
  • Fig. 51 A- 12 is a view similar to that of Fig. 51 A-2, but showing one layer of PDMS with OH groups exposed and an opposed layer of silicon based rigid materials with OH groups exposed facing the PDMS layer;
  • Fig. 51 A- 13 is a view similar to that of Fig. 5 l-A-2 but showing one layer of PDMS with OH groups exposed, and an opposed layer comprised of synthetic resin, carrying an intermediate bi-functional layer with OH groups exposed facing the PDMS layer;
  • Figs. 51 A14 toFig 51-A-16 show a fluidic channel, Fig. 16, having two different cross-sections, Figs 51A- 14 and 51A-15 taken, respectively on the detail lines A and B of Fig.51 -A- 16, the details correspondingly respectively with previous Figs. 51 A-5 and 51 A-7;
  • Fig. 51 -A- 17 and Fig. 51 A -18 show in respective cross-section and plan views, a fluidic channel arrangement including a channel that extends from 2inlets to an outlet, and, in communication with it, a 0 dead volume sample channel; the cross section shows a pneumatic chamber in which the membrane is shown in deflected open position in solid lines, and closed by dashed line;
  • Fig. 51 B (i) shows, diagrammatically, a pneumatic tool
  • Fig. 51 B (i) m being a magnified view of a portion of Fig. 51 B (i);
  • Fig. 51 B (ii) shows the pneumatic tool in cross-section with the fluidic layer pressed against it, and Fig. 51 B (iii) in plan view, with the connections to supply ports for selective application of vacuum and pressure to the pneumatic tool;
  • Figs. 51 B' (i), B'— (i) m , B'-(ii) and B'-(iii) correspond respectively with the foregoing for a system that is the same except the pressure controller is constructed to selectively apply vacuum at two values, -2 and -14 psi);
  • Fig. 51 C is a view similar to Fig. 51, illustrating stages of the system applied to multiple valves simultaneously;
  • Figs. 51 D-l and 51D-2 are graphs illustrating the selected pressures over time and the development of properties of the contacting surfaces during the make and break process;
  • Figs. 52A-52G and 53A to 53K concern another make and break protocol having similarities with that of Fig. 51 to Fig.51 D-2.:
  • Fig. 52 A is a cross-section similar to Fig. 51 A-6, but indicating an unbonded area beyond the limit lines B of the pneumatic chamber, while Fig. 52 A m is a magnified view of a portion of Fig. 52 A and Fig. 52 B is a plan view (top view), each denoting a leak path beyond the pneumatic chamber;
  • Fig. 52 C is a protocol flow diagram including cross-sectional views associated with states within the pneumatic chamber during the make and break protocol;
  • Figs. 52 D and- 52D m and Fig. 52-E are views similar, respectively, to Figs. 52 A, 52 A m and 52 B, but showing no leak path exists outside the chamber boundary;
  • Fig. 52 F and -G are similar respectively to Figs. 51 D 1 and51 -D 2 but have an initial phase using constant -3psi deflection pressure, followed by positive and negative pressure cycling.
  • Fig. 53 pictures diagrammatically a pumping and valve state sequence by which liquid flow can be drawn into the piston from the left and expelled to the right to produce a desired directional, pulsating flow.
  • Figs 53 A-53 I illustrate functions performable by the membrane layer:
  • Fig. 53 B close channel, fix micro-particle in channel, particle shown as round in cross-section
  • Fig 53 C close channel, fix micro-particle in channel, particle shown of another shape
  • Figs 53 D and E close channel,fix micro-particle element in the shape of a micro-length tube in channel
  • Fig. 53 F close channel,fix multiple micro-length tubes in channel, define flexible membrane for a valve and a piston
  • Fig. 53 G fix micro-length tube (GNR) in channel in fluidic layer and define valves and piston that constitute a pneumatically-actuated membrane-pump;
  • GNR micro-length tube
  • Fig. 53 H close channel and in conjunction with fixing micro-length tube in channel and defining flexible membrane of pneumatically actuated valve, form via for liquid to be pumped from or to reservoir;
  • Fig. 53 I in conjunction with closing fluidic channel and fixing micro-length tube in the fluidic channel, bounding a pneumatic channel and forming a pneumatic shunt at blockage of the pneumatic channel;
  • Fig. 53 J close channel, fix micro-length tube in channel, at other side, bound a pneumatic channel, pneumatic shunt formed; in the reservoir layer about a blockage of the pneumatic channel;
  • Fig. 53K in a fluidic layer of a device, close channel, fix tube in channel and form membrane portion of micro-valve, and by exposed portion of membrane beyond the fluidic layer, define planar compliant surface for engagement with narrow lip of boss at pneumatic interface;
  • Figs. 54 and 54 A illustrate, respectively, two positions of a microfluidic cartridge relative to a carrier plate upon which it is intended that the cassette be fixed while the plate is moved on a precision X, Y stage relative to a fixed, finely focused optical detection system;
  • Fig. 55 is a cross-section view of the cartridge of Figs. 54 and 54A now fixed to the carrier plate on the movable stage, to move over a fixed heater plate and optical detection system, the objective of which is exposed to the cartridge through a hole in the heater plate.
  • Fig. 55 shows solenoid-actuated three-way valves 9 on moving X,Y stage selectively apply Pneumatic Conditions at Supply Ports defined by raised bosses of Fig. 55A. Conditions supplied are: (1) atmospheric pressure, (2) positive (+) actuating pressure, (3) negative (-) actuating pressure. Only connections to moving ⁇ , ⁇ stage asssembly are positive and negative pressure line to manifolds feeding valves 9 and electrical control lines for solenoid coils of the valves;
  • Fig. 55A is a magnified view of a portion of Fig. 55;
  • Fig. 56 is an exploded view of the assembly of a bench top operating and scanning unit for scanning the microfluidic cartridge of Figs. 54 and 54A.
  • Figs. 57A, 57B and 57C are plan views of the microfluidic and pneumatic channel architecture of the cartridge of Figs. 54 and 54A;
  • Fig. 58 outlines the fluidic architecture of a single microfluidic subunit of the cartridge of Figs. 54 and 54A, and, in tabular form, presents the steps of an immunoassay protocol conducted within the cassette;
  • Fig. 61 diagrammatically illustrates the procedure of precisely determining the location of channels of a microfluidic cartridge, for instance when fixed within the precise X, Y stage system of Figs 54, 54A, 55 and 56;
  • Fig. 62 illustrates the fact that the precise position of channels, for monitoring, and the location of detection elements in the channel, for later reading of results, can be accomplished in the same system
  • Figs. 63 and 64 are representations repeated in the later Scanning drawings, illustrating signals obtained during position determination in the absence of trace;.
  • Fig. 65 General Schematic for Epi-fluorescent Scanning Microscope (similar to Fig. 101);
  • Fig. 73 Reading Code from Micro-length tube flow element
  • FIG. 74 Preferred Implementation of previous Figures first block;
  • Fig. 75 Reading code and analyte quantity from micro-length tube flow element simultaneously;
  • Fig. 98 depiction of a microfluidic system having microfluidic channels and monitor locations
  • Fig. 98A depiction of signals obtained in three phases at a set of monitoring positions under three different conditions, illustrating a properly running assay
  • Figs. 98B and 98C similar to Fig. 98A, depiction of signals obtained during improperly running assays;
  • Fig. 99 diagrammatically illustrates tracer signal during monitoring a microfluidic channel at a single location, over a brief period of time over which flow changes; time response at location 1 : I. Piston actuation (oscillating flow) @ full tracer concentration (no net flow); II. Pumping fluid with no tracer (in one direction, toward waste) to displace tracer laced fluid in channel; III. Oscillating flow, no tracer:
  • Fig. 100 is similar to Fig. 99, but diagrammatically illustrates monitoring a fluid to detect operation of a pump over many cycles of producing oscillating flow;
  • Fig. 101 similar to Fig. 65, illustrates, diagrammatically, the relation of a scanning system to a microfluidic device, shown aligned with a channel at a region that does not contain a detection element;
  • Fig. 102 illustrates the cross section of the region of interest (ROI) of the optical system of Fig. 101 in relation to the microfluidic channel and the cross-section profile of a fluorescence-exciting laser beam;
  • ROI region of interest
  • Fig. 103 outlines the fluidic architecture of a single microfluidic subunit of the cartridge of Figs. 54 and 54A, and, in tabular form, presents the steps of an immunoassay conducted within the cassette;
  • Fig. 104 is a diagrammatic view of a microfluidic channel containing micro- length tubes, on the insides of which are immobilized capture agents in the form of DNA and antibody, while Figs. 104 A and 104B are partially broken away cross sectional views of a device implementing Fig 104, taken at respective lines indicated in Fig 104;
  • Fig. 105 is a similar diagrammatic view of a microfluidic channel containing micro-length tubes, on the insides of which are immobilized capture agents;
  • Fig. 106 is a is a diagrammatic view of four parallel microfluidic channels containing micro-length tubes, on the insides of which are immobilized capture agent; in channels 1 and 2 the capture agent is DNA and in channels 3 and 4, the capture agent is antibody;
  • Fig. 107 is a is a diagrammatic view of four parallel microfluidic channels containing micro-length tubes, on the insides of which are immobilized capture agent, in channels 1 and 2 the capture agent is DNA and in channels 3 and 4, the capture agent is antibody, and with zones heated at different respective temperatures;
  • Fig. 108 is a diagrammatic view of four parallel microfluidic channels containing micro-length tubes, on the insides of which are immobilized capture agent, each channel containing micro-length tubes, two having inside surfaces
  • Figs. 109 and 109A are diagrammatic views from the end and side of a micro- length tube element being plucked by tweezers for removal from an open fluidic channel of larger width than the element, using tweezers the same as those shown respectively in Figs. 2 IB and 24 ; and
  • Fig. 1 10 is a diagrammatic view from the end of a micro-length tube element being plucked from an open fluidic channel of smaller width than the element, using a tweezer the same as shown in Fig. 50G.
  • a hollow flow element or a micro-length tube element i.e., a tube element having length less than 700 micron, preferably less than 500 micron, and in many cases about 200 micron, and a micro-bore diameter between about 75 +/- 50 micron that is fixed in a flow channel and exposed to flow of liquid sample, e.g., a glass nano reactor "GNR".
  • GNR glass nano reactor
  • micro-bore diameter at least 25 micron and preferably about 75 micron is similarly found to aid in the uniformity of the coating, provide increased immunity to clogging due to debris in the fluid, and still able to produce excellent assay results, while limiting the internal diameter to about 125 micron enables assays in many cases to approach the ambient assay condition.
  • Such devices are typically made of endlessly drawn micro-bore filament such as used to form capillary tubes, but in this case, the filament is finely chopped in length to form discrete, shorter micro-length flow elements. It is realized that capture agent immobilized on the surface of such a device, applied by immersion techniques, can raise a significant depletion problem.
  • any analyte in an ELISA or sandwich type of amino assay on antigen will bind to a capture antibody in a way that is governed by a kinetic reaction, a dynamic process.
  • analyte such as an antigen binds to capture agent such as an antibody
  • capture agent such as an antibody
  • the reverse also occurs, the bound analyte molecules unbind from the capture agent.
  • the kinetics concern an "on" rate and an “off rate— analyte being captured and analyte being released.
  • the capture reaction will continue, depleting the analyte in the ambient volume, and reducing its net rate of capture, until the system reaches equilibrium in which the rate of binding is equal to the rate of unbinding.
  • the gradual action occurs according to a substantially exponential curve.
  • the absolute value of the equilibrium condition depends on the original concentration of the analyte in the volume of sample being assayed. Increase in concentration results in a higher signal, decrease in concentration results in a lower signal. In cases in which assay depletion occurs, the concentration of the analyte in the sample is detrimentally decreased over time. It is realized that micro-length tubes fixed in flow channel may present an excess of capture agent in the volume of liquid sample to which the element is exposed, decreasing the effective concentration of the analyte. The concentration decreases at an excessive rate, relative to initial, starting point concentration sought to be measured.
  • micro- flow elements of various descriptions that are coated by immersion or the like and used in an immunoassay or sandwich assay or even a molecular diagnostic type of assay.
  • capture agent e.g. an antibody or some type of moiety that is a capture molecule for the analyte to be sensed or detected
  • One object of invention is to overcome this problem with respect to micro-length tube elements characterized by an inside surface and an outside surface, or often also with two end surfaces. Adding all surface area over which a density of capture molecules is coated can add up to a surface area on the order of over 100,000 square microns.
  • micro-length tube having on the order of about: a length of 200 microns, an external diameter or width of 125 microns, and an internal diameter or width of 70 microns.
  • a particular problem addressed here is to find practical approaches for accurately reducing active surface area of immersion-coated flow assay elements in general, and in particular, micro-flow or hollow elements, and in particular micro-length tube elements.
  • the discussion here refers to "diameter” and the presently preferred outside and internal profile of micro-length elements is cylindrical, it will be understood that tube elements of various outside and inside profile are easily manufactured and can work as well.
  • the use of the term "diameter' is used broadly here, to refer to the width of an element, no matter what its exact profile).
  • a further problem being addressed here concerns treated microflow-elements that are to be in fixed positions in channels for exposure to flow of sample. It is desirable to expose the elements in batch, in free state to an immobilization process for applying the capture agent or antibody to the element surface, and then transfer each element mechanically to its fixed position in a channel, for instance in a channel of a multiplex micro-fluidic "chip" (or"cartridge” or “cassette”). It is desired to use a quick and accurate placement process, for instance a pick and place device mounted on an accurate X, Y stage. For such purpose, it is desirable to physically contact the tiny element for picking it up from a surface and placing it in an open channel, which is then closed to form a micro-fluidic passage.
  • grippers e.g. a tweezer instrument that contacts the outer surface of the device.
  • the pick and place action is made possible by pre-aligning open channels to receive the micro-flow elements and the surface on which the free elements are supplied with the automated pick-and-place instrument. This enables the grippers to pick up and place the micro- flow elements precisely in desired flow channel positions in which they are to be fixed.
  • an active capture agent e.g.
  • Such a coating is susceptible to mechanical damage as a result of the mechanical manipulation process. Outside surfaces of micro- flow elements come in contact with (a) a supply surface, e.g. an aligning pocket or groove, (b) the transferring grippers, and (c) surfaces of the channel in which it is being deposited. All of these contact opportunities give rise to possible damage to the fragile coated capture agent, which typically is a very thin layer of antibody or the like adsorbed to the surface of the flow element. This coating is often only a few molecules thick, thickness of the order of nanometers or tens of nanometers, and is quite fragile. The net result of damaging a capture surface of the placed micro-element is seen during read out of the assay. If the surface has been scratched or perturbed in any way, that can give rise to an irregular concentration or presentation of captured analyte, the signal can be irregular, and contribute to irreproducibility or poor performance of the assay.
  • Discrete micro-flow elements are immersed in liquid containing capture agent, such as antibodies or antigens, and, after coating by the liquid, are picked, and placed into channels for flow-through assays.
  • the micro-flow elements are in preferred form of discrete micro-length tubes, defined as micro-flow elements of length less than about 700 micron, and bore diameter of 70 +/- 50 micron.
  • the flow elements are surface-treated so active capture agent, e.g. capture antibody, is not on the outside, or is of limited outside area.
  • micro-flow elements or in particular, micro- length tubes
  • a bath of active agent and violently agitated resulting in coating of protected inside surface, but due to extreme shear forces, a clean area on the outside surface, for instance the entire outside cylindrical surface of a round cross- section discrete micro-length tube.
  • a special filament-manufacturing process is conceived that results in preventing coating an exterior surface of flow elements with a predetermined capture agent. Capture agent on selected coated areas are ablated or deactivated with precisely positioned laser beam, such as can be produced by a mask for simultaneous treatment of a large number of elements, leaving residual active agent of defined area on the inside surface of micro-flow elements.
  • Residual capture agent itself, on the inside of the elements, usefully defines a readable code related to the desired assay.
