WO1997027324A1 - Parallel reaction cassette and associated devices - Google Patents

Parallel reaction cassette and associated devices Download PDF

Info

Publication number
WO1997027324A1
WO1997027324A1 PCT/US1997/000298 US9700298W WO9727324A1 WO 1997027324 A1 WO1997027324 A1 WO 1997027324A1 US 9700298 W US9700298 W US 9700298W WO 9727324 A1 WO9727324 A1 WO 9727324A1
Authority
WO
WIPO (PCT)
Prior art keywords
reaction
chamber
chambers
fluid
cassette
Prior art date
Application number
PCT/US1997/000298
Other languages
French (fr)
Inventor
Peter David Southgate
Zygmunt Marian Andrevski
William Ronald Roach
Peter John Zanzucchi
Original Assignee
Sarnoff Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sarnoff Corporation filed Critical Sarnoff Corporation
Priority to AU18251/97A priority Critical patent/AU1825197A/en
Publication of WO1997027324A1 publication Critical patent/WO1997027324A1/en

Links

Classifications

    • FMECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F15FLUID-PRESSURE ACTUATORS; HYDRAULICS OR PNEUMATICS IN GENERAL
    • F15CFLUID-CIRCUIT ELEMENTS PREDOMINANTLY USED FOR COMPUTING OR CONTROL PURPOSES
    • F15C5/00Manufacture of fluid circuit elements; Manufacture of assemblages of such elements integrated circuits
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0093Microreactors, e.g. miniaturised or microfabricated reactors
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273Containers 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
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502738Containers 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 integrated valves
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/505Containers for the purpose of retaining a material to be analysed, e.g. test tubes flexible containers not provided for above
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00351Means for dispensing and evacuation of reagents
    • B01J2219/00353Pumps
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00351Means for dispensing and evacuation of reagents
    • B01J2219/00389Feeding through valves
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00457Dispensing or evacuation of the solid phase support
    • B01J2219/00459Beads
    • B01J2219/00466Beads in a slurry
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00497Features relating to the solid phase supports
    • B01J2219/005Beads
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00585Parallel processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/0059Sequential processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • B01J2219/00722Nucleotides
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00783Laminate assemblies, i.e. the reactor comprising a stack of plates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00819Materials of construction
    • B01J2219/00822Metal
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00819Materials of construction
    • B01J2219/00824Ceramic
    • B01J2219/00828Silicon wafers or plates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00819Materials of construction
    • B01J2219/00831Glass
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00819Materials of construction
    • B01J2219/00833Plastic
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00873Heat exchange
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00891Feeding or evacuation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/0095Control aspects
    • B01J2219/00952Sensing operations
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0809Geometry, shape and general structure rectangular shaped
    • B01L2300/0816Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0861Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867Multiple inlets and one sample wells, e.g. mixing, dilution
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/12Specific details about materials
    • B01L2300/123Flexible; Elastomeric
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/18Means for temperature control
    • B01L2300/1805Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/18Means for temperature control
    • B01L2300/1838Means for temperature control using fluid heat transfer medium
    • B01L2300/185Means for temperature control using fluid heat transfer medium using a liquid as fluid
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406Moving fluids with specific forces or mechanical means specific forces capillary forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0475Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0481Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/06Valves, specific forms thereof
    • B01L2400/0633Valves, specific forms thereof with moving parts
    • B01L2400/0655Valves, specific forms thereof with moving parts pinch valves
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating 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
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/06Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178Special arrangements of analysers
    • G01N2035/00237Handling microquantities of analyte, e.g. microvalves, capillary networks
    • G01N2035/00247Microvalves
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00Chemistry: analytical and immunological testing
    • Y10T436/25Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/2575Volumetric liquid transfer

Definitions

  • the present invention relates to a disposable parallel reaction device for conducting reactions, which device can include a component containing all necessary supply and reaction chambers and connecting fluid exchange channels.
  • the parallel reaction device is particularly adapted for conducting polymerase chain reaction ("PCR") assays, and other scientific, forensic and diagnostic assays. Synthetic reactions, including combinatorial chemistry, can also be conducted in the device.
  • PCR polymerase chain reaction
  • the PCR assay has provided a powerful method of assaying for the presence of either defined segments of nucleic acids or nucleic acid segments that are highly homologous to such defined segments.
  • the method can be used to assay body fluids for the presence of nucleic acid specific for particular pathogens, such as the mycobacterium causing Lyme disease, the HIV virus or other pathogenic microbes.
  • the microbe diagnostic assay functions by adding, to a sample that may contain a target segment of nucleic acid from the microbe's genome, at least one pair of "primers” (i.e., relatively short nucleic acid segments or nucleic acid analogs) that specifically bind to (i.e., "hybridize” with) the target segment of nucleic acid.
  • the first primer of a pair binds to a first strand of the two-stranded target nucleic acid segment and, when hybridized, can prime the enzymatic reproduction of a copy of the second strand of the target nucleic acid segment in a direction arbitrarily designated as the downstream direction.
  • the second primer of a pair binds to the second strand of the target nucleic acid segment at a position downstream from the first primer hybridization site and can prime the enzymatic reproduction of a copy of the first strand of the target nucleic acid segment in the upstream direction.
  • the second primer will hybridize with the theoretical second strand determined with the Watson-Crick base-pairing rules.
  • To the sample are added the monomer building blocks of nucleic acid and an enzyme that specifically catalyzes nucleic acid reproduction from a single strand of nucleic acid to which the short primer is bound.
  • the enzyme is preferably highly resistant to destruction by elevated temperatures.
  • the sample is heated to a DNA melting temperature to separate the two strands of the sample nucleic acid and then cooled to a replication temperature.
  • the replication temperature allows the primers to specifically bind to the separated strands and allows the reproductive enzyme to operate.
  • the reaction mix contains two sets of the two stranded nucleic acid segment for each target nucleic acid segment that was originally present. Heating and replication temperature cycles are repeated until sufficient amounts of the nucleic acid segment are created through this exponential reproduction method. For instance, after 20 cycles the segment has been amplified as much as 2 20 -fold, or roughly 1 ,000,000-fold.
  • the degree of amplification achieved by the assay creates a large risk of contamination from foreign DNA from handling. Thus far, this risk has been dealt with in commercial, manual procedures by conducting the reactions in "clean" facilities that are extremely expensive to construct and maintain.
  • this risk implies that all the reagents needed and the reaction chamber for the amplification should be contained in a disposable platform in which the sample can be inserted in a controlled, one-time operation. This risk also implies that sample preparation steps should be minimized and, to the extent possible, conducted within a disposable platform.
  • the high temperatures needed to "melt" the nucleic acid so that the two strands separate imply that the reaction chamber must be well-sealed against vapor loss, even while allowing the insertion and removal of various reagent fluids. This goal is particularly hard to achieve on a suitable, disposable platform.
  • the reactions should be conducted in relatively small volumes, generally volumes of no more than about 100 ⁇ l, to conserve expensive reagents and minimize the amount of sample, which could be a precious sample fluid or tissue that must be conserved to allow for other types of testing or is available only in a small amount.
  • the present invention provides a device for conducting parallel reactions, comprising: (a) a cassette formed of a body having an upper surface, a lower surface, and an edge, and including an upper film or a lower film attached to the upper or lower surface, respectively, wherein the upper or lower film is formed of a flexible material; (b) two or more reaction flow-ways in the cassette, wherein each reaction flow-way comprises two or more fluid chambers which comprise a first supply chamber and a first reaction chamber having an upper wall and a lower wall, and wherein the fluid chambers are serially connected by first fluid exchange channels; (c) a valve for controlling the flow of fluid through a first fluid exchange channel; (d) a pump for moving fluids into or out of the fluid chambers; and (e) a first inlet port on the cassette connected to a first supply chamber in each reaction flow-way by a second fluid exchange channel.
  • the first supply chamber is preferably a supply chamber having a releasable seal blocking the outlet into the first fluid exchange channel connecting the first supply chamber to its reaction flow-ways; more preferably, the first supply chamber is an internal-outlet supply chamber.
  • the pump preferably comprises a foot-pad pump with foot-pads designed to push on the first supply chamber to open the sealed outlet and pump fluid into the connected first fluid exchange channel.
  • the first supply chamber is collapsible upon evacuation and fillable from a vacuum-collapsed state to a defined volume.
  • the second fluid exchange channel is releasably sealed so as to block the flow of fluids through the second fluid exchange channel.
  • the second fluid exchange channel is heat-sealed; more preferable, the second fluid exchange channel is sealed at multiple locations to prevent fluid communication between the first supply chambers.
  • the valve used in the context of the present invention is a plunger-type valve that is controlled by a pressure control means for: (i) applying a positive pressure to the plunger-type valve such that the plunger-type valve presses against the upper or lower film so as to impede the flow of fluid in a first fluid exchange channel, and (ii) releasing the positive pressure to the plunger-type valve such that the plunger-type valve releases from the flexible film so as to permit the flow of fluid in the first fluid exchange channel.
  • the plunger of the plunger-type valve is affixed to an instrument from which the cassette is detachable.
  • the cassette can be formed of a body that comprises recesses in its upper or lower surface which, together with an associated upper or lower film, form the first and second fluid exchange channels, and a plurality of fluid chambers.
  • a fluid chamber is formed in the upper or lower surface and at least one first or second fluid exchange channel is formed on an upper or lower surface located above or below that fluid chamber.
  • the cassette of the present invention further comprises: (f) at least one hole situated in the body so as to connect a first or second fluid exchange channel formed at the upper or lower surface of the body with a first or second fluid exchange channel formed at the other surface.
  • the portion of upper or lower film covering a said fluid chamber made up of a recess in the body is embossed to mirror the shape of the bottom of the fluid chamber such that when the chambers is evacuated the film portion will invert to match the shape of the bottom of the chamber.
  • one of the pumps is a foot-pad pump having a foot pad that fits against the surface of the inverted embossed film portion of said fluid chamber.
  • the cassette further comprises: (g) one or more second supply chambers, wherein two or more fourth fluid exchange channels connect the second supply chamber to two or more reaction flow-ways, which fourth fluid exchange channels include two or more said valves so that fluid from the second supply chamber can be directed to any one of the connected reaction flow-ways to the exclusion of the other connected reaction flow-ways; and (h) one or more second inlet ports on the cassette each connected to one of the second supply chambers by a separate third fluid exchange channel.
  • the device further comprises (i) a metering chamber interposed between the second supply chamber and the connected reaction flow-way.
  • the combination of elements (f), (g), (h), and optionally (i) forms a sample insertion device.
  • the cassette has more than one such sample insertion device and sufficient reaction flow-ways such that different experimental samples can be reacted in parallel.
  • each first reaction chamber are formed of an embossed portion of a said upper film and an embossed portion of a said lower film, wherein the embossing allows upper and lower walls of the first reaction chambers to be brought together to minimize the volume of the first reaction chambers.
  • at least one pump comprises a foot-pad pump with upper and lower foot-pads designed to push together the upper and lower walls of a first reaction chamber.
  • at least one of the pumps comprises gas pressure conduits for applying a positive pressure to the flexible upper or lower walls of a first reaction chamber so as to cause the flexible upper or lower wall to press inward thereby decreasing the volume within the first reaction chamber and impelling the flow of fluids therefrom.
  • the cassette further comprises (j) one or more waste chambers; and (k) an exhaust port for evacuating one or more of the first reaction chambers or the waste chambers.
  • Each embodiment of the invention can further comprise (I) a heater for heating one or more of the fluid chambers; (m) a cooler for cooling one or more of the fluid chambers; and (n) a temperature monitor for monitoring the temperature of one or more of the fluid chambers.
  • a foot-pad for pumping fluid out of the fluid chamber is associated with a heater and cooler for the fluid chamber; more preferably, the heaters and the coolers comprise a thermoelectric heat pump attached to a heat sink having a heater element.
  • the heaters and the coolers can change the temperature of a fluid chamber at a rate of at least about 5°C per second.
  • each embodiment of the invention can further comprise (o) a permanent magnet that can be positioned adjacent to one or more of the fluid chambers, or removed therefrom, wherein further the invention comprises means for moving the magnet adjacent to or away from the cassette.
  • Each embodiment of the invention can also comprise (p) a detection chamber or channel having a transparent wall.
  • each such - 6 - embodiment can include (q) a light source capable of directing light to the transparent wall of a chamber or channel; and also (r) a light detection device capable of detecting: ( 1 ) the light reflected from an illuminated chamber or channel having a transparent wall; (2) the light transmitted through an illuminated chamber or channel having a transparent wall; or (3) the light emissions emanating from an excited molecule in a chamber or channel having a transparent wall.
  • the invention includes at least one valve that comprises: ( 1 ) a shut-off means comprising a valve ball or pinch foot, and (2) switching means for positioning the valve ball or pinch foot so that the valve ball or pinch foot: (i) presses against the flexible film to cut off flow through a first or second fluid exchange channel, or (ii) releases away from the flexible film to allow flow through the first or second fluid exchange channel.
  • the switching means preferably comprises spring loaded levers.
  • At least one valve comprises: ( D a spacer, (2) a spacer spring means for normally pressing the spacer against the flexible film so as to cut off the flow of fluids through a first fluid exchange channel, and (3) an electromagnet effective when activated to sufficiently release the pressure against the flexible film to allow the flow of fluids through the first or second fluid exchange channel.
  • the invention provides a device for conducting assays in parallel using fluids that are confined to a disposable cassette
  • the disposable assay cassette which comprises (i) at least two reaction flow-ways, including a first reaction flow-way designed to receive and assay an experimental sample and a second reaction flow-way designed to receive and assay a negative control, (ii) for each reaction flow- way, at least one supply chamber connected thereto and containing fluids needed in the assay and at least one reaction chamber, (iii) a negative control supply chamber connected with the second reaction flow-way containing the negative control, and (iv) a test sample supply chamber connected with the first reaction flow-way designed to receive a test sample through an inlet connected with the test sample supply chamber, valves for controlling the flow of fluids in the cassette, and an instrument comprising a temperature control unit for controlling in parallel the temperature in a reaction chamber in each reaction flow-way, valve actuators for opening and closing the valves in the cassette, and one or more pumps for pushing fluid out of the various supply
  • the cassette further comprises (v) a third reaction flow-way designed to receive and assay a test sample and a positive control, (vi) connecting routes between the test sample supply chamber and both the first and third reaction flow- ways, wherein these connecting routes are controlled by valves that allow selective flow between the test sample supply chamber and either the first or third reaction flow-way, and (vii) a first positive control supply chamber connecting with the third reaction flow-way containing the positive control.