  • Flow channel shape is sized relative to flow elements fixed in the channel to allow (a) bypass channel flow along the exposed outside of a micro-flow element to reach and flow through later elements in the channel in case of clogging of the first element, along with (b) sample and assay liquid flow through the micro-flow element to expose the surface to capture agent and other assay liquids.
  • the element can simply be gripped, as by an elastomeric sheet pressed against the element. Electrostatic attraction between flow element and channel wall is employed to fix the element in position, overcoming any disturbing force of the placing instrument as it is drawn away after delivery of the element.
  • micro-flow element geometry After assay, fluorescence is excited and read by special scanning confined to micro-flow element geometry. Locators are seeded in the recorded data, and used to locate the regions of interest in detected fluorescence data, e.g. from micro-length tubes. Code, written with the capture agent substance inside the micro-flow element is read through a transparent wall of the element. Efficient assembly and tooling features are disclosed. All features are applicable to micro-length tubes, enabling their efficient use. A number of the features are or will be found to be useful with other hollow elements, for example, longer micro-flow elements.
  • the purpose of this invention is to deliver a method for performing a fluorescence measurement of multiple immobilized elements contained in a microfluidic chip.
  • This method provides for determining the paths to be followed during the scanning, as well as the proper focus, and camera exposure.
  • the method is based on a known general chip layout.
  • the method provided results in the ability to place the chip to be measured into the scanner and then start the scan without any additional manual settings required.
  • the method does the rest, and produces the desired fluorescence measurements as the results.
  • Certain aspects of invention involve eliminating or preventing the occurrence of active capture agent on outside surfaces of micro-flow elements, e.g. extended outside cylindrical surface, and/or end surfaces, while leaving active capture agent on the inside surface unperturbed, or of a desirable area or pattern.
  • Features addressing this aspect include techniques to selectively limit the capture agent on the interior surface and steps that act in combination with outside and inside surfaces to achieve the desired result.
  • a first technique is employed to eliminate or prevent capture agent, e.g. antibody, from immobilizing to the outside surface of hollow flow elements, especially, micro-length tube elements. That is done during a batch coating process, and involves suspending discrete hollow elements, especially micro-length tube elements, in an Eppendorff tube or other tube with the capture agent of interest and aggressively agitating fluid to impart disrupting shear forces to the exterior surface of the elements. Preferably, this is achieved by vortexing the fluid at high speed, for instance employing an instrument that orbits the container at approximately 2000 rpm of the orbiter, about an orbital path of the supporting shaft of diameter of about 25 mm.
  • capture agent e.g. antibody
  • the micro-tube elements are placed with a volume, e.g. a milliliter of capture agent, e.g. antibody.
  • a volume e.g. a milliliter of capture agent, e.g. antibody.
  • the appropriate vortexing speed is dependent e.g. on the nature of the suspension, e.g. the viscosity of the liquid chosen, and can be easily determined experimentally. It is set by observing whether the capture agent is effectively non-existent on the outside, long surface of the micro- length tube elements, e.g. the outside cylindrical surface in the case of the body being of circular cross-section.
  • the physical principle involved concerns shearing force on the outside surface of the micro-length tube element (or micro-tube element) that acts to prevent binding of the capture agent to the surface through an adsorption process.
  • the inside surface is environmentally shielded from this shearing by virtue of the geometry which is tubular, and the micro-size of the bore of the tube. This prevents vortexing from causing any turbulence to occur within the element.
  • the observed result of aggressive agitation is that fluorescence which is observed by performing a sandwich assay is completely absent from the outer cylindrical surface of a micro-length tube element, whereas it is present in an observable way on the inside surface.
  • fluorescence is also present on the end faces of elements.
  • Vortexing is the presently preferred technique for producing the shear forces.
  • the case showed here employs orbitally rotating the micro tube in a very rapid manner back and forth in small circles at a rate of approximately a couple thousand rotations per minute, and an excursion of about 25 mm.
  • any type of rapid oscillation that creates a high degree of turbulence can be employed, so a back and forth motion, a circular rotation, anything that would very rapidly mix the fluids and create high shear forces will suffice.
  • micro-length tube elements in the presence of aggressive agitation leads to removal of capture agent, e.g. antibodies, from outside surface of the elements, and prevention of their coating with the agent, but leaves the inside surface of the micro-length tube element in condition to immobilize capture agent, e.g. capture antibodies, for subsequent interaction with analyte of the sample.
  • capture agent e.g. antibodies
  • a non-stick coating e.g. sputtered gold, silver or graphite
  • Silane or similar coating must be applied to receiving surfaces before capture agent, e.g. antibodies will attach.
  • capture agent e.g. antibodies will attach.
  • the surface will not receive the silane or equivalent, then likewise, the active capture agent.
  • Another feature of invention concerns realizing the desirability and technique of removing coated capture agent from selected end surfaces of the flow elements and a margin portion or other portion of the interior surface.
  • the micro-length tube elements are further processed using a laser elimination process that removes or de-activates capture agent, e.g. antibodies, from surface from which the agent was not removed by the high shear process.
  • capture agent e.g. antibodies
  • Those surfaces include transverse end surfaces and a selected portion of the inside surface, leaving only an annular stripe on the inside surface sized sufficient to process the assay, but small enough to reduce depletion of the analyte from the sample.
  • an ablating laser is arranged transversely to the axis of elongation of the micro-length tube elements with the effect that the energy arrives though parallel to the end faces has a neutralizing or removal effect on the capture agent that is on those end faces, as a result of incidence of substantially parallel radiation, but also of internal reflection scattering of the radiation by the transparent substance that defines those end faces.
  • the net effect of two novel processes described, if used in novel combination, is to leave only a band of selected dimension, which can be small, of capture agent immobilized on the inside surface of the micro-length tube element. This can be done in a way that leaves one or more bands separated by a space of no capture agent. Thus one can generate a single band in the center or a single band closer to one end or multiple bands distributed along the length of the micro-length tube element. These bands can be of different widths, can have different spacing, and can be of the form of a code, e.g. a bar code, which is useful to encode the particular flow element, e.g. micro-length tube element.
  • a code e.g. a bar code
  • the short, hollow elements are first cut, i.e. chopped, from previously supplied continuous small-bore filament into the short, discrete elements. They are then treated in batch manner. A bulk of the micro-length tube elements (or micro-tube or hollow elements) is then exposed in an Eppendorff tube to wash buffer. After washing processing is performed, the buffer is removed, and replaced with a silane. By use of this simple, low-cost immersion step, the silane is allowed to bind to all of the surfaces of the micro-length tube elements. Excess silane after a period of time is washed away with water in a buffer. Then a capture agent, e.g.
  • the antibody, in solution is added to the Eppendorff tube with the bulk of elements and allowed to incubate overnight.
  • the incubation is performed on the orbital vortexer for approximately 16 hours at 2000 rotations per minute, with of the order of one-centimeter diameter orbital motion.
  • the orbital plate that contains the numerous Eppendorff tubes is approximately 6 inches in diameter, but the orbital motion is a circular pattern counterclockwise and then clockwise motion in a circular pattern of diameter of approximately 2 centimeters.
  • the net result is that the capture agent has been immobilized on the inside surface of the element and also on the end faces but it is not present on the outside cylindrical surface of the tubular element.
  • the capture agent solution is removed from the Eppendorff tube, which is replaced with a wash buffer solution, and the wash buffer solution is then further replaced with a stabilizing buffer, what is called a blocking buffer.
  • a stabilizing buffer what is called a blocking buffer.
  • a commercial material called StabilCoat® solution is used.
  • StabilCoat® blocking solution is introduced to the Eppendorff tube along with micro-length tube elements, then a portion of those elements is aspirated in a pipette along with some of the StabilCoat®, and dispensed onto an alignment plate.
  • the alignment plate contains a series of rectangular shaped pockets, each designed to accommodate and position a single micro- tube element within a small space, preferably with clearance tolerance sized in microns, a space of 10 to 50 microns between the micro-length tube element and the walls of the pocket. After the elements are allowed to roam on the plate, they fall into these pockets still in the presence of the solution of the buffer solution. The excess buffer solution is removed from the alignment plate, Fig.
  • the process of creating immobilized antibodies or other active biological species to serve as assay capture agent on micro-particles, and particularly the inside of hollow glass micro-particle elements, and then transferring the functionalized micro-particles to the channel of a microfluidic device, according to the invention, involves a number of critical steps.
  • Long spools of continuous capillary tubing of approximately 125 micron OD, 70 micron ID, with a few microns thick polyamide protective coating on the exterior are obtained from a manufacturer. Typically these are drawn, fiber-like filaments, with highly polished and accurately dimensioned inner and outer surfaces.
  • the spools are rewound onto a mandril in a tight mono-layer wrapping fashion such that each turn or strand, wound around the mandril is in contact with adjacent strands.
  • the wrapped series of strands consists of for instance a hundred strands. It is then wrapped with an adhesive tape by which the strands are captured in an assembled unit. The tape is then slit parallel to the mandrill axis and the tape removed bringing the strands with them. This produces a thin linear array of monofilaments in close contact with one another.
  • the relatively long array of monofilaments is then presented to a wafer dicing saw(as used in the semiconductor industry for dicing thin ceramic wafers), the filaments are diced at repeat distances of approximately 250 microns, producing cylindrical tubular micro-particles of that length.
  • the individual micro-particle elements still retained on the tape are liberated using a hot aqueous detergent solution which liberates the individual elements from the tape. They are allowed to settle into the bottom of a beaker, the tape is removed from the solution, then the aqueous solution is removed and replaced with a hot sulfuric acid and peroxide solution, used to dissolve the polyamide coating from the outside surface of the glass elements. This is followed by a substantial washing cycle, a number of flushings with de-ionized water to remove the residual sulfuric acid solution. After the hollow glass elements have been thoroughly washed, they are silanized using a silane reagent such as APTES which stands for aminopropyltriethoxysilane ("silane").
  • silane reagent such as APTES which stands for aminopropyltriethoxysilane ("silane").
  • micro-particles are allowed to incubate in the silane solution for approximately an hour after which they are rinsed and cured in an oven for another hour after which they're then stored in an ethanol solution.
  • silane reaches the inside surface of the tubular micro-particles, vigorous vortexing is used to uniformly distribute the silane throughout the interior of the hollow elements.
  • the micro-particles are then transferred to a reagent solution containing the capture molecule of interest, for example a capture antibody.
  • the silanized micro-particles are transferred to a vial containing the capture agent for example a capture antibody.
  • the capture antibody is allowed to bind to the active silane surface for a period of 16 to 24 hours after which a rinsing cycle is performed to remove any loosely bound capture agent and then finally the functionalized micro-particle elements are transferred to another vial containing a stabilizing compound such as SurModics' brand StabilCoat®. They remain in the StabilCoat® solution until it is desired for them to be transferred to a microfluidic device, such as a microfluidic cartridge. They are stored in the StabilCoat solution in a refrigerator until ready to be used.
  • a stabilizing compound such as SurModics' brand StabilCoat®
  • the particles are re-suspended in a stabilizing compound such as StabilCoat (trademark of SurModics, Inc.) for storage until needed.
  • a stabilizing compound such as StabilCoat (trademark of SurModics, Inc.) for storage until needed.
  • StabilCoat trademark of SurModics, Inc.
  • the purpose of the stabilizing compound is to protect the activity of the immobilized species when the micro-particles are taken out of the reagent and exposed to atmosphere.
  • the stabilizing reagent consists of high concentrations of sugars and proprietary compounds. We have found that when the water component is allowed to evaporate, thick residue of this sugary compound is left behind, which under low humidity conditions has a tendency to crystallize and become a fairly rigid structure which can cause particles in this compound to become almost irreversibly stuck to any surface that it comes in contact with and has been allowed to dry in.
  • a preferred step in the process of moving the micro-particles from the liquid state to the dry state involves dispensing the micro-particles in a solution of the protective coating liquid, e.g., StabilCoat, onto an alignment plate such as a precisely micro-machined grooved pocketed plate such as a silicon micro- machined plate with pockets configured to accept the micro-particles in an array pattern.
  • the shaped pockets are, e.g., rectangular in the case of short segments of capillary tubing forming hollow micro-particle elements.
  • the excess liquid compound is either spun off in a centrifuge or wicked away using an absorbent pad, leaving behind a small residue of protective compound about the micro-particle, e.g., both inside the hollow glass micro-particle element and around the outside of the element in the pocket capturing the element.
  • the exterior diameter have a diameter or width within the range of 1.2 and 4 times the internal diameter or width
  • length of the hollow flow elements best results are obtained with lengths of less than about 700 micron, and in many cases, less than 500 micron. In a presently preferred form, the length is 250 um.
  • the interior diameter have a diameter or width have a length to inner diameter of 20: 1.
  • Figs. 50A-50F This involves distributing the micro-particles in random fashion onto a flat surface, Fig. 50A, not having grooves or alignment pockets.
  • the advantage of this process is not requiring a micro machined component for the manufacturing process.
  • the disadvantage is that the micro-particles are randomly distributed in a pile. The tendency is for them to come to rest in a monolayer on the surface, but with random orientation. Further, as a result of removing the excess StabilCoat by centrifuge or wicking away the excess stabilizing solution, it has been observed that the micro- particles have a tendency to agglomerate into a dense pack of the randomly oriented elements.
  • a placement tool e.g., a vacuum tip, Figs. 50C, D that engages the top surface of the elements, in combination with a video system used to identify an individual element and a motion system (computer controller) responsive to the video system, which orients the relative relation of the vacuum pickup tip and the micro-particles in both X and Y coordinates, and angular orientation.
  • a placement tool e.g., a vacuum tip, Figs. 50C, D that engages the top surface of the elements
  • a video system used to identify an individual element
  • a motion system computer controller
  • the placement tool can be moved in minute movement, slightly laterally within channel, to bring GNR against one side wall.
  • a table carrying the fluidic layer preferably channel side up, moves in computer-controlled X, Y and Z, and the placement tool is stationary, with only the grippers (e.g. tweezers) moving under computer control.
  • the manufacturer temporarily secures the micro- particles in open channels prior to this fluidic component with open-sided channels being turned upside down for bonding to the flexible membrane.
  • the membrane is carried by the pneumatic component of the cartridge, to complete the assembly.
  • Electrostatic attraction draws the GNRs from the placement tool (enabling tool withdrawal) and holds them against one side of the over-sized channel with sufficient certainty that the assembly can be overturned for bonding against the membrane.
  • Compressive force of the elastically compressible membrane permanently then fixes the GNRs permanently in position.
  • the open channel in a resilient PDMs (silicone rubber) channel-defining layer is slightly undersize in width and may be oversize in depth relative to the GNR.
  • the placement tool thrusts the GNR down with force-fit into the channel, and the sides of the channel are slightly, resiliently deformed to accept the GNRs. The sides then grip the micro-particles tightly.
  • the GNRs may even be thrust so deep into the channels that they are submerged below the face plane of the fluidic layer.
  • the GNRs are short segments of fine capillary tubing, e.g., outside diameter, e.g., 125 micron, inside diameter 70 micron and length 250 micron).
  • the accepting microfluidic channels in one instance are (uniquely) wider than the micro-particles, and shallower. Successful placing depends upon electrostatic force associated with silicone rubber (PDMS, an electric insulator material ) to attract the micro-particles from the placing instrument, and retain them in position during the completion of the assembly process, which even involves turning the over-size channels upside down. In this case elastic deformation of the covering membrane at the sites of the raised top surface of the micro-particles permanently fixes the location of the micro-particles.
  • PDMS silicone rubber
  • an electric insulator material an electric insulator material
  • the microfluidic channels are undersize, widthwise, relative to the micro-particle width, Figs. 50 G, H, and the placing tool forces the micro-particles into the channels, to obtain a mechanical grip by elastomeric material forming the sides of the channel.
  • the microfluidic channels can have depth less than the micro-particles, so that the membrane stretches over them to further fix their location.
  • the channel depth may exceed the depth of the micro-particles, and the placement submerges the particles such that the overlying membrane is not locally disturbed by the presence of the micro-particles.