  • the cassette further comprises (viii) a fourth reaction flow- way designed to receive and assay a positive control, and (ix) a second positive control supply chamber connecting with the fourth reaction flow-way containing the positive control.
  • the cassette preferably comprises (v) a third reaction flow-way designed to receive and assay a test sample and a positive control, (vi) connecting routes between the test sample supply chamber and both the first and third reaction flow-ways, wherein these connecting routes are controlled by valves that allow selective flow between the test sample supply chamber and either the first or third reaction flow-way, and (VIII) a first positive control supply chamber connecting with the third reaction flow-way containing the positive control.
  • the pumps comprise one or more foot-pad pumps.
  • the temperature control unit preferably comprises a thermoelectric heat pump; and the thermoelectric heat pump preferably is attached to a heat sink having a heater element.
  • the valves of this embodiment comprise plunger-type valves.
  • the invention further provides a method of conducting assays, including chemical diagnostic assays, antibody-based assays and nucleic acid amplification-based assays, using one of the aforementioned devices, which method comprises (a) providing the device for conducting assays in parallel, wherein reagents and control materials are pre-loaded into the supply chambers; (b) inserting a test sample into the test sample supply chamber; and (c) reacting in parallel in separate reaction flow-ways ( 1 ) the test sample and (2) the negative control.
  • the reagents or control materials include binding domains derived from antibodies; alternatively, the reagents or control materials include fluids containing primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction.
  • the reacting comprises reacting in separate reaction flow-ways (1 ) test sample and (2) negative control with a suspension of nucleic acid-binding beads, wherein the suspension of nucleic acid-binding beads is provided by a separate supply chamber for each reaction flow-way; and replacing the fluid suspending the nucleic acid-binding beads with a fluid containing primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction.
  • the nucleic acid binding beads are paramagnetic beads and the replacing step comprises ( 1 ) magnetically locking the nucleic acid-binding beads in place while pushing the suspending fluid into a waste chamber, (2) resuspending the nucleic acid- binding beads in a wash fluid, wherein wash fluid is introduced from a separate supply chamber for each reaction flow-way, (3) magnetically locking the nucleic acid-binding beads in place while pushing the suspending fluid into a waste chamber, and (4) resuspending the nucleic acid-binding beads in the fluid containing primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction.
  • the invention relates to a method of conducting nucleic acid amplification reactions using the aforementioned device, which method comprises (a) providing the device for conducting assays in parallel, wherein reagents and control materials are pre-loaded into the supply chambers, wherein the reagents or control materials include primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction; (b) inserting a test sample into the test sample supply chamber; and (c) reacting in parallel in separate reaction flow- ways ( 1 ) the test sample, (2) a negative control and (3) a mixture of the test sample and a positive control.
  • the present invention further preferably relates to a method of conducting nucleic acid amplification reactions using the aforementioned device, which method comprises: (a) providing the device for conducting assays in parallel, wherein reagents and control materials are pre-loaded into the supply chambers, wherein the reagents or control materials include primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction; (b) inserting a test sample into the test sample supply chamber; and (c) reacting in parallel in separate reaction flow-ways (1 ) the test sample, (2) a negative control, (3) a mixture of the test sample and a positive control and (4) a positive control.
  • the invention still further provides a device comprising a cassette suitable for conducting reactions therein, which cassette comprises a body having one or more recesses and one or more embossed films covering the recesses.
  • the cassette includes a hole extends through the body, further comprising a fluid exchange channel in communication with a valve, which valve is in communication with the hole, and a film having an embossed portion sealed to the body such that the hole and the fluid exchange channel are covered.
  • the device further comprises preferably a pneumatically driven plunger for pressing the embossed film portion at or about the valve, and pressure control means for (i) applying a positive pressure to the pneumatically driven plunger such that the plunger presses against the flexible film so as to close the valve, and (ii) releasing the positive pressure to the pneumatically driven plunger such that the plunger releases from the flexible film so as to open the valve.
  • Figures 1A, I B and 1 C show a top, side and bottom view of a cassette of the invention.
  • Figure 2A shows a side view of a Bursapak supply chamber.
  • Figure 2B illustrates a method for sealing closed a fluid exchange channel.
  • Figure 2C illustrates how pressure can be used to open a Bursapak supply chamber.
  • Figures 2D and 2E illustrate a foot-pad that can be used to pressurize the fluid in the Bursapak supply chamber.
  • Figure 3 schematically diagrams a parallel reaction device of the invention.
  • Figure 4A illustrates a cassette of the invention.
  • Figures 4B-4E show the cassette of Figure 4A with various subsets of the features thereof illustrated and numbered.
  • Figure 5A and 5B show a plunger-type valve mechanism for regulating fluid flow through a cassette.
  • Figure 6 shows in perspective view the part of a plunger-type valve located in the body of a cassette.
  • Figure 7 shows the parts of a plunger-type valve located outside the cassette (i.e., in the instrument).
  • Figures 8A, 8B and 8C show various configurations of valve mechanisms for regulating fluid flow through a cassette.
  • Figures 9A and 9B show a magnetic spring valve mechanism for regulating fluid flow through a cassette.
  • Figure 10 shows a support device for rapidly heating and cooling a reaction chamber and providing a foot-pad for a foot-pad pump.
  • Figures 1 1 A and 1 1 B show the operation of a foot-pad pump on a reaction chamber.
  • Figure 12 shows a schematic of accessory support devices for rapidly heating or cooling a reaction chamber.
  • Figure 13 shows another mechanism for rapidly heating or cooling a reaction chamber.
  • Figure 14 shows yet another mechanism for rapidly heating or cooling a reaction chamber.
  • Figures 15A and 15B show two side views of a detection channel.
  • Figure 16 shows an example of a magnet useful for locking paramagnetic beads at a certain location in a cassette.
  • Figures 17 A and B show a device for mounting a septum to the cassette.
  • PCR protocols and other nucleic acid amplification protocols often use an "annealing temperature" less than the replication temperature to accelerate the rate at which the primers bind to (i.e., hybridize with) the sample nucleic acid; this annealing temperature is typically between about 45°C and about 72°C, often about 55°C. Generally, the annealing temperature will be about 5°C below the lowest T m for the interaction between (a) one of the primers used in reaction and (b) the target nucleic acid segment.
  • Bursapak chamber a chamber formed in a solid support and having a film formed of a flexible material that is sealed to the support at the edges of the chamber and has an outlet channel that is blocked by a portion of the film which is sealed over the outlet channel, wherein the seal over the outlet is broken or removed by pressurizing the fluid contents of the chamber at a pressure that does not affect the seal at the edges of the chamber; preferably, the film is on one face of the cassette body and the outlet is oriented toward the other.
  • cassette a disposable device for conducting reactions therein having a cassette body, one or more upper membranes or one or more lower membranes which individually or in combination define one or more supply chambers, one or more reaction chambers and fluid exchange channels connecting the supply chambers to reaction chambers.