  • micro-particles and particularly on the inside of hollow micro- particle elements (micro-length tube elements) will now be described by reference to a specific example. This will be followed by examples of novel transfer of functionalized micro-particles to an operative position within a microfluidic device, for instance to a microfluidic channel.
  • long spools of continuous glass capillary tubing of approximately 125 micron OD, 70 micron ID, with a few microns thick polyamide protective coating on the exterior are obtained from a manufacturer. Typically these are drawn, fiber-like filaments, with highly polished and accurately dimensioned inner and outer surfaces.
  • the spools are rewound onto a mandrel in a tight mono-layer wrapping fashion such that turns or strands wound around the mandrel are in contact with adjacent strands.
  • the wrapped series of strands comprises, for instance, one hundred strands.
  • the strands are then wrapped on their outside with an adhesive tape by which the strands are captured in an assembled unit. The tape is then slit at one point parallel to the mandrel axis and the tape with the strands is removed. This produces a thin linear array of
  • the relatively long array of monofilaments is then presented to a wafer dicing saw (as used in the semiconductor industry for dicing thin ceramic wafers).
  • the filaments are diced at repeat distances of the order of 1000 micron or less, to produce micro-length tube elements.
  • the repeat distances are less than 700 micron, and in preferred instances, of the order of 250 microns, producing cylindrical micro-length glass tube particles of that length.
  • Those tubes having internal volume of the order of a nanoliter and are referred to as "glass nano reactors", or "GNR"s).
  • the individual micro-length particles or elements are liberated from the tape using a hot aqueous detergent solution. They are allowed to settle into the bottom of a beaker and the tape is removed from the solution. Then the aqueous solution is removed and replaced with a hot sulfuric acid and peroxide solution, to dissolve the polyamide coating from the outside surface of the glass elements. This is followed by a substantial washing cycle, employing a number of flushings with de-ionized water to remove the residual sulfuric acid solution. After the hollow glass elements have been thoroughly washed, they are silanized using a silane reagent such as APTES
  • micro-particles are allowed to incubate in the silane solution for approximately an hour. To ensure the silane reaches the inside surface of the tubular micro-particles, vigorous vortexing is used to uniformly distribute the silane throughout their interior to form a silane coating.
  • the micro- length tubes are then rinsed and cured in an oven for another hour after which they are stored in an ethanol solution, ready for being functionalized, (i.e., treated to surface- immobilize a capture molecule of interest, for example a capture antibody).
  • the silanized micro-particles are transferred to a vial containing the capture agent, for example a capture antibody.
  • the capture agent for example a capture antibody.
  • vigorous vortexing is used, which is found to be capable of substantially uniformly distributing the capture agent throughout the tubular interior of the micro-length particles to produce substantially uniform immobilization of the capture agent over the length of the interior surface.
  • the capture antibody under these conditions is allowed to bind to the active silane surface for a period of 16 to 24 hours.
  • the vortexing conditions of the process prevent immobilization of the capture agent to occur to longitudinally-extending outer surfaces of the violently agitated micro-length particles.
  • the functionalized micro-particle elements are re-suspended by transfer to another vial.
  • the vial contains a stabilizing compound such as SurModics brand StabilCoat tm .
  • the purpose of the stabilizing compound is to protect the activity of the immobilized species when the micro-particles are taken out of the reagent and exposed to ambient conditions.
  • the functionalized micro-length tube elements remain in the stabilizing solution stored in a refrigerator until ready to be used, i.e. until it is desired for them to be transferred to a dry state within a microfluidic device, such as a microfluidic cartridge.
  • a preferred step in the process of moving the micro-particles from the liquid state to the dry state involves dispensing the micro-length particles in a solution of the protective coating liquid, e.g., StabilCoat, onto a so called "pick-up plate", for presenting the micro-particles for subsequent picking and placing in microfluidic channels.
  • a pick-up plate is a precisely micro-machined grooved locator plate, preferably a pocketed locator plate such as a silicon micro- machined plate, with pockets configured to capture individual micro-particles in an array pattern.
  • the shaped pockets are, for example, rectangular in the case of short segments of capillary tubing forming hollow micro-particle elements (micro-length tubes).
  • the excess liquid compound is either spun off in a centrifuge or wicked away using an absorbent pad, as described herein. Either technique leaves behind a small residue of protective compound about the micro-particle, e.g., both inside the hollow glass micro-particle element and around the outside of the element, i.e. in the pocket capturing the element. In cases later described in using a planar pick up plate, a small residue remains with the elements in a puddle on the plane surface on which the elements are displayed. In mass manufacture, it is typically desired, after applying the micro-particles to a pick up plate, to store the plate and particles prior to installation into the microfluidic device.
  • the stabilizing reagent consists of high concentrations of sugars and proprietary compounds.
  • the water component is allowed to evaporate, thick residue of this sugary compound is left behind, which under low humidity conditions has a tendency to crystallize and become a fairly rigid structure which can cause micro-particles in this compound to become almost irreversibly adhered to any surface with which it comes in contact and allowed to dry.
  • Figs. 15-21 ; 42-50 When ready to assemble, the pick and place technique elsewhere described herein (Figs. 15-21 ; 42-50) is employed in which the micro-particles are first disposed in a groove or pocket of a locator plate, from which they are picked by tweezer (Figs. 15-21C; 43; 45) or vacuum tool (Figs. 46-49A).
  • An alternative, novel technique involves distributing the micro-particles in random fashion onto a flat surface, Fig. 10D, not having grooves or alignment pockets.
  • the advantage of this process is not requiring a micro machined plate component for the manufacturing process.
  • the disadvantage is that the micro- particles are randomly distributed in a pile. The tendency is for them to come to rest in a monolayer on the surface, but with random orientation. Further, as a result of removing the excess stabilizing reagent (StabilCoat) by centrifuge or wicking away the excess stabilizing solution, it has been observed that the micro-particles have a tendency to agglomerate into a dense monolayer concentration of randomly oriented elements. However we realize that there are solutions to this problem.
  • Individual micro-particles can be picked from this dense concentration by use of an automated vacuum tip that engages the top surface of the elements, such as previously described herein, in combination with a computer-control vision system used to identify an individual element and its orientation from a detected image, and a motion system responsive to the vision system, which orients the relative relation of the vacuum pickup tip and the plate supporting the micro-particles in both X and Y coordinates and angular orientation.
  • automated tweezers may be operated with controlled motions in X and Y coordinates, and angular orientation, as described herein.
  • the GNRs immediately after immobilization are stabilized in a reagent that is capable of protecting or stabilizing the catcher antibody on the surface of the GNRs, e.g., StabilCoat®.
  • GNRs are transferred to an alignment plate and dispensed on to the alignment plate in a puddle with the stabilizing reagent and the liquid form of the stabilizing reagent is used to aid in the assembly or the self-assembly of the GNRs into certain alignment pockets.
  • the stabilizing compound has a very high sugar and salt content it is desirable to remove as much of the mass of the liquid from the surface or around the surface of the GNRs prior to allowing any residual reagent to evaporate.
  • a capillary working process is used to soak up residual stabilizing reagent (StabilCoat®) into the absorbent pad and draw out of the channels through the channels and out of the channels and away from the GNRs.
  • StabilCoat® residual stabilizing reagent
  • Typical dimensions for the channels are now given for the device shown in Fig. 10 A-C.
  • the plane view, Fig. 10A shows the channels retaining the GNRs.
  • the GNRs are located in pockets approximately 125 microns wide by 400 microns in length separated from each other by a narrower channel approximately 75-80 microns in width by 200 microns in length.
  • the Figures are broken away, showing microtubes 1, 2 and n, where n may be as large as about 50.
  • the narrow channel is provided to aid in the flow of the excess reagent through the channels and away from the GNRs to the wicking (absorbent) pad and narrower to prevent GNRs from migrating out of the retaining pocket that they are intended to fall into.
  • the length of the channels can range typically from 5 millimeters to 25 millimeters, or even 75 millimeters depending on the size or the scale of the manufacturing process involved.
  • the number of GNRs ranges from a few hundred to a few thousand depending on the cross-section area. Per channel, the number of GNRs is on the order of 50.
  • Fig. 10B is a side view of the GNRs on a surface in a channel with a puddle of stabilizing reagent (StabilCoat®) surrounding the channels and contained on and around the plate. It depicts a wicking (absorbent) pad in the process of being moved toward the end of the channels full of the stabilizing solution.
  • StabilCoat® stabilizing reagent
  • Fig. IOC illustrates a wicking pad shown on the left top surface of the plate and having drawn the excess stabilizing reagent (StabilCoat®) away from the GNRs through the channels.
  • StabilCoat® stabilizing reagent
  • the micro-length tube elements (or hollow elements) while still in the plate can be further processed with a laser, preferably an ultraviolet laser, which could be an excimer laser, fluoride or krypton fluoride laser, with two beams that are spaced such that the ends and a an end margin portion or section of the element are exposed perpendicular to the element axis by a laser beam in a way that either ablates or denatures the capture agent, e.g. antibody, from the ends of the element as well as a section of the inside surface of the element.
  • a laser beam preferably an ultraviolet laser, which could be an excimer laser, fluoride or krypton fluoride laser, with two beams that are spaced such that the ends and a an end margin portion or section of the element are exposed perpendicular to the element axis by a laser beam in a way that either ablates or denatures the capture agent, e.g. antibody, from the ends of the element as well as a section of the inside surface of
  • the advantage of so treating the surfaces of the micro-length tube element, in reducing depletion, and in particular, with regard to the considerations of by-pass flow may be understood by considering the total surface area exposed to the coating solution as represented by the sum A+2B+C where A is the internal cylindrical surface, B is the end face, and C is the external cylindrical surface area. It is pointed out that according to the concepts articulated that it is possible to reduce the depletion area to A only, that being the only area that carries meaningful information. Indeed the reduction can be extended by suitably sizing the laser beams to treat the margins of the inner cylindrical surface from which the active agent has been removed or has been deactivated, which can be of preselected length.
  • A, B, and C where C is the exterior surface area of the micro-length tube element having a length dimension of approximately 250 microns, interior diameter of 75 microns and an outside diameter of 125 microns, C being the exterior surface area is approximately 98,000 square microns.
  • B which is the two-times the end face surface area is approximately 15,600 square microns, and A being the inside surface area approximately 58,000 square microns.
  • the total of A, 2B, and C is approximately 171,000 square microns.
  • Assays run in the microfluidic device can use various types of micro-length tube elements with different capture agent present on the inside surface of the elements.
  • one type of micro-length tube element would contain a capture antibody associated with the antibody interleukin-6.
  • Another one could be interleukin-2, and yet a third would be interleukin-12.
  • Each of the micro-length tube elements of respective types can be placed into different channels and or locations within that channel, thereby determining at the time of performing the assay what type or what particular antibody was used in that particular location. That is a method for identifying the type micro-length tube element for what has bound onto that surface.
  • striped code pattern created by selectively ablating or selectively denaturing antibody functionality along the length of the micro-length tube element for providing for a code, a bar code, which is then used to identify that particular type.
  • a wavelength in the range of about 193 nanometers to 250 nanometers is presently preferred.
  • One possible way is to establish a single laser beam of a particular width and then translating micro-length tube element, then dispensing a fluence of laser to the element, turning the laser off, translating to a new position then dispensing another amount of fluence thereby eliminating or denaturing only portions of a particular tubular element.
  • Another method is to use an opaque mask to establish a particular pattern, and illuminate simultaneously the entire micro-length tube area with the ultraviolet light.
  • Yet another method is to scan the micro-length tube element using a synchronized system of X, Y galvanometers.
  • micro-length tube elements Besides writing the code, one could scan simply the ends of the micro-length tube elements to ablate capture agent. It is anticipated that a feature size as small as 30-40 microns is possible with an ultraviolet laser. For example, a 250-micron long micro-length tube element having a feature size of 30 microns would result in 8 possible stripe zones and with 8 possible stripe zones, one could produce a number of patterns. For example, a pattern using a binary coding system would lead to a total number of combinations of 28, which are 256 possible combinations.
  • Step (1) provide micro-length tube elements.
  • Step (2) apply coating, the manufacturing process, provide a coating particular of a capture antibody
  • Step 3 a manufacturing process that coats only the inside surface of the micro- length tuber element.
  • a capture agent e.g. antibody
  • the second means of coating only the inside of the micro-length tube element.
  • the agitation process would prevent coating of the antibody on the outside surface only, but not the end faces. So further reducing surface coating on the end faces would involve using a laser process.
  • the second means of preventing coating on the outside surface concerns the manufacturing process at the stage of drawing and coating endless micro-bore tubing, prior to chopping to form discrete micro-flow elements or micro- length tube elements.
  • a-bond-preventing coating prior to the usual polymer coating prevents silanization of the exterior surface, preventing silane from adhering to the outside surface, and therefore defeating the ability for many capture agents, for instance antibodies and antigens, from adhering to the surface.. This would further prevent antibodies from being bound to the outside surface.
  • the polymer coating is added to the glass filament during its manufacturing process to maintain the intrinsic strength of the glass filament.
  • the process for providing or manufacturing the micro-length tube elements, the raw elements without a coating involve chopping the micro-bore tubing into the particular length element, which for the preferred configuration is approximately 250 microns, then removing the polymer coating using a sequence of acid and basic baths. Subsequent to the manufacturing process that coats only the inside surface of the micro-length tube element, the micro-length tube elements are then secured into a channel of a microfluidic device.
  • the channel of the microfluidic device is configured such that a portion of the flow is allowed to bypass the outside of the micro-length tube element.
  • Micro-length tube elements are placed into a channel in a way that, preferably, approximately two times the flow volume proceeds around the outside of the tubular element as compared to the volume proceeding through the inside area. So the ratio of the cross- sectional areas of the by-pass flow versus the inside diameter is approximately 2: 1.
  • Micro-length tube elements are then secured into the channel whereby the channel wall is an elastomer, which allows the micro-length tube element to be placed in the channel, and the grippers used to place the element into the channel can be released because the adhesion of the elastomer is sufficient to secure the element into the channel.
  • a top is then secured over the open channel containing the micro-length tube element.
  • the top also includes an elastomeric material. That elastomeric material is used to compress and thus secure the element in the channel because the open channel duct is of smaller depth than the outside diameter of the tubular element.
  • the elastomeric "roof or top thus provides a means of securing the micro-length tube elements in their locations in that channel.
  • the membrane or the flexible layer that is actuated by vacuum or pressure to operate the valves and the pistons is made from elastomeric material and different, advantageous techniques are used to fabricate the device.
  • the two subassemblies prior to aligning. Once the subassemblies are aligned, the two subassemblies are brought together under bonding conditions to form one completed assembly, and fixing the embedded location of the elements. Then the two subassemblies are brought together to complete the fluidic channels. Bringing them together completes the valve and piston devices as well as embedding the detection elements. These features occur with a non-permanently bonded implementation.
  • End of arm tooling (tweezer or vacuum probe)
  • Source / Target X, Y table (moves in X and Y coordinates)
  • the subassembly 46 i.e. the controls/reservoir layer 46, is comprised of two elements, the upper injection molded or machined plastic component 56 with a PDMS membrane sheet 38 bonded to its lower surface.
  • the bottom fluidic layer or subassembly 50 has detection elements, e.g. hollow short cylindrical flow elements 32.
  • the fluidic subassembly consists of a thin glass sheet 42 with a PDMS gasket or sheet 38 permanently bonded face-wise to its upper surface, the sheet 38 having cut-outs defining fluidic channels between channel walls 44, the channel bottomed on the glass sheet 42, Fig. 31C.
  • the detection elements are dispensed, in the embodiment shown, by pick and place action, into fixed positions in the channels of the fluidic layer 48.
  • the two subassemblies 46 and 50 are brought together and bonded in a way that provides fluid-tight and leak- free operation, but also enables the actuation of valves and pistons by portions of membrane 38.