  • cassette body a solid portion having sufficient depth and sturdiness to allow cavities formed therein to provide the depth for fluid chambers and fluid exchange channels.
  • connection between fluid chambers, inlets or detection channels
  • two fluid chambers, inlets or detection channels are “connected” or have a “route of connection” therebetween if there is one or more fluid exchange channels joining the two such that fluid can move from one to the other.
  • DNA strand separation temperature the temperature used in a nucleic acid amplification protocol to separate the complementary strands of nucleic acid that may be present in a sample; this temperature is typically between about 92°C and about 97°C, preferably about 94°C.
  • the inserted fluid volume is within about 10% of the first volume, more preferably within about 3% of the first volume.
  • the first volume is the maximum volume of fluid that can be inserted into the chamber without affecting the integrity of the chamber.
  • fluid chamber encompasses reaction, supply, waste metering and sample storage chambers, and other fluid containing chambers.
  • contents of the chambers can be pumped out using a foot-pad having a shape that conforms to a covering film that is inverted to match the shape of the bottom of the chamber, the chamber can be closed by maintaining the foot-pad pressed against the inverted covering film.
  • fluid-tight a space or chamber is fluid-tight if it retains an aqueous fluid in the space at a temperature of 99°C for one hour; a seal between two materials is fluid- tight if the seal is substantially no more permeable to water than the most water-permeable such material.
  • foot-pad a plunger having a shape designed to conform to the inverted shape of the covering film of a supply chamber; when the plunger presses against the flexible film it pressurizes the fluid in the supply chamber and, if an exit is available, pushes the fluid out of the supply chamber.
  • foot-pad pump a mechanical, electromechanical or pneumatic device that uses a one or more, preferably two or more, foot-pads to press on one or more fluid chambers such as supply chambers or reaction chambers to pressurize the contents and push the contents out through an unobstructed connected fluid exchange channel.
  • integral parts or elements of a valve are integral to a body layer or to a cassette if they cannot be facilely and reversibly detached from that body layer or cassette.
  • internal outlet Bursapak supply chamber a Bursapak supply chamber wherein the outlet channel is located away from the edges of the supply chamber such that fluid-containing space is interposed between the sealed outlet channel and the edges chamber.
  • negative control a material designed to be comparable to a sample to be assayed but lacking the substance to be assayed for, such that a positive result upon assaying a negative control would indicate a problem with the assay protocol or assay reagents.
  • nucleic acid melting temperature or T m the transition temperature for two-stranded duplex of nucleic acid at which the equilibria shifts from favoring the base-paired duplex to favoring the separation of the two strands.
  • positive control a material designed to generate, in the absence of a problem with the assay chemistry such as the presence of an interfering substance, a positive assay result.
  • reaction flow-away a series of two or more serially connected fluid chambers through which fluids can move.
  • reduced pressure a pressure less than ambient atmospheric pressure.
  • replication temperature the temperature used in a nucleic acid amplification protocol to allow the nucleic acid reproductive enzyme to reproduce the complementary strand of a nucleic acid to which a primer is bound (i.e., hybridized); this temperature is typically between about 69°C and about 78°C, preferably about 72°C, when using a heat stable polymerase such as Taq polymerase.
  • serially connected two or more fluid chambers are serially connected if there are fluid exchange channels by which fluid from a first of the serially connected chambers can pass to a second of the serially connected chambers, and from there to a third of the serially connected chambers, and so on until the fluid passes to the last of the serially connected chambers.
  • target nucleic acid segment a segment of nucleic acid that is sought to be identified or measured in a sample, such as a sequence intended, if present, to be amplified in a nucleic acid amplification reaction such as a PCR reaction, strand displacement assay or ligase chain reaction; the target segment is typically part of a much larger nucleic acid molecule found in the sample.
  • thermoelectric heat pump a device for heating and cooling fluid chambers that is made up of one or more thermoelectric blocks.
  • the cassette of the present invention includes at least one reaction chamber and at least one supply chamber in combination with interconnecting fluid exchange channels.
  • the cassette comprises a body into which the aforementioned chambers and channels are formed such that when covered by a film and sealed, as described below, the formed body with film can hold fluids.
  • the shape of the body can be any shape, although preferably it is a flat square, rectangular or circular structure of length and width or diameter substantially greater than its depth, such as, for example, 3 cm x 3 cm x 3 mm, inter alia, and the length and width or diameter can be further described with respect to a top or bottom surface, and the depth can be further described with respect to an edge.
  • the chambers and channels prior to covering by the film can be open to any surface of the body, preferably is open to the top or bottom, more preferably is open to the top and bottom, although each chamber or channel preferably is open to one side only.
  • Figures 1 A, 1 B and 1 C show a top view, cross-sectional view and bottom view of a portion of one embodiment of a cassette 1 00 according to the invention.
  • the cassette 1 00 has a body 1 05 in which are defined inlet 1 30, first fluid exchange channel 141 , supply chamber 1 50, second fluid exchange channel 1 42, reaction chamber 1 60, third fluid exchange channel 1 43 and waste chamber 1 70.
  • the body 1 05 has first upper film 1 10A, second upper film 1 1 OB, third upper film 1 10C and lower film 1 20.
  • first seal portion 1 1 1 A (shaded area), second seal portion 1 1 1 B (shaded area) and third seal portion 1 1 1 C (shaded area) show where first upper film 1 1 0A, second upper film 1 1 0B and third upper film 1 1 0C, respectively, are sealed against body 105.
  • shading 1 21 shows where lower film 1 20 is sealed against body 1 05.
  • Inlet 1 30 has a septum 1 31 .
  • First, second and third upper films 1 10A-C are collectively referred to as "upper films 1 1 0.
  • Septum 1 31 can be, for instance a bilayer material formed of an outer layer of silicon or neoprene rubber and an inner layer of chemically inert material such as tetrafluoroethylene homopolymer (e.g. , Teflon, E.I. duPont de Nemours and Co., Wilmington, DE) facing the body 105.
  • Second upper film 1 1 0B and lower film 1 20 are embossed or shaped at positions 1 61 and 1 62 to help form reaction chamber 1 60, as will be described in greater detail below with reference to Figures 1 1 A and 1 1 B.
  • First upper film 1 1 0A is embossed or shaped at the location of supply chamber 1 50 so that first upper film protrudes above the upper surface of body 105, creating a greater volume for supply chamber 1 50 and facilitating the mechanism by which supply chamber 1 50 is emptied, as described further in the text below with reference to Figures 2A and 2B.
  • Third upper film 1 1 0A is embossed or shaped at the location of waste chamber 1 70, which embossing facilitates the mechanism by which the waste chamber is filled.
  • a valve 1 80 is formed in third fluid exchange channel 143. The outlet 1 51 of supply chamber 1 50 is sealed by a portion of first upper film 1 10A.
  • Supply chamber 1 50 is a Bursapak supply chamber, which type of supply chamber is a particularly useful type of supply chamber for use in the cassette of the invention. Because many of the cassettes described below make use of this preferred type of supply chamber, Bursapak supply chambers are described in more detail in the following section.
  • FIG 2A shows a side view of a Bursapak supply chamber 1 50 having supply cavity 1 55, which can contain a fluid.
  • the Bursapak supply chamber 1 50 has an inlet first fluid exchange channel 141 , which is preferably sealed, for instance by heat sealing at sealing location 1 41 A, after the Bursapak supply chamber 1 50 has been filled with fluid, and an outlet second fluid exchange channel 1 42 which is initially sealed with a fourth seal portion 1 1 1 D of first upper film 1 1 0A.
  • Figure 2B shows the use of die 1 300 to heat seal first fluid exchange channel 1 41 , at sealing location 1 41 A.
  • Figure 2C illustrated how pressure -- indicated by the arrows -- applied to the fluid in Bursapak supply chamber 700 is effective to pull the seal portion 1 1 1 away from the outlet second fluid exchange channel 142.
  • Figure 2D illustrates a foot-pad 210 that can be used to apply pressure to the fluid in Bursapak supply chamber 1 50 and pump it through outlet second fluid exchange channel 1 42 Foot-pads can be fabricated of any suitably sturdy material including, without limitation, aluminum, plastics, rubber, alumina, copper, sintered beryllia, and the like.
  • Upper films 1 1 0 and lower films 1 20 are preferably constructed of a flexible film such as a polyethylene, polyvinylidene fluoride or polyethylene/polyethylene terephthalate bi-layer film Suitable films are available from Kapak Corporation, Minneapolis, MN or E.I duPont de Nemours and Co., Wilmington, DE. Polyethylene/polyethylene-terephthalate bi-layer film such as 3M No. 5 or 3M No 48 (3M Corp., MN) or Dupont M30 (DuPont de Nemours, Wilmington, DE) are particularly preferred.
  • the polyethylene layer is preferably positioned against body 1 05.
  • Figure 2E shows the foot-pad used to pump fluid out of Bursapak supply chamber 1 50
  • the first upper film 1 1 0A is embossed or shaped, for instance by applying suitably shaped, heated dies to the first upper film 1 10A, so that it can protrude away from the body 1 05 when the supply chamber 1 50 is filled and will rest, without substantial stretching, against the bottom of supply chamber 1 50 when the supply chamber 1 50 is evacuated.
  • Bursapak chambers operate as illustrated in Figures 2A- 2C. To assure proper functioning, in some embodiments it may be necessary to seal fourth portion 1 1 1 D relatively more weakly, for instance using a weaker adhesive or a lower temperature sealing die.
  • Body 105 is preferably formed of a molded plastic, such as high density polyethylene, but other materials that are suitably resistant to the chemistries sought to be conducted on the parallel reaction device, such as glass and silicon-based materials, can be used.
  • body 1 05 is plastic, it is preferably formed by a molding process that is used to form cavities and channels that will be sealed with upper and lower films 1 1 0 and 1 20 to form fluid chambers and fluid exchange channels. Such cavities and channels are formed in glass and silicon materials by chemical etching or laser ablation.
  • Upper and lower films 1 1 0 and 1 20 typically have a thickness of from about 0.3 mils to about 5 mils, preferably from about 1 mil to about 3 mils.
  • Reaction chamber 1 30A typically has a thickness, between upper and lower films 1 10 and 1 20, of from about 0.1 mm to about 3 mm preferably of from about 0.5 to about 1 .0 mm and an area, defined by the inner surface of upper or lower films 1 1 0 or 1 20, of preferably from about 0.05 cm 2 to about 2 cm 2 , more preferably from about 0.1 cm 2 to about 1 cm 2 , yet more preferably about 0.5 cm 2 .
  • the dimensions of reaction chamber are preferably sized small enough to permit rapid thermal cycling (on the order of about 10 seconds) .
  • Fluid exchange channels typically have a diameter between about 200 and about 500 ⁇ m.
  • Supply chambers 1 50 typically have a volume between about 5 and about 500 ⁇ l, preferably from about 1 0 to about 200 ⁇ l, more preferably from about 30 to about 1 60 ⁇ l.
  • Metering chambers preferably have a volume between about 5 and about 50 ⁇ l.
  • the total volume of each reaction chamber 1 60 is between about 5 ⁇ l and about 200 ⁇ l, more preferably, between about 10 ⁇ l and about 1 00 ⁇ l.
  • each reaction chamber has a thickness (i.e., distance between upper film 1 10 and lower film
  • Upper and lower films 1 1 1 0 and 1 20 preferably are resistant to temperatures as high as about 1 20°C and are between about 1 and about 6 mils in thickness, more preferably, between about 2 and about 4.
  • the thinness of the membranes facilitates rapid heat exchange between the reaction chamber and an adjacent heating or cooling device.
  • Figure 3 illustrates schematically a parallel reaction device 301 according to the invention having five reaction flow-ways, each such flow-way, respectively, used for analyzing (A) a sample 300, (B) a positive control 31 0, (C) a negative control 320, (D) a positive control 330 combined with sample 300, and (E) a sample 300.
  • Each of these samples and controls is introduced into one of first through fifth lysing chambers 340A-E (collectively, lysing chambers 340). Lysing reagents and washing buffer can be distributed from first supply chamber 350 and second supply chamber 360, respectively, to all five lysing chambers 340. Waste can be emptied from lysing chambers 340 into a single waste chamber 370.
  • each of lysing chambers 340 can then be transferred to one of first through fifth reaction chambers 380A-E, respectively (collectively, reaction chambers 380).
  • Amplification reagents are added to each of reaction chambers 380 from a third supply chamber 390. Waste can be emptied from reaction chambers 380 into waste chamber 370.
  • the remaining contents of each of reaction chambers 380A-E can then be transferred into one of first through fifth storage chambers 399A-E, respectively.
  • Each valve which regulates the flow of fluids into and out of the various chambers is separately diagrammed in Figure 3 as an encircled letter "v. "
  • lysing chambers 340 and reaction chambers 380 preferably have flexible upper film 1 1 0 and lower film 1 20 that can be manipulated with a foot-pad pump or a gas pressure flow control means. If both upper and lower walls of a fluid chamber are formed with films 1 10 and 1 20, then channels passing through the region of the device occupied by the lysis chambers 340 or reaction chambers 380 must pass adjacent to such chambers rather than above or below the chambers.
  • FIG. 4A Another cassette 200 is illustrated in Figure 4A.
  • the illustrated cassette 200 has planar dimensions of 3 1 ⁇ inches by 5 5/1 6 inches, although other sizes are contemplated, including for instance in circumstances where the sizes of the fluid chambers and other components of the cassette differ from those illustrated.
  • Figures 4B - 4E show the body 205 of the cassette together with illustrations of various subsets of the components of body 205.
  • the solid lines connecting inlets, valves or fluid chambers represent fluid exchange channels. Those fluid exchange channels represented by dark lines are formed in the upper surface of body 205, while those represented by lighter lines are formed in the lower surface of body 205.
  • At the top of Figure 4B are illustrated the symbols used to represent an inlet 230 or a supply chambers 250 of various sizes (sizes recited for illustrative purposes only) .
  • Alpha first reaction chamber 261 A can receive fluid from any of seven supply chambers 250, which supply chambers 250 are alpha first supply chamber 251 A, alpha second supply chamber 252A, beta second supply chamber 252B, alpha third supply chamber 253A, beta third supply chamber 253B, alpha fourth supply chamber 254A and beta fourth supply chamber 254B.