  • One novel a feature of this construction is that the two subassemblies as described, using a PDMS gasket, enables capture or embedding detection elements, here extremely short hollow flow elements, (Micro-length tube elements) into channels. Combining those two subassemblies into a single assembly provides the functionality of having microfluidic channels that contain the hollow flow elements as well as functioning valves and pistons.
  • the fluidic subassembly is assembled by covalently bonding PDMS to glass, and then upper assembly, the reservoir assembly is formed by covalently bonding PDMS to plastic.
  • the dominant advantage is the placing the discrete, small detection elements, the hollow flow elements, into open channels prior to assembling.
  • the importance of the technique also relates to enabling the immobilization of capture agent, e.g. antibody, onto a solid substrate in an efficient batch process, thereby allowing many thousands of these elements to be fabricated in one very simple batch process, which is cost effective and highly reproducible.
  • capture agent e.g. antibody
  • features of the concept include bringing together subassemblies to capture elements in a fixed position, the capture (or detection) elements having been pre-prepared in batch process, with the final assembly, which employing a bonding process, especially the permanent plasma bonding process to join the subassemblies, and doing it in a selective way at the valve seats by repeatedly locally deflecting and bringing in contact the valving surfaces, which will now be described.
  • Valve Break-In Process Connect pneumatic control input ports to externally controlled pneumatic line/s
  • Native PDMS comprised mainly of repeating groups of -0-Si(CH3)2 - is hydrophobic in nature, and, without special treatment, has a tendency to adhere to, but not permanently bond to other like surfaces such as PDMS, glass and silicon.
  • Oxygen plasma and similar techniques have control parameters such as pressure, power, and time all of which determine the concentration of surface OH groups. Higher concentrations of OH groups lead to more covalent bonds between the two surface and therefore higher mechanical bonds.
  • the hydrophilic surface will undergo “recovery” back to its native hydrophobic state via migration of short, mobile polymer chains from the bulk to the surface. Full “recovery” occurs over a period of hours at room temperature and can be accelerated with increased temperature and retarded by storage in vacuum and/or low temperatures. This is accommodated by storing activated substrates at -50C in vacuum bags for several days to lock-in the hydrophilic surface treatment prior to bonding.
  • the bonding mechanism follows a fairly slow condensation reaction, which involves the liberation of, water over a period of several minutes to a few hours before completely consuming the available OH sites, it is possible to interrupt this process before completion.
  • the bond strength between the interfaces is comparable to the bulk tear strength leading to an irreversible attachment of the two materials. Attempts to separate the layers at this stage will lead to bulk damage of one or both of the layers.
  • interruption of the bonding process by mechanically separating the surfaces during the early stages of the bonding cycle is found to irreparably damage only the small number of formed bonds between the two surfaces.
  • the tear strength of the bulk is considerably higher than the interface bond, therefore separation produces no irreparable damage to the bulk.
  • microvalves are formed between layers of PDMS by surface activating, e.g. plasma activating, the PDMS or similar surfaces, bringing them into contact and then activating the valves to open and close in such a manner that permanently disrupts bonding between the flexible membrane and the valve seat, but results in complete and robust bonding elsewhere over broad surfaces to hold the device together.
  • surface activating e.g. plasma activating
  • the PDMS or similar surfaces bringing them into contact and then activating the valves to open and close in such a manner that permanently disrupts bonding between the flexible membrane and the valve seat, but results in complete and robust bonding elsewhere over broad surfaces to hold the device together.
  • a product employing the concepts described is a consumable microfluidic cartridge for the purpose of quantifying antibody concentrations in human plasma samples.
  • the cartridge such as shown in Fig. 30, contains on board provisions for sample inlets, in other words, a reservoir that will receive a sample to be analyzed, e.g. a blood plasma or serum sample.
  • a completed cartridge 20 contains sample inlet wells 22 for receiving patient plasma or serum sample or other type of bodily fluid, including cerebral spinal fluid, or urine. It will also contain a buffer inlet well 24, buffer being a reagent used during the processing of the assay, a waste reservoir well 26 designed to contain all of the reagents and sample that flow through the microfluidic channels and that are no longer needed all self-contained on the microfluidic cartridge, also containing a reservoir well 28 which has contained in it a detection antibody with a fluorescent label. The preferred embodiment, the detection antibody will be dried down in the channel or in the reservoir and rehydrated during operation using the buffered contained in buffer well 24.
  • Fig. 31 shows the microfluidic channels containing 4 independent microfluidic channel groups containing the extremely small hollow fluidic flow elements, referred to hereafter as elements.
  • Fig. 31 shows those four channel groups each containing six channels 30.
  • the extremely small hollow flow elements are formed in a batch process with a capture antibody provided on the inside surface of the elements and those elements are placed into channels 32.
  • Example of dimensions of the hollow elements The length of the preferred embodiment is approximately 250 microns, the inner diameter approximately 75 microns, and an outer diameter of approximately 125 microns.
  • Fig. 32 is a blown up schematic of the hollow elements shown in two example channels parallel example channels.
  • the channels are wider than the elements, and the elements are attracted by near electrostatic force to adhere to one channel wall, defining by -pass flow paths on the other side.
  • Fig. 34 shows a cross-sectional view of a hollow flow element in channel 30 with space surrounding hollow element on the outside of the element.
  • Fig. 34 depicts hollow element 32 in microfluidic channel 30 with flow arrows 40 depicted, the hollow element as captured by the top surface elastomer membrane 38 and on the bottom surface by glass substrate element 42.
  • Typical dimensions for the glass substrate layer 42 are 200 microns thick of boro- silicate glass and the elastomer membrane layer element 38 has typical thickness of 100-200 microns. Also providing the channels are an elastomer, PDMS material typical 100-150 microns tall thus forming the microfluidic channel. Also shown in Fig.
  • the elastomer membrane layer continues both to the left and to the right as well as the glass substrate continuing to the left and to the right and on either side containing one or more parallel microfluidic channels also containing hollow glass elements, glass layer element 42 is bonded to elastomer wall, a micro-fluidic channel wall 44, previously formed in a subassembly process using a covalent bonding technique involving plasma activation of the PDMS surface and subsequent contacting and therefore bonding to the glass layer, the hollow element is inserted into that channel.
  • Channel depth is less the diameter of hollow element that are picked and placed against one of channel walls such that electrostatic forces between the element and channel walls release the placing device, e.g. tweezers or vacuum pickup, from the element.
  • the placing device e.g. tweezers or vacuum pickup
  • Channel 30 enclosed by bringing into contact both ends of elastic membrane 38 of control/reservoir 46.
  • Fig. 32 shows schematically two example channels containing a series of four spaced apart elements 32 and by -pass flow space 41.
  • Fig. 35 is a top view of the fluidic layer sub-assembly 48 with elements 32 in channels 30.
  • the assembly 50 contains the elements.
  • FIG. 35 four sets of microfluidic single sample, i.e., four analyte networks 52 are shown, each network is designed to perform an assay with its own respective sample.
  • Fig. 33 is a blowup schematic of a single channel 30 containing four elements 32 and microfluidic piston chamber 36, and valve 54 having seat 34, Fig. 31.
  • Fig. 33 depicts by arrowheads, flow through the bypass flow path 41 around the hollow element 32 as well as through the element.
  • the channels 30 are formed by glass substrate 42 and micro-fluid channel walls formed by knife cutting sheet of PDMS of 110-micron thickness 32.
  • Fig. 30 shows forming the fluidic area 48 by bringing together glass sheet 42 and the unique cut-patterned PDMS sheet 42 using known techniques.
  • Reservoir/control plastic member 56 (containing fluidic reservoirs for sample, 22, assay buffer 24 and reagent waste 26) is bonded to PDMS membrane 38 to form control/reservoir layer 46.
  • Fig. 24 is a top view of the fluidic layer sub-assembly 48 with elements 32 in channels 30.
  • the assembly 50 contains the elements.
  • FIG. 24 four sets of microfluidic single sample, i.e., four analyte networks 52 are shown, each network is designed to perform an assay with its own respective sample.
  • the channels 30 are formed by glass substrate 42 and micro-fluid channel walls formed by knife cutting sheet of PDMS of 110-micron thickness 32
  • Fig. 37 is a top view depicting final assembly 46.
  • Pneumatic interface ports 58 are adapted to match with computer-controlled pneumatic control lines that provide pressure and vacuum actuation to valves 54 (formed by membrane 38 and microfluidic value seat 34) and pistons 55 (the pistons being formed by elastomer membrane 38 lying over piston fluidic chamber 36 and piston pneumatic chamber) piston control lines 60 and valve control lines 62.
  • the piston pump formed by membrane 38 sandwiched between 37 and 36 is activated by vacuum in one direction and pressure in the other.
  • the assay cartridge having macro sized features for containing patient sample (e.g. blood serum, urine), reagents such as buffers and secondary antibodies, and waste.
  • patient sample e.g. blood serum, urine
  • reagents such as buffers and secondary antibodies
  • a portable microfluidic assay device of overall ("footprint") dimensions of, e.g., 5 inches by 3 inches.
  • footprint overall dimensions of, e.g., 5 inches by 3 inches.
  • the microfluidic channels closely packed with other channels and features, of a distance of the order of four to eight times the width of a pneumatic micro-channel, with, for instance, channel sizes on the order of 100 microns or less, for instance, channel widths approximately 100- 150 microns and depths of 100-150 microns, with precision in features of 10-20 microns of tolerance.
  • macro-size features such as sample wells and reagent reservoirs are desired to be incorporated in the portable device, typically of several millimeters in dimension, i.e. several millimeters cross-wise and several millimeters deep.
  • the invention is especially useful in microfluidic assay devices which have a pneumatic channel component, a fluidic channel component, and a flexible membrane joining the two.
  • an excellent starting material for forming the pneumatic channels comprises a double sided pressure sensitive adhesive sheet having a non-fluorescent central layer formed of rigid material, non- fluorescent adhesive on each side, and peelable liner layers protecting the adhesive and we found a process for forming the pneumatic channels and features by processing this sheet by using a C02 laser, Fig. 23 A, followed by very simple bonding process.
  • the laser is used to ablate pneumatic micro-channels and other structures by cutting entirely through the core, the adhesive layers and the liners to form the sidewalls of the desired channels and other features.
  • a pneumatic channel has a side wall formed partly of the inner core layer of the adhesive sheet, and partly by the adhesive itself, at both sides of the core layer. This technique is found to form fine precise features having sizes on the order of 100 microns or less, with channel widths approximately 100-150 microns, and depths ofl00-150 microns, with precision in features of 10-20 microns of tolerance.
  • fluorescence is available commercially with size up to 27 inches, for instance. It is important that the material have very low tendency to fluoresce when exposed to an excitation laser such as green laser or red laser used to excite fluorescence in the conduct of epi-fluorescent reading of fluorescent-tagged analyte at the capture sites of an assay.
  • Mylar TM polyyester
  • a material often used in the manufacturing of pressure sensitive adhesive sheets as a structural component upon which the adhesive is applied has a high degree of auto-fluorescence which interferes with the process of taking a measurement in the cartridge, and is inappropriate for use with assay cartridges intended to be read by an epi-fluorescence or other stimulated fluorescent emission process.
  • a core layer of polypropylene is excellent for this purpose.
  • a polypropylene layer of approximately 2 mils (50 micron) thickness with the adhesive on each side of 1.8 mils or 45 microns thickness is found to perform very well with silicone adhesive layers.
  • a suitable product is sold by Adhesive Research under the product designation AR 90880 having layers of silicon based adhesive, known as SR26 silicon adhesive.
  • a product formed by the techniques described is a portable consumable immunoassay cartridge (cassette) constructed of several layers, the pressure sensitive adhesive with channels formed by through-laser cutting, Fig. 23B, 23 C being one of the layers integrated into the cartridge. That layer, with its peel strip removed, is attached to the bottom flat surface of a rigid reservoir layer, Fig. 23D that defines the macro features previously mentioned, i.e. sample wells and buffer and reagent reservoirs.
  • Laminated to the bottom surface of the pneumatic channel layer by the second pressure sensitive adhesive sheet, with peel strip removed is a membrane layer which is formed on a 100 micron thick PDMS membrane containing fluidic vias that are aligned with vias in the reservoir layer and the adhesive sheet layer.
  • This assembly is bonded to the fluidic layer, elsewhere described, and see Fig. 23D, that bonding being effective to capture discrete detection elements that have been introduced to the micro-fluidic channels and connect the microfluidic channels of the cartridge, though the vias, to the sample wells and reservoirs elsewhere described.
  • the rigid reservoir cartridge layer is either a machined plastic body approximately 6 to 14 millimeters thick or an injected molded plastic body approximately 6 to 14 millimeters thick having reservoir macro-features located on its top surface, the features approximately 3 to 6 millimeters in dimensions, and with fluidic vias from the reservoirs penetrating through the bottom of the reservoir layer, aligned with vias laser-cut in the pressure sensitive adhesive layer.
  • the adhesive sheet with laser-through-cut pneumatic channels, vias and other features is laminated to the bottom surface of the rigid reservoir layer in alignment with the vias in the reservoir layer so that the fluidic vias are arranged to transport sample and reagents from the reservoir layer to the fluidic layer through the vias in the reservoir layer through vias in the pressure sensitive pneumatic layer and vias in the following membrane layer,, to then enter into the fluidic layer
  • Also contained in the pneumatic layer are features associated with valves and pistons in addition to the fluidic vias.
  • These features can be ovals approximately 800 um long and 500 um wide in the case of valves, or 3000 um long and 800 wide in the case of pistons with long channels connecting these features along the entire surface of the substrate and terminating at the pneumatic actuation ports located at the end of the device in a series of pneumatic vias, Fig 23B.
  • One of the challenges in creating a highly functional disposable immunoassay cartridge is in constructing one that has a large number of features which therefore provides a high degree of functionality, for example is, one which is capable of running multiple samples, preferably 16 to 48 different samples, and for each sample the ability to precisely quantify several analytes, 4-8, on one cartridge.
  • Such requirements often drive the complexity and the need for a high density of fluidic features, including valves, vias and piston pumps.
  • the independent fluidic circuit For each sample there is on the cartridge an independent fluidic circuit having the ability to perform measurements of up to 8 unique analytes.
  • the independent fluidic circuit is fluidically isolated from all other fluidic circuits on the cartridge and is used to perform the same measurements in parallel on different samples.
  • the current design squeezes as many as 20-30 different fluidic features, such as valves, piston pumps and vias into an area of approximately 200 square millimeters (10 x 20 mm), as well as the pneumatic channels that connect the features.
  • a cartridge that measures 16 individual samples would have 16 independent fluidic circuits. However the functionality of each circuit is identical which means that every circuit is architecturally identical. So across the cartridge there would be 16 buffer inlet valves (one for each circuit) as well as 16 valve banks associated with each of the detect reagents, waste outlets and pistons. Since the circuits are identical copies it ispossible to share pneumatic control lines across all circuits and use a small set of independently controlled pneumatic channels, limiting the complexity of the instrument that runs the cartridge. So for example, a cartridge with 16 samples running up to 8 different analytes, could for example have as few as 7 pneumatic channels where each of those pneumatic channels connects the same set of functional features located in each of the independent fluidic circuits.
  • the functional sets would include banks of valves for example a bank of valves that allow the detect reagents to flow at a particular time or a bank of valves designed to close off and isolate a set of fluidic channels from one another in the manifold region of the fluidic circuit, or a bank of valves located at the output or a bank of pistons.
  • Sets of functionality are connected to each other through a single contiguous pneumatic channel which terminates at one end at the pneumatic interface and the other end at the last feature in the string of connected features.