  • Beta first reaction chamber 261 B, gamma first reaction chamber 261 C and delta first reaction chamber 261 D each can receive fluid, in a manner parallel to the arrangement for alpha first reaction chamber 261 A, from seven supply chambers 250 as illustrated.
  • Alpha first reaction chamber 261 A, beta first reaction chamber 261 B, gamma first reaction chamber 261 C 5 and delta first reaction chamber 261 D connect to alpha second reaction chamber 262A, beta second reaction chamber 262B, gamma second reaction chamber 262C and delta second reaction chamber 262D, respectively, via alpha first fluid exchange channel 241 A, beta first fluid exchange channel 241 B, gamma first fluid exchange channel 241 C and delta first fluid exchange
  • Alpha second reaction chamber 262A, beta second reaction chamber 262B, gamma second reaction chamber 262C and delta second reaction chamber 262D connect to first waste chamber 271 under the control of alpha first valve 281 A, beta first valve 281 B, gamma first valve 281 C and delta first valve 281 D, respectively.
  • alpha seventh inlet 237A and beta seventh inlet 237B which are connected to alpha fifth supply chamber 255A and beta fifth supply chamber 255B, respectively.
  • Alpha fifth supply chamber 255A and beta fifth supply chamber 255B are connected to alpha second reaction chamber 262A and beta second reaction chamber 262B.
  • Exhaust port 275 allows the first reaction chambers 261 , second reaction chambers 262, first waste chamber 271 , second waste chamber 272, metering chamber 290 and detection channels 295 to be evacuated prior to use. This evacuation is possible because all of the first reaction chambers 261 , second reaction chambers 262, first waste chamber
  • Alpha sealing position 276A and beta sealing position 276B can be heat sealed when the evacuation process is complete to lock the first reaction chambers 261 , second reaction chambers 262, first waste chamber 271 , second waste
  • sixth supply chamber 256 is filled using alpha eighth inlet 238A and is connected to metering chamber 290 under the control of alpha second valve 282A. Seventh supply chamber 257 is filled
  • metering chamber 290 35 using beta eighth inlet 238B and is connected to metering chamber 290 under the control of beta second valve 282B. From metering chamber 290 fluid can be directed to either gamma second reaction chamber 262C or delta second reaction chamber 262D under the control of gamma second valve 282C and delta second valve 282D, respectively.
  • fluid from alpha second reaction chamber 262A can be directed to alpha detection channel 295A under the control of alpha 5 third valve 283A.
  • Alpha eighth supply chamber 258A, beta eighth supply chamber 10 258B, and so on, are respectively connected to alpha detection channel 295A, beta detection channel 295B, and so on.
  • Alpha eighth supply chamber 258A, beta eighth supply chamber 258B, and so on are filled through ninth inlet 239.
  • the first supply chambers 251 can be used to store fluid having suspended paramagnetic beads used in preparing nucleic acid from biological samples, which paramagnetic beads are
  • a foot-pad pump operates propel in parallel the fluid and suspended beads from the first supply chambers 251 to the connected first reactions chambers 261 . To assure that the beads are suspended the foot-pad pump operating on the first supply chambers 251 and foot-pad pump operating on the first reaction chambers 261 can alternately be
  • the second supply chambers 252 can contain a buffer solution, such as a buffer solution used to wash the paramagnetic beads.
  • associated foot-pad pump has four foot-pads designed to interact with either ( 1 ) alpha second supply chamber 252A, gamma second supply chamber 252C, epsilon second supply chamber 252E and eta second supply chamber 252G or (2) beta second supply chamber 252B, delta second supply chamber 252D, zeta second supply chamber 252F and theta second supply chamber
  • the pump has two sets of four pads designed to interact with second supply chambers 252.
  • the third supply chambers 253 alternate in size between supply chambers 253 having volumes of 1 00 ⁇ l and supply chambers 253 having volumes of 30 ⁇ l.
  • the 1 00 ⁇ l supply chambers 253 can be used to store cell lysis solutions while the 30 ⁇ l supply chambers 253 can be used to 5 store solutions of primers.
  • Alpha, gamma, epsilon and eta fourth supply chambers 254A, 254C, 254E and 254G can be used to store a solution containing the appropriate nucleotide triphosphates for a nucleic acid amplification assay.
  • 10 254H can be used to store solutions containing the polymerase enzyme for the nucleic acid amplification assay.
  • a desirable feature for a cassette such as that illustrated in Figures 4A-4E is the ability to incorporate a positive control in one or more, but not all, of the reaction flow-ways 265 (not identified in Figures, first
  • reaction flow-way 265A includes alpha first and second reaction chambers 261 A and 262A
  • second reaction flow-way 265B includes beta first and second reaction chambers 261 B and 262B, and so on) .
  • a material that should generate a positive assay result can be inserted into sample that otherwise may or may not produce a positive signal (i.e., experimental 0 samples) or in samples that should not produce a positive signal (i.e. , negative controls) . In this way, the source of any substances that interfere with the assay can be determined.
  • Controls e.g., fluids that have a predetermined amount of a component to be tested for or that are known to lack the component, can be inserted into alpha and beta second reaction chambers 262A and 262B from
  • Bursapak supply chamber is avoided simply by not pumping its contents into the connected reaction chambers.
  • the illustrated cassette 200 has a first waste chamber 271 and a second waste chamber 272 (collectively waste chambers 270) of sufficient volume to accommodate all the fluids introduced into the cassette.
  • Waste chambers 270 are prepared in an evacuated state such that the films forming the outer wall of the waste chambers 270 (see film 1 10C of Figure 1 ) rest against the inner surfaces of the waste chambers 270. As fluid is pumped into the waste chambers 270, the film will flex outwardly to provide room for the inserted fluid.
  • Supply chambers 250 are also evacuated in like manner prior to filling. Most supply chambers 250 will, in a preferred embodiment, be pre- filled prior to shipment to the laboratory where the assay will be conducted. Of course, the test sample will be inserted at the lab site. Fluid insertion is best described with reference to Figure 1 B. A needle can be inserted into septum 1 31 and used to evacuate supply chamber 1 50, causing film 1 1 0A to collapse onto the floor of supply chamber 1 50. Then, fluid can be inserted through the septum into supply chamber 1 50. The first fluid exchange channel is then blocked, for instance by heat sealing or by crimping.
  • delta reaction flow-way 265D Focusing on delta reaction flow-way 265D, note that experimental sample from sixth supply chamber 256 is first relayed to delta second reaction chamber 262D while flow to delta first reaction chamber 261 D is blocked by operating a foot-pad pump minimize the volume of delta first reaction chamber 261 D. Typically, the first reaction conducted on the experimental sample will occur in delta first reaction chamber 261 D. To move the experimental sample from delta second reaction chamber 262D to delta first reaction chamber 261 D, delta second valve 282D is closed, the foot-pad pump acting on delta first reaction chamber 261 D is released, and the foot ⁇ pad pump acting on delta second reaction chamber 262D is operated to pump the experimental sample into delta first reaction chamber 261 D.
  • Foot-pad pumps that operate to drain a supply chamber 1 50 can remain engaged with the supply chamber 1 50 to prevent back-flow into the supply chamber 1 50.