  • Pneumatic channels in an effort to intersect with the sets of active features at every circuit, are required to serpentine back and forth across a microfluidic cartridge, never overlapping one another, in an effort to cover all of the features located on the surface of the cartridge:long continguous channels, as long as 10 to 20 inches in length. And also as a result of the high density of pneumatic channels located on the devices it is necessary to keep channels, pneumatic channels as tightly packed as possible in order to accommodate the high degree of functionality required to run such an assay. As a result of these long channels tightly packed and located on a cartridge, and having a serpentine like path nature it was discovered that laser cutting a PSA based film for the purposes of creating these pneumatic channels had the deleterious effect of being structurally unsound. During the manufacturing process or immediately following the laser cutting process it was discovered that the substrate with such formed channels was unable to structurally support itself and retain the required necessary dimensional tolerances.
  • the channels now having interruptions in them and not having the continuity of air flow from the pneumatic input to the final terminal structure at the end of the channel as a result of the bridges, is made functional again by deploying shunts either directly underneath the bridges in the membrane layer or directly above the bridges in the reservoir layer, as shown in figure 23Dand 23E.
  • shunts being formed in the membrane layer shown in figure 23 D a hole or a via is cut in the membrane similar to those used for the fluidic vias.
  • a small pocket is machined into the bottom surface of the reservoir layer or it can be formed in the process of injection molding a piece of plastic involving the formation of the reservoir layer.
  • the channel network of pneumatic channels and the shunts whose alignments overlap with the bridges form a contiguous pneumatic channel capable of actuating all of the features, such as pistons and valves, located throughout the area of the cartridge.
  • Such channels have cross sectional dimensions of approximately 150 microns by 150 microns.
  • the benefits of the approach include lower cost of manufacturing and higher precision in feature locations, because the raw material for the pressure sensitive adhesive is relatively inexpensive per cartridge ( ⁇ $ 1/ cartridge) and because the relatively high speed of manufacturing these channels also results in a relatively low cost yet high precision structure necessary to implement the precision actuated pistons and valves in a pneumatically actuated microfluidic device.
  • the same equipment, the laser set up can be used with a different program to execute drawings that have been made.
  • the implementation of the membrane layer which is an integral part of the fluidic cartridge as it is responsible for a number of functions. It is responsible for closing off the channels, and and making them closed fluidic channels, for containing elements placed into such channels for the flexibility associated with forming microvalves and pistons Fig 23F..
  • a PDMS membrane is necessary as an integral part of the microfluidic cartridge and needs to be permanently adhered to the pneumatic channel surface whatever that pneumatic channel surface is formed in.
  • the bonding process between the PDMS membrane and the PDMS is difficult and costly as it involves multiple steps typically, and also is limited to a small subset of plastics such as polystyrene, polycellphone, COC and COP. Some of those plastics are unsuitable for the formation of a reservoir layer that is formed thick such as 6-12 millimeter thick, having both macro features on one side and micro features on the other side. In the case of COC and COP, the cost of such plastics makes the formation of a device such as that prohibitively expensive. Which then relegates one to a very few number of available plastics such as polystyrene.
  • Figs. 51A 1-4 illustrate PDMS bonding to PDMS.
  • Fig. 5A 4 shows deflection of the two PDMS layers in opposite directions by elastic deformation, and in Fig 51A dashed lines indicate the layers in region Ri relaxed, un-deflected. Adjacent regions R 2 of the layers are retained in contact, either solely by initial surface-to-surface bonding, or with the added benefit of outside confinement or compression, indicated by arrows P.
  • the Figures illustrate the two conditions achieved cyclically during exercise of the make and break protocol previously described (Fig 51), enabling bonding at contiguous regions, but bond prevention in a selected region of the potentially bondable surfaces.
  • differential gas pressure to produce the deflections. For instance, thus can be avoided the need for design of special mechanical moving devices or the need for drying associated with the use of liquid pressure.
  • the gas pressure differential can be achieved by application of positive gas pressure to the interstital space, as by a special channel, with the benefit of being able to use high values of pressure to speed the operation or for use where bonds are formed rapidly or are of high strength.
  • gas pressure differential be produced by application of vacuum to the outside of a layer to produce its deflection.
  • One advantage is that the manufacturer can employ channels and cavities of the device itself, such as those associated with pneumatic operation of the device during its normal use. By this the manufacturer can reduce the need for special, costly tooling and extra manufacturing steps. An example is the manufacture of micro fluidic cartridge device described herein.
  • Fig. 51A 5 in the case of applying vacuum on the backside (outside surface) of a PDMS layer to produce gas pressure differential, the cavity is a closed vacuum chamber engaged upon the backside of the PDMS layer.
  • Fig. 51A 5 illustrates such a deflection chamber on each side of the pair of contacting membrane surfaces.
  • the vacuum-actuated deflected state of region Ri is shown in solid lines while the dotted lines illustrate the natural relaxed and undeflected state when there is no vacuum is applied to the vacuum chambers.
  • positive pressure is applied to both chambers, having the effect of enhancing the momentary contact, and therefore lessening the time needed before another break phase of the cyclical process is performed, thus speeding the neutralization of the layers in regions Ri.
  • Fig. 51A 6 illustrates a deflection chamber on only one side of the assembly, deflecting a single membrane.
  • the opposing membrane is shown rigidly backed against a planar surface to which it may previously have been adhered, to ensure that it remain stationary when portion Ri pulls away.
  • Fig. 51 A 7 illustrates a single deflection chamber and an opposing valve seat on planar surface in construction similar otherwise to that of Fig. 51 A 6.
  • the deflection chamber may be formed by manufacturing tooling constructed only for that purpose and then removed.
  • the deflection chamber is in fact part of the final microfluidic product, with numerous obvious advantages with respect to tooling cost and economy of manufacturing steps.
  • Fig. 28 diagrammatically illustrates a microfluidic cartridge having a complex microfluidic (liquid) channel network, a large number of microfluidic valves formed by the make and break protocol, and other pneumatically controlled features that are all actuated simultaneously with the valves.
  • One of the advantages of employing positive pressure relates to the fact that in employing vacuum to deflect the membrane, the maximum pressure that can be applied is 1 atmosphere, approximately -14 psi, which limits the deflection forces applied to a membrane whereas a positive pressure applied between the sheets is nearly unlimited in magnitude to cause separation to occur between the sheets.
  • deflection cavity In respect of applying positive pressure to produce the outward membrane deflection, two alternatives for the deflection cavity will be described, that of a deflection slot defined by walls that engage the outer surface of the layer, but that is open and un-limiting with respect to the deflection distance for the layer, and that of a chamber that also has the walls, but is closed with a ceiling.
  • the walls of an open or closed cavity for pressurized deflection define the physical perimeter of the area of the deflection, thus defining regions Ri and R2, and hence the area over which the make or break process occurs.
  • the limit of deflection is a function of the elastic properties of the membrane and the pressure applied, and may be most useful in the use of moderate positive pressures, or where there is ample margin for error regarding the strength of the membrane.
  • the use chamber which, in addition, has a ceiling creates an additional physical limit for outward deflection,which can potentially protect the membrane from deleterious effects of tearing or bursting the membrane, useful e.g. where particularly thin membranes or particularly high outward forces are to be employed.
  • the flexible, deflectable sheet has been monolithic, and in the examples of Figs. 51 A 1 to 7, it has been PDMS.
  • a composite sheet may be used instead, comprised of multiple layers, the basic requirements being that the overall sheet is flexible to be capable of elastic deflection, and that the inner surface be bondable. While not necessary according to broad aspects of the invention, it is advantageous that the bondable surface of the composite still be surface-activated PDMS, either pre-formed as a separate sheet or as a coating on a carrier sheet which itself may be monolithic or a composite.
  • the composite comprises a bondable pre-formed layer joined to a layer of another substance.
  • the bondable layer may be surface-activated PDMS and the back layer of a pre-formed layer of a material other than PDMS, for example a sheet of PDMS laminated to a second pre-formed flexible sheet of a compatible substance, or one that can be rendered compatible by use of a treatment, such as flame or plasma treatment or by use of an intervening layer that can be bonded to both.
  • the second layer may be a pre-formed sheet of Mylar TM (PET), Polycarbonate or Polyurethane produced as blown or cast film sheet, as appropriate for the resin and the
  • Figure 51A 10 illustrates a flexible pre-formed back sheet coated with a thin coating of PDMS material typically ranging from approximately one to three microns in thickness, having its surface activated in preparation for a bonding process.
  • the back layer for instance may be Mylar TM (PET) of thickness approximately 5 to 15 microns.
  • PET Mylar TM
  • an essentially monolithic layer of PDMS may carry an exterior coating of another substance, for purposes such as improving the gas-barrier properties of the composite.
  • Fig 51A 11 illustrates a laminate structure similar to that of Fig. 51 A 10, but with a flexibility-increasing feature. It is useful for the condition in which a flexible outer sheet bonded to PDMS sheet (or it could be a flexible sheet having a PDMS surface coating) wherein the flexible sheet is less flexibile than PDMS and it is desired that the composite exhibit increased flexibility, e.g. to increase the deflection capability and thus the flow capacity of a valve or pump formed by the deflectable membrane.
  • the principle being illustrated is use of an interruption or reduction in thickness of the back layer.
  • the interruption can be a single moat in the back layer, extending around the perimeter of the defined area Ri, or, as shown, a series of concentric moats that allow a greater displacement to be generated as a result of allowing the flexibility of the PDMS to perform the majority of the stretching during an activation of either a vacuum deployed or pressure deployed activation protocol.
  • PDMS has the advantages of being a low cost material that is easily bonded, flexible and easily machined or otherwise easily formed into channels surfaces to cooperate with the deflectable membrane features described.
  • laminated structures including that of Fig. 51A His the ability of a material other than PDMS to block gair passage or reduce the overall gas permeability coefficient of the structure.
  • PDMS is known to have a high degree of gas permeability other plastic material such as polyester, polycarbonate and polyethylene exhibit extremely low permeability relative to PDMS. This can be of great benefit in microfluidic type devices in which positive gas pressures are used for actuating valves, preventing gas permeation through the membrane into the fluidic channels thereby creating bubbles that can have a deleterious effect.
  • air bubbles that enter the reagent stream can attach to the capture agent and prevent binding; air bubbles can prevent complete wetting of surfaces, and thus inhibit the capture agent from capturing an antigen of interest; and air bubbles can also displace fluid in the microfluidic channels causing the microfluidic system to become less stiff from a fluidic point of view and also increasing the variability of flow rate, producing uncertainty of flow rate that can impair quantification assays.
  • Fig.51A It employs a series of narrowly defined moats or channels that are cut into the more rigid yet somewhat flexible air-impermeable backing sheet part of the flexible sheet/ PDMS laminate.
  • the moats allow the stiffer component, e.g. Mylar TM (polyester) or polyethylene to deflect using the underlying flexible PDMS to act as an expansible spring, therefore achieving greater deflection.
  • a fixed bondable surface opposite to a flexible membrane PDMS bondable face is not limited to the same material, and there are circumstances in which advantages are obtained by using a different bondable surface.
  • the deflectable membrane has a PDMS bondable surface, but the opposite bondable surface is provided by a rigid silicon based material including crystalline or amorphous silicon, amorphous silica, silicates and ceramics.
  • Benefits of different thermal conductivity, electrical conductivity, or the ability to add electrical contacts, as in the case of silicon or having the beneficial properties of silica in the form of optical clarity low autofluorescence optical smoothness or the special hardness properties and insulating properties of ceramics can be of advantage.
  • Figure 51A 13 illustrates the accommodation of a flexible membrane with a surface-activated PDMS bondable surface to a synthetic resin or metal based device employing an intermediate bifunctional layer.
  • surfaces of well-known plastics including COC (cyclical olefin polymer), COP (cyclical olefin copolymer), polycarbonate, polysulfone, polystyrene can be surface-activated or metals such as aluminum or iron that either readily form oxide layers can be employed.
  • Such surfaces can be modified with an intermediate bifunctional layer such as an organol, to create an oxide layer.
  • a portable microfluidic cartridge 2 is placed into an operating and scanning instrument by the user. It enters in a receptacle or reception area 6 at which the cartridge is retained for conducting the assay while scanning.
  • FIG. 54 A suitable receptacle is shown in Figs. 54 and 54a, and the relationship of the receptacle and cassette when in assay/reading position is shown in Fig. 55 and the detail of Fig. 55a.
  • An implementation of the overall system is shown in exploded view, Fig. 56.
  • Fig. 56 includes x, y precisely movable stage 13 that moves the cartridge on its carrier relative to the stationary objective lens.
  • cartridge 2 and the cartridge receptacle 6 are shown with a clamping mechanism 12 and pneumatic interface 8.
  • a series of computer-operated solenoid valves 9 that move on the stage with the cassette apply positive and negative air pressure to ports that interface with the positioned cassette.
  • Fig. 57 illustrates a microfluidic configuration within a cartridge, illustrating both a series of fluidic networks and a pneumatic channel network to actuate on-board membrane micro-valves and micro-pistons in the fluidic network.
  • the fluidic network in Fig. 57A comprises eight discrete microfluidic circuits closed by an overlying elastic membrane, e.g., a continuous layer of PDMS. Each of those circuits has a number of microfluidic channels, valve locations, and piston locations. Portions of this membrane are located at formations in the channel that define valve and pump cavities. The corresponding portions of the membrane define movable elements of the valves and the piston.
  • the pneumatic channel network Fig. 57B is shown as an overlay in in Fig. 57C. It matches the fluidic network with respect to the various features that need to be actuated.
  • sample step 58 starts with the prime flow step, and is followed by sample step, wash step, secondary antibody step, another wash step, a dye step for reacting to attach reading dye to the captured moiety, and finally another wash step.
  • sample step wash step
  • secondary antibody step wash
  • reactive dye is caused to flow from its respective inlet well by activation of the pumps formed by each piston and upstream and downstream valves, with the end result of captured moieties at the detection elements that are labeled with the reactive dye, ready for reading to quantify the result of the assay.
  • volumes employed on this device include the sample at 20 microliters, a buffer of 150 microliters (as shown in the table)— the total volume of the microfluidic circuit is approximately 1.8 microliters.
  • Fig. 98 four micro channels on a portable microfluidic cartridge are illustrated, each having two monitor positions. The further discussion relates to the first set of monitor positions 1, 2, 3, 4 in respective channels.
  • Fig. 98a three different operations of an illustrative assay with discrete phases are represented by times tl, t2, and t3.
  • phase 1 at time tl at four different locations on a cartridge to sample four channels the tracer signal is detected to be at the expected nominal value within the acceptance rate. It is therefore considered a successful phase 1 disposition.
  • Phase 2 at time t2 the nominal level is near zero, which might indicate that a buffer or some fluid that intentionally had no tracer was properly flowing in the channels at that particular phase.
  • phase 3 at time t3 third reagent or fluid in the channel has a tracer level that is different from that of phase one but is detected to occur at its nominal value within its acceptable and expected range. So the entire operation considering phases 1, 2, and 3 would be considered successful. This represents proper operation with no failures.
  • Fig. 98b illustrates another run of the same assay.
  • phase 1 time tl, a failure has occurred wherein the detected tracer signal occurs outside (here, below) the acceptance range.
  • channel 1 the tracer signal is shown present, but lower than the acceptance range, whereas in channel 4 the detected tracer value is shown as not present.
  • Fig. 98c at time tl of the assay run all four channels are shown as having a detectable tracer signal below acceptable range. But note that all four signals are equal and uniform. This indicates that there is not an independent failure mode within that cartridge but probably indicates that an improper dilution had been used to create the reagent that was used.
  • a hierarchy of signal modes can be constructed, e.g., in the simplest case a signal versus no signal, a simple digital response, and in other cases where the quantitative value of the signal does not meet expectations.
  • a further level of complexity involves quantifying the level and comparing that quantity to an acceptable level where there is range of acceptable levels (acceptance range) not just on or off. That quantification technique might be used to determine whether a proper dilution or proper concentration or proper reagent was used in the proper location.
  • Another advantageous level of sophistication is in monitoring and analyzing the signal structure over time, to obtain the temporal response of the signal relative to an expected temporal response. That requires a more detailed explanation.
  • a detected tracer signal is shown while monitoring over time a fixed location in a microfluidic channel, for example a channel approximately 100 microns wide by 100 microns deep in a length section of approximately 20-50 microns long.