  • FIGS 5A, 5B, 6 and 7 illustrate yet another embodiment of the invention that utilizes plunger-type valves to control the flow of fluids in the cassette 1 00 or cassette 200.
  • the operation of such a plunger-type valve in a cassette 1 00 or 200 is illustrated above with reference to Figures 5A and 5B.
  • Plunger 810 has a plunger rod 81 1 and a piston 81 2.
  • plunger rod 81 1 is withdrawn away from such that third film 1 1 0C, which is embossed to protrude away from the seat 1 81 of valve 1 80, does not interfere with fluid flow from alpha third fluid exchange channel 143A, into valve 1 80, and out through beta third fluid exchange channel 1 43B.
  • plunger rod 81 1 presses film 1 1 0C against valve seat 1 81 , blocking fluid flow.
  • Figure 6 shows a three-dimensional view of valve 1 80, including valve seat 1 81 and valve trough 1 82.
  • the plunger 81 0 can be constructed of numerous durable materials including without limitation a plastic such as polycarbonate or metal such as stainless steel or aluminum or the like.
  • the diameter of plunger rod 81 1 is typically from about 20 to about 1 00 ⁇ m, preferably about 60 ⁇ m, while piston 81 2 typically has a diameter from about 100 to about 300 ⁇ m, preferably about 200 ⁇ m.
  • the ratio of the cross-sectional area of the piston 81 2 to that of the plunger rod 81 1 is at least about 1 0-fold, thereby providing a corresponding mechanical advantage.
  • Instrument 900 (not shown) has a pneumatic device 800 formed of first portion 800A and second portion 800B which can be joined together, for instance, by bolts, rivets, adhesives or snap-fitting pieces.
  • flexible gasket 820 Interposed between the first and second portions 800A and 800B is flexible gasket 820, which can be formed of a suitable film such as poly (2-chloro-1 ,3- butadiene) (e.g., Neoprene, DuPont de Neumours, Wilmington, DE) or silicon rubber.
  • Flexible gasket 820 can be held in place by the clamping action of first and second portions 800A and 800B, which adherent force can be supplemented using heat sealing or adhesive.
  • Pneumatic cavity 830 is formed in both first and second portions 800A and 800B and has a cavity inlet 831 .
  • Fluid preferably a gas
  • pump induced pressure in third fluid exchange channel 1 43A is sufficient to displace (a) third upper cover into displacement cavity 840 and (b) plunger 81 0 from the valve seat 1 81 , thereby allowing flow.
  • Pneumatic device 800 can be formed of numerous durable materials including without limitation a plastic such as polycarbonate or metal such as brass or aluminum or the like.
  • Valve ball 620 is used to press lower film 1 20 flush against the lower surface of first body layer 601 so as to block fluid flow through hole 632.
  • Valve ball 620 can be fabricated of any suitably material such as nylon, high density polyethylene, polycarbonate and the like.
  • Lower film 1 20 is sealed to portions 601 A and 601 C of first body layer 601 , but typically is not sealed to portion 601 B.
  • the sealing between lower film 1 20 and portions 601 A and 601 C can be done using, for instance, adhesives or by clamping the membrane between body layer 601 and second body layer 602.
  • First body layer 601 , second body layer 602 and third body layer 603 can be joined together using, for instance, by bolts, rivets, adhesives or snap-fitting pieces. Pressure can be applied to valve ball 620 to press it against or release it from lower film 1 20 in a number of ways. Note that the valve is designed so that valve ball 620 will automatically center itself to properly seat itself against first layer 601 .
  • Figure 8A shows a spring loaded lever 640 that allows a push motion to open the valve, where force is applied as indicated by arrow "B" .
  • a push rod 643 (not illustrated) can be used to so engage spring loaded lever 640.
  • Figure 8B illustrates another embodiment that uses pull rod 641 to open the valve.
  • both spring loaded level 640 and pull rod 641 depend on the spring 642 formed from third body layer 603. Both types of rods can be activated by a cam 650 that is driven by a shaft 652 (not illustrated).
  • liquid flow is, for instance, in the direction indicated by arrow "A" and proceeds by first conduit 631 and second conduit 632.
  • valve ball 620 is seated against first body layer 601 , the valve is closed and flow is stopped.
  • lower film 1 20 deforms in response to fluid pressure, into cavity 633 to form third conduit 633A (not shown) linking second conduit 632 with fourth conduit 634.
  • Fourth conduit 634 connects with fifth conduit 635.
  • Figure 8C illustrates the use of a cam 650 to activate a pull rod 641 that is spring loaded with pull rod spring 651 .
  • All of the various pull rods 641 and pull rod springs 651 can be contained in a single base plate 604, such as that shown in Figure 8C, which can be attached to the instrument 900.
  • the valve of Figure 8C also differs in employing a pinch foot 621 instead of a valve ball 620 and in seating the pinch foot 621 against portion 601 B instead of against the opening of second conduit 632.
  • the valves are normally in the closed position. The positioning of the valves can be programmed and activated by controller 960 (not shown) .
  • membranes or seals can be employed to maintain the various fluids in their chambers. These membranes could be broken by applying a light pressure. Alternatively, the fluids could be frozen prior during storage to attaching the parallel reaction device to the base plate.
  • second and third body layer 602 and 603, respectively, can be designed to be separable from first body layer 601 , which contains fluid exchange channels and fluid chambers.
  • first body layer 601 which contains fluid exchange channels and fluid chambers.
  • the valve locations are not strongly closed to fluid flow, although the lower film 1 20 can rest securely enough against portion 601 B to prevent inadvertent fluid flow.
  • a ball retention film 61 5 is usefully sealed to the upper side of second body layer 602 to assure that the value ball 620 does not fall out of the device. The advantage of separating these pieces is that the portions of the parallel reaction device containing mechanical elements can be re-used while the fluid-handling portion can be disposed of.
  • Figure 9B shows a closed electromagnetic valve 380 for use in controlling the flow of fluids in a cassette 300.
  • the electromagnetic valve 380 Located in a portion 700 of instrument 900, the electromagnetic valve 380 has a spacer 730 that is pressed against a flexible upper film 1 1 0 by first spacer spring 731 and second spacer spring 732.
  • the first and second spacer springs 731 and 732 or the spacer 730 are sufficiently magnetic or magnetically permeable that they can be drawn away from upper film 1 1 0 by activating electromagnetic coils 740.
  • the electromagnetic valve 380 is shown in the open position with spacer 730 electromagnetically drawn away from valve seat 381 .
  • Figure 10 illustrates a part of instrument 900, reaction cell servicing device 400, having upper auxiliary block 400A for moving fluids into or out of a reaction chamber 1 60.
  • upper auxiliary block 400A is honeycombed with upper conduit 430A.
  • Upper conduit 430A has an upper inlet 431 A and an upper outlet 432A.
  • First upper portion 401 A of upper auxiliary block 400A is fabricated of any suitably sturdy material, but is preferably constructed of the same material as third upper portion 403A.
  • Second upper portion 402A is preferably fabricated of a heat-insulating material, such as, without limitation, nylon, polycarbonate and the like.
  • Third and fourth upper portions 403A and 404A are preferably fabricated of a heat-conductive material, such as, without limitation, aluminum, copper, sintered beryllia, and the like. Upper portions 401 A-404A can be joined using, for instance, bolts, rivets, adhesives or snap- fitting pieces. Upper electrical heaters 440A are positioned adjacent to the reaction chamber 1 60.