  • a very specific isolated location within the channel is monitored over three different phases.
  • the first phase shown depicts the condition of no flow occurring within the channel but the channel has present in it a reagent laced with a tracer of a certain concentration that provides a detected signal of any type, e.g., detected fluorescence.
  • Flow may be introduced to the channel in an oscillatory fashion.
  • the purpose of oscillating the flow in normal operation of an assay is to enhance the interaction of the analyte present in the unknown concentrations sample with a capture moiety, e.g., an antigen in the sample with an immobilized capture antibody.
  • a typical defined volume (“slug") of liquid is used, of fixed volume that is much larger than the volume exposed at the detection point. When portions of this oscillating slug of liquid move away, this allows time for diffusion to take place in those portions to bring the material into equilibrium before it comes back for exposure to the capture site, and back and forth.
  • Fluorescent dye is used that is subject to photo bleaching.
  • the negative actuating pressure value is greater than the positive pressure value and therefore induces a greater rate of displacement of the piston.
  • the negative pressure for actuating the membrane diaphragm of the pump is about -8 or -10 psi, while the positive pressure actuation of the piston is under about 4 psi.
  • the signature shown in Fig. 100 is indicative of normal operation. In the case where a pneumatic interface was improperly sealed or seated, then these peaks heights would occur at different levels, outside of normal acceptance range, so this is a type of failure mode that could be detected. Another factor involved here is the flow rate, which depends not only on displacement volume of the piston, but also on the impedance of the fluid in the microfluidic channel. If the impedance is increased by the addition of blockage from a contamination source or some other problem, such as a detection element being misplaced in the channel, then the nature of the signature structure would be different from what is expected and shown in this graph.
  • the trace shown in Fig. 100 and the detected values in Figs. 98a-99, are acquired by capturing the fluorescence intensity during steps of the assay by an imaging system shown diagrammatically in Fig 26. It has an objective lens and a series of optical elements.
  • An excitation beam from a laser is introduced to the monitoring location at a microfluidic channel, Fig. 102.
  • the optics transform the stimulated fluorescent object, see Fig. 102, to an image plane shown as a photo detector (but in a preferred implementation, a CCD camera).
  • the intensity of the pixels within a region of interest (ROI) captured on the camera are summed
  • Fig. 102 the laser beam is shown in cross-section as an oval while a rectangular box circumscribing that oval illustrates the region of interest (ROI) over which the pixel intensities are integrated to produce a single resultant signal point. That value at this point in time is plotted as a point on the graph in Fig. 100.
  • the scanning system is adapted, during reading of assay results, to interrogate a detection element on which assay capture agent is immobilized, see the Scanning Figures described later herein. But in monitoring mode, as depicted in Fig. 102, by relative movement between optics and microfluidic system, the system is focused on a selected monitoring point on a fluid-carrying channel at a point in which the detection element is not present.
  • Figs. 101 and 102 The optical arrangement of Figs. 101 and 102 is used to generate the signal trace in Fig. 100 or the "snap shot" at a monitoring point in Figs. 98a-98c, or the measurements over time of Fig. 99.
  • a portable microfluidic cartridge 2 is placed into an operating and scanning instrument by the user. It enters in a receptacle or reception area 6 at which the cartridge is retained for conducting the assay while scanning.
  • FIG. 54 A suitable receptacle is shown in Figs. 54 and 54a, and the relationship of the receptacle and cassette when in assay/reading position is shown in Fig.55 and the detail of Fig. 29a.
  • An implementation of the overall system is shown in exploded view, Fig. 56.
  • Fig. 56 includes x, y precisely movable stage 13 that moves the cartridge on its carrier relative to the stationary objective lens.
  • cartridge 2 and the cartridge receptacle 6 are shown with a clamping mechanism 12 and pneumatic interface 8.
  • a series of computer-operated solenoid valves 9 that move on the stage with the cassette apply positive and negative air pressure to ports that interface with the positioned cassette.
  • a suitable instantaneous image size it is appropriate to use an excitation beam imaged through the objective lens to a spot size of approximately 12 microns wide by 250 microns long.
  • the region of interest (ROI) of the camera that includes that spot has an area of approximately 35 microns width by 250 microns length.
  • the micro fluidic channel of the cassette that will be monitored has a channel width of approximately 180 microns.
  • Three scenarios for monitoring the tracer dye with such a beam are: (1) Continuous Scanning Modality.
  • the microfluidic channels are scanned e.g., with substantially constant velocity across all channels of a microfluidic system, such as on a cartridge. In that case, the beam crosses over the channels and measures the fluorescence intensity as a function of position or as a function of time, as the channels are crossed.
  • the Scanning Modality is especially useful. It is used to scan across all channels of a microfluidic system, e.g., on a cartridge, repeating this during each phase of execution of the assay. It can also stop at various locations for a short period of time to collect a trace at that location, and stop long term for staring to characterize the flow over time even in the case of monitoring usual assay function.
  • Fig. 57 illustrates a microfluidic configuration within a cartridge, illustrating both a series of fluidic networks and a pneumatic channel network to actuate on-board membrane micro-valves and micro-pistons in the fluidic network.
  • the fluidic network in Fig. 57A comprises eight discrete microfluidic circuits closed by an overlying elastic membrane, e.g., a continuous layer of PDMS. Each of those circuits has a number of microfluidic channels, valve locations, and piston locations. Portions of this membrane are located at formations in the channel that define valve and pump cavities. The corresponding portions of the membrane define movable elements of the valves and the piston.
  • the pneumatic channel network Fig. 57B is shown as an overlay in in Fig. 57C. It matches the fluidic network with respect to the various features that need to be actuated.
  • Fig. 58 is a magnified view of one of the circuits of Fig. 58, illustrating a number of the micro-features including valves, pistons, and the various reagent or reservoir inputs including the sample, the buffer (wash), the assay reading dyes, the secondary antibodies and the waste.
  • the four elements GNR shown in black in each of the four individual (isolatable) channels represent glass nano-reactors (GNRs) embedded in those channels. This illustrates the basic micro fluidic unit replicated a number of times in the cartridge depending on the number of samples that the cartridge is designed to accommodate.
  • the upper and lower black and white traces shown in the center illustrate the fluorescence intensity being high in channels 1, 2, 3, and 4 based on the peaks shown on the traces. This illustrates that the scanner while scanning across that path encounters high fluorescence because of benign tracer in liquid in each of the channels.
  • the locations of the channels are then determined very precisely by taking the encoder information superimposed on that trace. The precise location of those channels is thus determined relative to the absolute coordinate frame of the scanning system. It is possible now to produce high resolution and highly aligned scans because the precise locations of the scans are now determined with respect to the x, y stage.
  • the primary benefit of the approach described, of precisely identifying the location of the channels, is to relax the requirement that the cartridge be precisely aligned on the stage by the user.
  • Fig.54 assay cartridge (cassette) 2 is shown above carrier plate 4, in preparation for being placed into receptacle area 6.
  • the cartridge will make intimate contact with pneumatic interface 8, so that pneumatic controls (solenoid valves 9) can actuate appropriately to apply air pressure and vacuum via interface 8, to actuate the micro- valves and pistons on board the cassette and thus perform the assay.
  • the cartridge is retained in the receptacle interface 6 by retaining clamp 12.
  • Fig. 55 clamp 12 is shown with the cartridge in place in receptacle area 6.
  • Fig. 54A the retaining clamp is shown in the process of being closed.
  • Figs. 55, 55A, and the exploded view of Fig. 56 illustrate the relationship between the carrier plate 4 and cartridge 2 in it and the rest of the mechanical assembly of the instrument in an exploded view.
  • the precision x, y stage 12, chassis 16, heat plate 14, and optic subassembly 18 are shown. Not shown is an enclosure for the system that excludes ambient light form the cartridge or other microfluidic assay system, and from the optical system, such that ambient light does not interference with fluorescent excitation and detection during performance of the assay and during the reading of assay results.
  • FIG. 55 A a further magnified view, the pneumatic interface and the clamping pneumatic interface 8 are shown with the cartridge 2 in intimate contact with pneumatic interface 8 while the clamping anvil 26 is resiliently compressed, providing a force compressing the cartridge against the pneumatic interface.
  • the clamp is held in its down position by a latch.
  • Figs. 62, 63 and 64 again illustrates four isolation channels and the path of the scanning sweep performed to identify the precise location of each of the channels.
  • Fig. 63 shows a trace obtained by such a scan using white light illumination as opposed to using fluorescence with benign tracer, by laser-based epi-fluorescence process.
  • the signal shown in Fig. 63 illustrates a high level of signal followed by fairly small dropouts or spikes illustrating where the edge of the channel or shadow is formed as a result of white light illumination as it impinges upon a channel.
  • a simple technique to implement, having significant value uses a scanner during the assay protocol to simply detect the presence or absence of the tracer dyes at the various phases of performing the assay protocol.
  • the scanning process generates signal patterns that are compared to predetermined anticipated levels associated with the normal performance of the assay on the cartridge.
  • the invention provides an entire system of monitoring methods that can be employed in coordinated fashion to address the previously described failure modes, and others. For example, another failure mode not previously discussed is in the controls based in the bench top operating and scanning instrument itself. If a control fails, e.g., a pneumatic pressure controlling solenoid valve, this failure is also detected.
  • a control fails, e.g., a pneumatic pressure controlling solenoid valve, this failure is also detected.
  • the invention provides a generic means of detecting a host of potential failure modes during a microfluidic assay system run and especially determining whether a reagent is present or not in that channel, and if it is the proper reagent in that channel.
  • the invention enables simply scanning across channels with benign fluorescent dye for the purpose of precisely locating the microfluidic channels, for setting up scan parameters, e.g. for the purpose of identifying optimal focus location, another important feature of significant value. This is in the set up process during the execution of the assay protocol that is described further within, in relation to the Scanning Figures. While the cartridge is running the assay protocol, e.g., under pneumatic protocol, the scanner system can simultaneously be used to perform a number of measurements useful to the later detection phase when the assay is completed. Those measurements include locating the channels based on the fluorescence properties of the channels containing liquid with the tracer dye.
  • Process controls are routinely used in the art in measurements, for example, in ELISA plates, controls are used as individual wells on an ELISA plate.
  • researchers when running any type of instrument always want to know or have positive verification that the measurement that they made is believable, and it is performed the way it is expected to be performed.
  • the present invention is a means to producing that confidence in a microfluidic system and the data that the system produces.
  • the fluorescent dyes used for benign tracers inherently should not interact with the components of the assay. However if chemical interaction were found, it would be routine to chemically modify the dye to make it more inert or more benign with respect to interferences in the system. There are known conjugations that can be performed on the dye mark for such purpose.
  • the invention has special utility in respect of complex microfluidic based systems that run a sequence of reagents, assays where quantification is the primary outcome of the measurement
  • GNR's may be useful in stationary, non-portable microfluidic systems, to make very accurate measurements.
  • Techniques of monitoring described have applicability in such instances, e.g. in high throughput blood testing.
  • a unique assay system will now be described, which involves pneumatically actuated valves and pistons for delivering precise volumes of reagents throughout a microfluidic disposable cartridge.
  • the instrument needs to have significant robustness in terms of the useful life of the instrument.
  • a key requirement is that the cartridge must interface with a pneumatic control component on the instrument in reliable fashion so that no pneumatic leaks occur between the cartridge and the pneumatic actuation system.
  • the valves and pistons on the cartridge are controlled by pressure and vacuum provided by the instrument, and if a leak were to occur at the interface between the cartridge and the reader, those valves would not actuate precisely and reliably. The result would be imprecise control of the flow of reagents on the cartridge and uncertain results with regard to the assay.
  • the assay depends upon precisely timed actuation and metering of reagents, precise volumes and precise times for exposure of the unknown sample to the capture agent, the subsequent flushing and washing of the sample prior to mixing, and exposure with secondary capture agent and then followed by the subsequent washing of that component followed by exposure to a fluorescent dye, or regarding the last two steps, alternatively, exposure to a fluorescently labeled secondary agent.
  • One of the important features is the novel arrangement by which both a compliant component and a rigid component are provided, that are brought together under force to form an airtight seal.
  • the beneficial relationship is that the compliant component is located on the disposable element and not on the non-disposable side of the reader.
  • the benefit is that the rigid component has a much longer life than a compliant component, as the rigid component would not undergo deformation over time, whereas a compliant component such as silicone rubber and other forms of rubber or even plastics would undergo inelastic deformation which eventually would lead to failure mode in the form of a pneumatic leak.
  • the rigid component located on the operating instrument is metal, either aluminum or steel, and the compliant component is PDMS or silicone rubber carried by the assay cartridge.
  • the rubber is exposed and advantageously is provided as an extension of one of the layers within the cartridge. It is provided on the bottom surface of the cartridge, while the wells and reservoirs of the cartridge are provided on the top surface.
  • the thickness of the silicone rubber is approximately 100 microns. In the preferred implementation it spans the entire surface area of the cartridge which could be 120 millimeters by 85 millimeters, and its durometer is about 30 shore A.
  • a clamping system is provided to place pressure to bring the two together to form a seal that is maintained at numerous pneumatic vias. In the example, there are seven vias positioned in close proximity. For example the spacing between vias is approximately 2 millimeters and the via diameter is approximately one millimeter.
  • X, Y direction constraint is provided by a set of four corner retaining stands, these being tapered to enable easy insertion of the cartridge into the thus-formed receptacle pocket.
  • Pressure is applied to the cartridge only in one location to obtain stability in the Z coordinate.
  • the force is applied downward through the cartridge in one embodiment by a roller connected to a leaf spring, the roller arranged to contact the top surface the cartridge and provide a downward force which compresses the compliant material on the cartridge against the pneumatic manifold.
  • a simple releasable clamp applies the pressure.
  • a pneumatic solenoid or an electrical solenoid can similarly apply force to maintain the connecting pressure.
  • a motorized roller is a convenience from a user point of view, but a swing bar as shown is simple, effective, and avoids potential failure modes.
  • the other significant feature on the movable stage is the pneumatic interface manifold.
  • the pneumatic control lines are controlled by solenoid valves that are carried on the X, Y stage and connected via flexible hose to a pneumatic manifold in pneumatic communication with a vacuum pump and a pressure pump.
  • the speed at which that occurs is directly proportional to the dead volumes in those channels. For example, switching from pressure to vacuum requires the vacuum to be completely drawn on whatever volume is contained downstream of the solenoid valves. Using the features just described, all of the downstream channels from the solenoid valves are extremely small, and the distance between the solenoid valves and the chip is maintained in a very short distance. Yet another advantage of having the pneumatic interface as a fixed machined component mounted on the stage having a low dead volume is in the ability to use low flow rate, and therefore inexpensive vacuum and pressure pumps. Since the volume of the pneumatic lines downstream of the solenoid valves and the rate of states changes determine the average flow rate, it is desirable to keep the dead volume low so as to allow the use of smaller, low flow rate pumps.
  • Speed is important because a large number of actuations must occur throughout the assay protocol. There can be tens of thousands of actuations and even tens of milliseconds or hundreds of milliseconds difference can add up to a substantial loss of time.
  • valves There are two different active components on the cartridge, valves and pistons.
  • the purpose of the valves is to determine which reagents flow and to which channels they flow.
  • the pistons are the primary components for motivating the fluid. They provide the positive and negative displacement to the reagents located on the cartridge, and so are the primary elements for motivating fluid.
  • the features contained on the stage are the pneumatic interface with the solenoid valves and cartridge and the clamping device. That is all that is on the stage that moves.
  • the cartridge on the movable stage is exposed to a fixed heater plate underneath, supported only 4 or 5 millimeters below the surface of the cartridge and exposed face-to-face for radiant heat transfer.
  • the bottom surface of the cartridge where the active capture elements are contained in the microfluidic channels must be maintained at a constant temperature between 35 and 37 degrees C.
  • the heater plate extends in the dimension that is actually slightly larger than the surface area of the cartridge to cover its range of travel. A uniform temperature profile is thus maintained over the surface of the cartridge.
  • the biggest advantage of that is that the temperature of the cartridge is easily maintained without having to control the temperature of the entire reader enclosure. Thus there is no concern about heating the electronics and other sensitive components within the enclosure. Temperature control and stability is only provided at the critical surface.
  • One of the features of the instrument is to excite a fluorescence signal using a laser and then capture that fluorescence with an objective lens while the stage is translated.
  • a well-known epifluorescence configuration is employed in which the excitation signal, provided by a laser or laser diode, is sent through an objective lens and then the returning fluorescence is captured by the same objective lens and sent to an imaging CCD camera.
  • the heater plate which held fixed directly underneath the cartridge is provided with a hole that allows both the excitation and the emission signal to propagate through to the fixed optical system.
  • All objective lenses have a so-called "working distance.” Key features of an objective lens include numerical aperture and magnification, which will determine the ability of the objective to capture the fluorescence intensity in a very efficient manner and image that back to the CCD camera.
  • the working distance for typical lOx objectives is somewhere between 5 and 12 millimeters. It is important to maintain a distance of somewhere between 5 and 12 millimeters between the objective and the bottom surface of the cartridge.
  • the total distance between the objective and bottom of the cartridge is approximately 12 millimeters, the thickness of the plate is a small fraction of that, it is 4-6, and the plate itself has a hole that allows the light to transmit through.
  • the hole is sized such that the objective is brought up into the hole itself, fitting partially into the plate.
  • the sequence begins with placing the cartridge onto the cartridge receptacle pocket, then sealing that cartridge using a clamping mechanism, then after warm-up, actuating the pneumatic valves which forces the reagents including the buffers and the samples and the detection antibodies to flow in a very specific sequence for a specific period of time allowing incubation to occur.
  • the incubation results in the binding of the unknown antigen in the sample to the capture moieties contained in the cartridge.
  • the various reagents flow in a given sequence with intermediate wash steps followed finally by a fluorescence scanning process
  • the bench top unit has just a few key subsystems.
  • the cartridge is placed into a little receptacle area and located in that receptacle area is the pneumatic interface boss that has limited end surface area ("lip area") for contact with the cartridge. It protrudes off of the surface, that is the highest surface.
  • One end of the cartridge sits on that boss.
  • the other end of the cartridge sits on a small rail on the other side of this containment area.
  • corner guides that make it easier to place the cartridge.
  • a small arm contains on it a little spring loaded containment clamp. The spring loaded clamp bar comes down and rests on the top surface of the cartridge, and pushes the cartridge down on to the pneumatic boss.
  • the epi-fluorescent optical system is a laser diode, red laser diode, a collimator lens, a cylindrical lens, and three filters.
  • An excitation filter ensures any of the excitation light is within a certain wavelength band.
  • There is a dichroic beam splitter which has a high reflectivity for the red of excitation 640 nanometers, but very low reflectivity for the deeper red that comes back as a result of the fluorescence, around 680-690 nanometers.
  • the 680 coming through hits another filter, the emission filter. This allows only a small band - it blocks all red, and it allows a small band. Following this is a focusing lens onto a camera, called a tube lens.
  • a cylindrical lens in the infinity space between the collimator and the injector provides a stigmatic beam at the target. This produces a laser beam at target of very long elliptical profile.
  • the beam is approximately 500 microns long by about 8 microns thick. It is like a line.
  • the instrument scans that line down the channels to illuminate the whole width of the channel.
  • novel arrays of elements described above are useful only if effectively read after the fluid assay is performed.
  • the following scanning apparatus, procedures and methods for automatically scanning a microfluidic chip effectively solves the problem with arrays of micro-flow elements, and in particular, micro-length tube elements.
  • the scanner utilized in this method is a fixed, inverted epifluorescent microscope equipped with a three axis (x, y, and z) stage for motion of the chip to be read, a CCD camera for bright field imaging and fluorescence detection, a diode- pumped solid-state laser for excitation, and a white LED for bright field illumination.
  • a cylindrical lens is used in the laser optical path prior to any filters to expand the beam size. This allows the excitation of a larger surface area in a single pass and allows for some flexibility when placing the elements in the flow channel during chip manufacturing. All of these scanner components are controllable via a computer as follows: on command x, y, z motion, image acquisition / imaging settings, laser on/off, and LED on/off.
  • the method described herein uses various sequences and combinations of the scanner control / acquisition to orchestrate the automatic scan. See Scanning for a general schematic of the scanner.
  • a scan is comprised of a sequence of steps con Figured with start / end (x, y) positions, z (focus) position, velocity, and a segment number.
  • start / end (x, y) positions, z (focus) position, velocity, and a segment number When the end position of a step in the sequence is not the start position of the next step, the stage will make a full speed move to the x, y, z position of the start of the next step in the sequence, and no data is collected during this rapid move. While executing one of the steps (moving from start to end in x, y at a fixed z) data is collected versus time.
  • the data that is collected includes time, the step segment number (assigned while configuring a scan step), the present x and y positions, the camera settings (gain, exposure etc.), and information extracted from the images in the video stream from the camera.
  • the information extracted from the camera video is based on a region of interest (ROI).
  • An ROI is defined as a rectangle somewhere in the image.
  • the pixels within the ROI are processed to extract information from the image. For example the mean, median, standard deviation, max, min, etc. of the pixels inside the ROI from a given image are computed and included in the data collected during a step move.
  • the data collected throughout the sequence of steps that comprise the scan is written to a file (see sample in) that may then be processed to extract desired information.
  • x, y positions based on a previously complete homing of the scanner stage are sufficient for guaranteeing that the scan executed will, in fact, pass over all the channel edges, given that the chip is mechanically referenced to the stage.
  • the issue is then to determine precisely where the channel edges are relative to the x, y stage positions.
  • a 'find channels' scan is executed following the horizontal lines in the middle of the channels shown in Scanning . More specifically, the find channels scan is broken into distinct steps such that there is a step across each individual channel at the 'top' and the 'bottom' of the scan zone that is tagged with a unique segment number in order to facilitate subsequent data processing. Further, the scan is done in bright field (i.e.
  • the ROI used is very narrow in width and extends the full height of the image (as show in Scanning), and the z position is intentionally 'defocused' from a nominal focused z position of zero, as established by homing the stage.
  • the 'Find Channels' routine can be done during the 'detect' flow phase of the chip assay.
  • the channels are filled with fluorescent dye.
  • the scan to find the channels is then done as described above but the scanner is in fluorescence mode (i.e. laser on, LED off). This has the advantage that the signal to noise ratio is very high.
  • An example of the data collected during a 'find channels' scan is given in Scanning and a zoom in to a single channel scan is given in Scanning.
  • the 'find channels' scan has eight steps, one for each channel crossing above and below the elements.
  • a scan data file from a find channels scan is processed on a per scan segment basis. Consequently, the data processing operates on a set of data as depicted. The data processing proceeds as shown in Scanning. In the Figure, the data processing will produce a scan configuration that can be used for the find micro-length tube elements procedure, or it will throw an error that will halt the auto-scan procedure. The information collected during this procedure is useful for:
  • This procedure takes as input the x, y positions of the first micro-length tube element in each channel as determined by the 'find elements' procedure. For each of these positions the procedure moves to the given x, y position and conducts a sweep of z from a negative position thru zero to a positive position. While the z sweep is taking place the full images from the camera video stream are run thru a Sobel edge detect filter and then the resulting image standard deviation is computed. The end result is a set of data as shown in Scanning. For each segment (i.e. at the x, y position of an element), the resulting z position versus standard deviation plot is then used to find the z position at the maximum value of the standard deviation (See Scanning for details). The z positions, at the maximum standard deviation, from each segment are the 'in focus' z positions for each channel on the chip. The information collected during this procedure is useful for: Setting the focus for the Fluorescence Scan,
  • the purpose of this procedure is to select the appropriate camera exposure setting in order to efficiently utilize the range of the camera given the fluorescence level of the micro-length tube elements. Too short of an exposure will lead to dark images, poor signal to noise, and underutilizes the camera range. Too long of an exposure will lead to saturated images that cannot be used for collecting a fluorescence measurement.
  • the degree to which a micro-length tube element fluoresces is dependent on the concentration of the targeted capture agent, e.g.
  • the best exposure setting must be determined in-situ, for each fluid channel in the chip.
  • a scan as depicted in Scanning, is constructed.
  • the auto- expose procedure then follows the sequence given in Scanning.
  • the ROI used for this procedure is the same as that used for the fluorescence scan discussed in the next section.
  • This procedure is done with the LED off and the Laser on. To avoid significant photo-bleaching effects the velocity of this scan is selected to minimize the laser exposure incurred by the element.
  • the end result of this process is the optimal exposure setting per channel to be used in the fluorescence scan discussed next.
  • the Fluorescence Scan(FS) can be constructed.
  • an ROI is used to collect pixel intensity values.
  • the ROI for the fluorescence scan is a rectangle oriented with the long side perpendicular to the flow channel and positioned in the image on top of the laser cross section (Scanning & 28.
  • the size of the ROI is determined by the size of the laser spot, the width of the fluorescent region on the micro-length tube element, the width of the channel containing the element, the number of pixels needed for a measurement and the scan speed.
  • the data collected from the FS is loaded into memory and the mean ROI value is plotted vs. time (Scanning). This Figure depicts the mean ROI value for all the channels scanned vs. time.
  • each segment consists of one flow channel's worth of data ().
  • a peak detection algorithm is used to determine the element positions in the channel with respect to the background signal.
  • the micro-length tube element positions found during the 'Element Find' can also be used to locate the elements in the segment. Thresholding
  • k-means clustering can be used to separate the pixels associated with the background from the pixels associated with the micro- length tube element.
  • the outputs of the clustering algorithm are centroids representing the mean background value and the mean element value. The mid-point between these two centroids is used as a threshold. Thresholding must be done on a channel-by-channel basis due to differing background and exposure settings per fluid channel.
  • the mean time history gets filtered using a Savitzky-Golay (SG) filter and the peak detect algorithm identifies all threshold crossings larger than a predetermined width, thereby rejecting of most of the high frequency noise in the data.
  • the time history is further broken up into an element signal component and a background signal component.
  • the element signal component comes from the section of the channel with the fluorescent micro-length tube element in it.
  • the background component comes from the 'empty' section of the channel adjacent to, but downstream of the fluorescent element (). This allows each micro-length tube element to have its own background-offset correction.
  • the center of each component is found and the data points +/- 25% of the element width are then extracted to create an average value for each component (See the highlighted points in Scanning). Only points about the center of the element and background are used in order to eliminate element edge effects. Since the signal rides on a background offset, the average of the background points is subtracted from the average of the element points and the result is normalized for camera exposure and finally stored as that element's mean RFU (Relative Fluorescence Unit). This is performed for each micro-length tube element in each channel on the chip.
  • RFU Relative Fluorescence Unit
  • This hybridized DNA is typically a synthetic molecule custom synthesized from any of the commercially available oligo supply houses (e.g. Integrated DNA Technologies IDTDNA.com), or an amplicon, the end-product of an amplification reaction such as PCR, or various isothermal reactions (Hyberbranched Amplification, Helicase reactions, qPCR, cold-PCR, etc), as these methods can easily generate the relatively high concentrations of DNA required for fluorescent detection. These higher concentration DNA hybridization events can be visualized either by the use of an intercalating agent such as Sybrgreen (Life Technologies) or Ethidium Bromide.
  • an intercalating agent such as Sybrgreen (Life Technologies) or Ethidium Bromide.
  • DNA strands in solution can be directly fluorescently labeled either during commercial synthesis, or using any of the chemical or enzymatic methods known to those practiced in the art (e.g. PCR with labeled nucleotides, etc). In this manner DNA strands that are sufficiently
  • DNA immobilized on the GNR is modified to generate signal following hybridization.
  • DNA strands known in the art as hairpin probes are specifically designed to fluoresce, or increase in fluorescence, following the hybridization of a complimentary molecule.
  • a typical design includes a DNA molecule that possess a fluorescent dye and a quencher molecule located on different bases of the molecule, typically at the distal ends of the strand.
  • These probes are typically self-complementary; hence normally found in a closed, hairpin confirmation wherein the fluor and the quencher are in close proximity and non-fluorescent. When the probes binds to a complimentary sequence, however, the molecule becomes linear and double stranded, separating the fluor from the quencher and fluorescence results.
  • a similar application utilizes a single sample flowing through multiple parallel channels, in which at least one channel is devoted to only one or more protein assay GNRs, but another channel has one or more GNRs functionalized to DNA for monitoring purposes, that channel having only one or more GNRs functionalized for DNA or also including GNRs for other purposes, such as protein assay mentioned earlier.
  • the DNA is employed as a signal calibrator, where the fluorescent signal from the DNA is compared to an expected signal, and the observed signal is either corrected to match the expected signal (the difference presumably due to differences in fluid flow rates, laser intensity or focus), or is simply confirmed to be within a predefined range, and hence acceptable, or outside the range, and thus unacceptable, thus alerting the user of possible poor quality data.
  • GNRs are produced, each complimentary to an individual population of DNA targets. These distinct GNRs can all be placed in the same channel, or can be spread across multiple channels.
  • the data is used to generate a calibration curve. This calibration curve is either used in conjunction with pre-determined relationships between DNA signal and protein concentration to determine the concentration of proteins detected by antibody -coated GNRs, or can be used to calibrate the signal from one instrument with another, thus ensuring that data generated on one system is equivalent with that generated on another.
  • GNR-based DNA capture also enables the detection or monitoring detect of the levels of a specific DNA sequence or sequences in a sample, whether it be solely DNA detection, or a hybrid system in which protein and DNA are simultaneously detected using respective GNRs in a single system.
  • populations of GNRs are made with capture strands complementary to the sequence of interest, for instance a sequence in circulating blood or in cell lysate.
  • multiple different populations of GNRs, specific for different DNA target populations can be generated and employed within a single channel, or cartridge provided sufficient discrimination exists between hybridization conditions for the various target sequences. In situations where a high concentration of DNA is available, DNA amplification is not required.
  • non-amplified systems depends upon how many copies are present in the sample. For example when one is looking at specific viral loads, depending on the level of a virus present in a patient, one may encounter suitably high levels of DNA. Also in the case of transgenic organisms, multiple copies of specific genes introduced into the transgene are often present a high level.
  • the process can be employed to ensure organic crops are free of engineered seed. This is of particular interest to the organic farming profession as a whole, and certain geographic areas such as Europe.
  • This invention addresses the requirement to test multiple samples and analytes (DNA or protein) simultaneously with a relatively small footprint and high ease of use.
  • This type of system is employed at transportation hubs, ports of entry and other areas where the crops are concentrated after harvest (grain silos, etc.).
  • Diagnostic applications include the ability to monitor disease progression via the patient's biological response (protein production, e.g. cytokines) and the level of the infectious agent (by DNA signature). For example, in the case of flu outbreaks, one can simultaneously monitoring a patient's cytokine levels while quantifying the relative abundance of flu strains as indicated by GNR's specific diagnostic DNA sequences for the major flu subtypes.
  • the DNA-based GNR system can also be used as an enrichment application, where capture probes are immobilized to the GNR, and a sample is flowed through the channel, with complimentary sequence binding to the GNRs. The capture can be employed for one of two purposes.
  • Either the captured material can be released in a subsequent wash step, and recovered for downstream manipulation of the enriched nucleic acid, such as next-gen sequencing.
  • the capture process can be employed to REMOVE unwanted sequence from a sample, as a form of subtractive hybridization.
  • a high concentration, possibly confounding DNA species can be removed from the sample to enable downstream manipulation of the residual nucleic acid.
  • the ability to intermingle the DNA and antibody GNRs in the same channel or place them in discrete channels provides the ability to either maximize plexity (the number of distinct analytes measured in one assay) or to employ substantially different reagents for the different detection systems if required. For example, if DNA detection requires buffers and conditions that are not amenable to antibody based detection, separate channels can be employed, while if the two systems utilize similar conditions the two assays can be combined, saving real estate and enabling more assays to be run per cartridge. DNA binding rates and specificities can be controlled through buffer composition and the presence of additives such as salt, DMSO, TMAC/TMAO and free Mg++ to mention a few. Individually addressable channels permit the use of channel-specific additives or hybridization buffers designed to ensure maximal hybridization conditions for the DNA probes.
  • Hybridization temperatures of given nucleic acids are determined by a number of factors, mainly the sequence length and base content (the relative amount of A,C,T and G nucleotides). If the temperature is too far above the hybridization temperature, the complimentary strands will not anneal. If it is too low, non-specific binding of non-complimentary strands can result. Thermal control is allows the hybridization temperature of a given channel to be tailored to the optimized temperature for the nucleic acid hybridizing in the channel.
  • the channel temperature be controlled, but the spatial channel separation, possibly in conjunction with thermal isolation/insulation strategies including air gaps, insulating foam or rubber or peltier cooling pads, allow the temperature of a given channel to be shifted dramatically relative to the temperature of a neighboring channel.
  • This provides a variety of benefits. Reaction temperatures are easily adjusted to ensure optimal DNA hybridization temperatures, providing optimal specificity for DNA capture probes in each individual channel.
  • thermal control of the individual channel permits combination of thermally stabile and thermally labile assays. Assays that require or benefit from elevated temperature (such as nucleic acid hybridizations) can be run concurrently with assays that require stabile or reduced temperatures such as protein assays by isolating thermal exchange from one channel to the next.
  • Novel systems employ the PDMS-confined micro-length tube elements with nucleic acid, antibody or antigen capture agent (i.e., probe) immobilized on internal surfaces of the elements: Besides those previously described in the many examples above, are the following:
  • a combination of (a) one or more micro-length tubes are internally functionalized with nucleic acid capture agent and (b) one or more micro-length tubes are internally functionalized with capture agent for antibody-antigen binding.
  • the agents are selected, and present within the micro- length tubes in sufficient number of each type element, with active agent in sufficient concentrations, to enable the nucleic-acid functionalized elements to detect a complementary tracer and serve as an assay control or in a monitoring system for an antibody- antigen assay conducted by successive back-and forth flows within the microfluidic channel.
  • An example arrangement is illustrated in Figs. 104, 104 A and 104B.
  • That implementation uses a tracer for antibody-antigen binding with respect to a nucleic acid assay.
  • a tracer of one nucleic acid can be employed with respect to an assay for another nucleic acid, using appropriately functionalized micro-length tubes for capture of the tracer and for the assay.
  • micro-length tube elements are provided for the assay than for tracer detection.
  • the micro- length tubes of each functionalization are pre- formed en masse, as by dicing long drawn tubing, batch functionalized with respective capture agents, and micro-length tube elements from the batch are located in the micro-fluidic channel. This can be done by a pick-and-place instrument, such as tweezer or vacuum tip instrument, which may be manual or under automated control as previously described.
  • the microfluidic system may be provided in a portable cartridge, devoted to a single sample, or multiple microfluidic networks may be provided, having respectively different sample wells or sources.
  • the capture protocol is preferably implemented with flows of successive sample, wash and reagent(s), each flow phase including a succession of back and forth movements of a given slug of a given fluid, slug dimension of the order of 100 times the length of a micro-length tube element, with sufficient number of successive slugs of that fluid to carry out the intended phase of the assay, before the next fluid of the assay sequence is introduced.
  • micro-length tube elements immobilizing a nucleic acid capture agent to their interior surfaces are provided for passive (i.e. without amplification) analytical detection of a native nucleic acid (or more than one) in a sample.
  • An example is given in Fig. 105.
  • a tracer as described in (1) is also included.
  • the micro-length tubular elements are pre-formed en masse, batch functionalized with respective capture agents, and elements from the batch are located in the channel, e.g. by a pick-and-place instrument, which may be manual or under the automated control shown.
  • the microfluidic system may be provided in a portable cartridge, devoted to a single sample, or multiple microfluidic networks may be provided, having respectively different sample sources;
  • a plurality of parallel, isolated micro-channels connected to receive portions of the same sample differing sets of internally functionalized micro-length tube elements are provided, for conducting multiple independent assays on the same sample, each as described for (2), with or without a tracer as described in (1), at least some of the micro-length tube elements being functionalized with nucleic acid.
  • An example is shown in Fig. 106.
  • two or more parallel channels receive sample from the same source and discharge to a common waste receptacle, as shown previously.
  • multiple channels are provided with nucleic acid immobilized within micro-length tubes, the nucleic acids being different species in the respectively different channels, and provisions are made for applying different reaction conditions to the respectively different channels, for instance, different temperature conditions.
  • An example is shown in Fig. 107.
  • nucleic acid probes may be incorporated in each channel to detect a tracer, according to feature (1) above, thus to obtain indication of proper operation of each channel, which may be conducting a different assay from the rest.
  • An example is given in Fig. 108.
  • nucleic acids illustrated in the figures are shown as DNA (i.e. single strand DNA) , but like examples can be employed with other forms of nucleic acid, for instance single strand RNA or mRNA.
  • immobilized capture agent for antibody- antigen binding are shown as an antibody, but antigens can alternatively be immobilized as capture agents against antibody targets.
  • Micro-length tubes of various compositions can be used, for instance transparent plastic with low fluorescence, but the presently preferred form is glass, forming glass nano-reactors (GNRs), and those are shown in the following examples.
  • the tubes are preferably sections of drawn form, with the smooth internal surface characteristics of the drawing process, in which the material is progressively drawn from a heated ingot or progressively emerges through a stationary die.
  • the substance is chosen to be transparent to the wavelengths of fluorescence passing outwardly, and in the case of stimulated fluorescent emission, also to the wave length of the stimulating radiation passing inwardly.
  • the micro-length tubes need not be transparent.
  • Figs. 104, 104A and 104B illustrate a microfluidic channel that is part of a microfluidic network having micro-valves and micro-pistons, all as previously described herein, that produce flows in the channel.
  • the microfluidic channel is of width W, for instance 180 micron.
  • GNR's are held immobilized in the channel by a PDMS membrane that forms the top of the channel.
  • the GNR's may have an outside diameter O.D. of 125 micron, inside diameter I.D. of 75 micron, and length L. of 250 micron.
  • Other regions of the same PDMS membrane form pneumatically deflectable portions of pneumatically actuated valves and pistons that produce the indicated channel flow in response to positive and negative pneumatic pressure applied to respective deflection chambers, controlled by a network of pneumatic channels connectible to a pneumatic controller, all as previously described.
  • the inner surface of the first two GNRs carry immobilized DNA for capturing a target tracer and the following four GNRs carry immobilized antibodies, e.g. for assay.
  • This arrangement is useful in an antibody detection platform in which the DNA-immobilized GNRs are used as a control for the execution of the assay, i.e. to determine that the right fluid has flowed at the right rate and right duration.
  • This arrangement is also useful to passively detect (i.e. without amplification) native DNA occurring at high enough concentrations not requiring biological or signal amplification. In this particular case both DNA and antibody detection occur in a single channel using immobilized GN's.
  • a tracer is spiked into the sample containing a complementary nucleic acid strand, e.g. a DNA strand to bind to an immobilized DNA strand on the internal surface of the GNR
  • nucleic acid Another use with respect to nucleic acid, concerns immobilizing nucleic acid to the surface of the GNRs for capture of a native nucleic acid in a sample.
  • a series of 6 GNRs are placed in a single channel, the internal surface of each GNR having immobilized DNA for capture of native nucleic acid in a sample.
  • the nucleic acid species may be the same for the set of GNRs, e.g. for purpose of assay redundancy, or different, to detect different species.
  • the GNR-immobilizing PDMS membrane be permanently bonded to structure forming the walls of the channel, to achieve a robust assay device.
  • PDMS activated surface bonding may be by use of PDMS activated surface bonding previously described herein, using the make and break technique at the associated micro-valves.
  • An alternative use for the arrangement shown is to capture DNA for extraction and assay by other means or for further processing.
  • the PDMS membrane is not surface- activated during manufacture, and forms a removable bond with a cooperating surface such as another layer of un-surface-activated PDMS in which channels sides are cut or glass forming the channel sides.
  • Fig. 106 instead of having a single channel with immobilized nucleic acid GNR's, a series of channels is provided, illustrated here by 4 parallel channels, in which a selected combination of nucleic acid-immobilized GNRs and antibody- immobilized GNRs are placed.
  • Fig 106 illustrates channels 1 and 2 having nucleic acid-immobilized GNRs and channels 3 and 4 having antibody- immobilized GNRs.
  • Fig. 107 again has 4 parallel channels with nucleic acid- immobilized GNRs in channels 1 and 2 and antibody-immobilized GNRs in channesl 3 and 4, with the addition of selected channels being uniquely heated.
  • channel 1 is shown to be heated to a temperature of 50 degrees Celsius
  • channel 2 to a temperature of 37 degrees Celsius
  • the remaining two channels heated to 32 degrees Celsius.
  • the specific elevated heating of channels 1 and 2 are for the purpose of optimizing the specificity of the nucleic acid binding properties for the particular nucleic acids that are used in those channels.
  • Fig. 108 illustrates 4 channels with 3 GNRs in each channel, the first 2 GNR's in each channel having antibodies immobilized on the GNRs while the 3 rd GNR in each channel has nucleic acid immobilized on the GNR for the purpose of running control using the nucleic acid GNRs or, in the alternative, for the purpose of passive detection of native DNA.
  • Figs. 109, 109A and 110 illustrate use of the same tools previously described with respect to placing the elements, Figs. 109 and 109A illustrating removal of a micro-length element with tweezers in the case of channel width being greater than width of the micro-tubes, while Fig. 110 illustrate removing a GNR with tweezers for a channel in which the micro-length elements have been force-fit.
  • Figs. 109, 109A and 110 illustrate use of the same tools previously described with respect to placing the elements
  • Figs. 109 and 109A illustrating removal of a micro-length element with tweezers in the case of channel width being greater than width of the micro-tubes
  • Fig. 110 illustrate removing a GNR with tweezers for a channel in which the micro-length elements have been force-fit.

Abstract

L'invention concerne un dispositif microfluidique comprenant un réseau de canaux microfluidiques scellé sur un côté par une feuille de membrane, la feuille ayant un PDMS qui délimite au moins la surface scellant le canal. La feuille de membrane sur son côté opposé scelle un côté d'un canal pneumatique. Le canal pneumatique est agencé pour permettre à la déformation pneumatique d'une partie déformable de la feuille de membrane d'être en contact avec une surface opposée pour réguler l'écoulement dans un canal du réseau. La feuille de membrane confine dans un canal du réseau au moins une microparticule, un tube de longueur micrométrique ou un nano-réacteur en verre, fonctionnalisés avec un agent de capture, qui a été inséré dans ce canal.
PCT/US2013/033610 2009-11-23 2013-03-22 Acide nucléique confiné dans une membrane pdms et éléments de capture à tube de longueur micrométrique fonctionnalisés avec des anticorps/antigènes, et systèmes les utilisant WO2013142847A1 (fr)

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US14/479,286 US9700889B2 (en) 2009-11-23 2014-09-06 Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results
US14/479,283 US9500645B2 (en) 2009-11-23 2014-09-06 Micro-tube particles for microfluidic assays and methods of manufacture
US14/479,290 US9651568B2 (en) 2009-11-23 2014-09-06 Methods and systems for epi-fluorescent monitoring and scanning for microfluidic assays
US14/479,288 US9759718B2 (en) 2009-11-23 2014-09-06 PDMS membrane-confined nucleic acid and antibody/antigen-functionalized microlength tube capture elements, and systems employing them, and methods of their use
US14/479,291 US9546932B2 (en) 2009-11-23 2014-09-06 Microfluidic assay operating system and methods of use
US14/479,285 US10065403B2 (en) 2009-11-23 2014-09-06 Microfluidic assay assemblies and methods of manufacture
US14/479,284 US10022696B2 (en) 2009-11-23 2014-09-06 Microfluidic assay systems employing micro-particles and methods of manufacture
US14/479,287 US9855735B2 (en) 2009-11-23 2014-09-06 Portable microfluidic assay devices and methods of manufacture and use
US15/105,297 US10401463B2 (en) 2012-09-24 2014-12-20 Breath-hold detection for magnetic resonance imaging
US15/340,661 US10220385B2 (en) 2009-11-23 2016-11-01 Micro-tube particles for microfluidic assays and methods of manufacture
US15/477,902 US10786800B2 (en) 2009-11-23 2017-04-03 Methods and systems for epi-fluorescent monitoring and scanning for microfluidic assays
US15/581,526 US10076752B2 (en) 2009-11-23 2017-04-28 Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results
US15/638,526 US10209250B2 (en) 2009-11-23 2017-06-30 PDMS membrane-confined nucleic acid and antibody/antigen-functionalized microlength tube capture elements, and systems employing them, and methods of their use
US16/118,985 US10414143B2 (en) 2009-11-23 2018-08-31 Microfluidic assay assemblies and methods of manufacture
US16/570,127 US11292237B2 (en) 2009-11-23 2019-09-13 Microfluidic assay assemblies and methods of manufacture
US17/711,601 US11938710B2 (en) 2009-11-23 2022-04-01 Microfluidic assay assemblies and methods of manufacture

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PCT/US2013/030054 Continuation-In-Part WO2013134742A2 (fr) 2009-11-23 2013-03-08 Particules micro-tubulaires pour analyses microfluidiques et procédés de fabrication
PCT/US2013/030056 Continuation-In-Part WO2013134744A2 (fr) 2009-11-23 2013-03-08 Ensembles d'analyse microfluidique et leurs procédés de fabrication
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PCT/US2013/030052 Continuation-In-Part WO2013134740A1 (fr) 2009-11-23 2013-03-08 Procédés et systèmes de surveillance et d'analyse épi-fluorescents destinés à des essais microfluidiques
PCT/US2013/000062 Continuation-In-Part WO2013133899A1 (fr) 2009-11-23 2013-03-08 Systèmes d'analyse microfluidiques utilisant des micro-particules et procédés de fabrication
PCT/US2013/030057 Continuation-In-Part WO2013134745A1 (fr) 2009-11-23 2013-03-08 Dispositifs d'essai microfluidique portables et procédés de fabrication et d'utilisation
PCT/US2013/030056 Continuation-In-Part WO2013134744A2 (fr) 2009-11-23 2013-03-08 Ensembles d'analyse microfluidique et leurs procédés de fabrication
PCT/US2013/030053 Continuation-In-Part WO2013134741A2 (fr) 2009-11-23 2013-03-08 Procédés et systèmes de fabrication de systèmes d'analyse de microréseaux, de mise en oeuvre d'analyses microfluidiques, et de surveillance et de balayage pour obtenir des résultats d'analyse microfluidique
US14/479,288 Continuation-In-Part US9759718B2 (en) 2009-11-23 2014-09-06 PDMS membrane-confined nucleic acid and antibody/antigen-functionalized microlength tube capture elements, and systems employing them, and methods of their use
US14/479,285 Continuation-In-Part US10065403B2 (en) 2009-11-23 2014-09-06 Microfluidic assay assemblies and methods of manufacture
US14/479,290 Continuation-In-Part US9651568B2 (en) 2009-11-23 2014-09-06 Methods and systems for epi-fluorescent monitoring and scanning for microfluidic assays
US14/479,284 Continuation-In-Part US10022696B2 (en) 2009-11-23 2014-09-06 Microfluidic assay systems employing micro-particles and methods of manufacture
US14/479,286 Continuation-In-Part US9700889B2 (en) 2009-11-23 2014-09-06 Methods and systems for manufacture of microarray assay systems, conducting microfluidic assays, and monitoring and scanning to obtain microfluidic assay results

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US11278886B2 (en) 2010-09-07 2022-03-22 Lumiradx Uk Ltd. Assay device and reader
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