  • the upper and lower heaters 440A and 440B are generally thin layers of conductive material that is separated from the heat-conductive upper and lower sections 402A and 402B of upper and lower auxiliary blocks 400A and 400B by a thin electrical insulation layer.
  • Such an insulation layer is formed, for example, by direct deposition onto the substrate.
  • silicon nitride can be deposited from the gas phase or aluminum oxide can be deposited using a liquid carrier.
  • the conducting layer forming upper and lower heaters 440A and 440B are, for example, deposited by vacuum evaporation (e.g., for a nichrome conducting layer) or by deposition from the vapor (e.g., for an indium tin oxide conducting layer) .
  • pre-formed heater sheets are cemented to the substrate, for instance using an epoxy cement or the adhesive recommended by the vendors.
  • Appropriate heaters can be obtained from Elmwood Sensors Inc. (Pawtucket, Rl) or from Omega Engineering Inc. (Stamford, CT) .
  • individual heater elements have planar dimensions appropriate, alone or in combination with electrically coupled heater elements, to match the size of the reaction chamber to be heated.
  • Fourth upper portion 404A constitutes an upper foot-pad 404A' for a foot-pad pump that operates to pump fluid out of a reaction chamber 1 60.
  • a foot-pad is associated with a heating and cooling device, it is preferably fabricated of a material with high thermal conductivity such as aluminum, copper, sintered beryllia, and the like.
  • the operation of the foot-pad pump 460 which includes lower foot-pad 404B', is illustrated in Figures 1 1 A and 1 1 B. When the upper and lower foot-pads 404A' and 404B' are withdrawn away from reaction chamber 1 60, the reaction chamber 1 60 can be filled with fluid (see Figure 1 1 A) .
  • instrument 900 has a device for pumping and controlling reaction cell temperature, such as reaction cell servicing device 400, for each reaction chamber 1 60 in the cassette 1 00 or 200.
  • a device for pumping and controlling reaction cell temperature such as reaction cell servicing device 400, for each reaction chamber 1 60 in the cassette 1 00 or 200.
  • FIG. 1 2 shows a schematic of the accessory support devices for the upper auxiliary block 400A of Figure 1 1 .
  • Water is propelled through upper and lower conduits 430A and 430B, respectively, from pump and water cooler console 950.
  • Pump and water cooler console 950 further includes fluid valves operating under the control of controller 960. Electrical current is supplied to upper and lower heaters 440A and 440B, respectively, by power supply 970, which is controlled by controller 960.
  • Controller 960 receives input from upper and lower thermal sensors 450A and 450B, respectively.
  • upper auxiliary block 500A includes a set of paired first and second upper thermoelectric blocks 51 1 A and 51 2A, respectively, while lower auxiliary block 500B has a set of paired first and second lower thermoelectric blocks 51 1 B and 51 2B, respectively.
  • First upper and first lower thermoelectric blocks 51 1 A and 51 1 B, respectively, are made of p-type semiconductor material, while second upper and second lower thermoelectric blocks 51 2 A and 51 2B, respectively, are made of n-type semiconductor material.
  • the thermoelectric blocks 51 1 and 51 2 are electrically connected in series by upper and lower connectors 51 3A and 51 3B as illustrated to form thermoelectric heat pumps.
  • Such thermoelectric heat pumps are available for instance from Tellurex Corp. , Traverse City, Ml and Marlow Industries, Dallas, TX.
  • Upper and lower gas inlet/outlets 510A and 51 OB are connected to upper and lower manifolds 520A and 520B, respectively, formed by the space between the upper and lower thermoelectric 5 blocks 501 A and 501 B.
  • Upper and lower manifolds 520A and 520B (which are made up of the space between thermoelectric blocks) are connected, respectively, to an upper plurality of passageways 521 A or a lower plurality of passageways 521 B.
  • the outer portions of upper and lower auxiliary blocks 500A and 500B are upper and lower heat sinks 504A and 504B, respectively,
  • First upper air-tight collar 506A, second upper air-tight collar 507A, first lower air-tight collar 506B and second lower air-tight collar 507B help form upper and lower manifolds 520A and 520B.
  • Upper and lower thermal sensors 570A are preferably constructed of a heat-conductive material such as, without limitation, aluminum, copper, sintered beryllia, and the like.
  • 15 and 570B are connectable to a controller or a monitoring device by upper and lower leads 571 A and 571 B, respectively.
  • upper end-plate 502A viewed from underneath or lower end-plate 502B viewed from above would have a series of holes which are the outlets of upper and lower passageways 521 A and
  • thermoelectric blocks 500A and 500B Another attribute of the auxiliary blocks 500A and 500B is that the thermoelectric blocks typically are arrayed in three dimensions rather than two.
  • Heating is achieved by applying voltage of the proper polarity to upper first and second leads 508A and 509A and to lower first and second
  • upper and lower first end-plates 502A and 502B are preferably constructed of a material of high thermal conductivity, such as sintered beryllia. Other suitable materials
  • the thermoconductivity of end-plates 502A and 502B is at least about 0.2 watt cm " 1 K ⁇ 1 , more preferably at least about 2 watt-cm " 1 -K "1 .
  • the upper and lower temperature sensors 570A and 570B can be, without limitation, thermocouples or resistive sensors.
  • the upper and lower sensors 35 570A and 570B can, for example, be deposited on upper and lower first end-plates 502A and 502B as thin films or they can be in the form of thin wires embedded into holes in upper and lower first end-plates 502A and 502B.
  • Upper and lower auxiliary blocks 500A and 500B provide an alternate method of applying pressure to second upper film 1 1 0B and lower film 1 20 to push fluid out of reaction chamber 1 60.
  • gas pressure is applied through upper gas inlet/outlet 51 OA and corresponding lower gas inlet/outlet 51 OB (not shown) of lower auxiliary block 500B, the gas exiting upper and lower pressurized fluid channels 521 A and 521 B (not shown) forces upper and lower films 1 1 0 and 1 20 together, thereby forcing fluid from reaction chamber 1 60.
  • Upper or lower auxiliary block 500A or 500B can contain a plurality of upper or lower pressurized fluid channels 421 A or 421 B, respectively, which are used to operate a gas pressure flow control means.
  • the fluid within these channels typically is a gas such as oxygen or nitrogen.
  • Gas of higher than atmospheric pressure can be applied to the upper or lower pressurized fluid channels 421 A or 421 B from, for instance, a pressurized gas canister or a pump applied to upper or lower gas inlet/outlet 41 OA or 41 OB.
  • a vacuum usually a partial vacuum, can be applied to the upper or lower pressurized fluid channels 421 A or 421 B using, for instance, a vacuum pump.
  • Figure 14 illustrate another upper auxiliary block 1 500A and lower auxiliary block 1 500B that use thermoelectric heat pumps but use a foot-pad pump instead of a gas-pressure mediated pumping device.
  • Upper and lower foot-pads 1 505A and 1 505B are used to pump fluid out of reaction chamber 1 60.
  • Thermoelectric blocks 1 51 3 are used to heat or cool as described above.
  • Upper and lower heat sink thermal sensors 1 592A and 1 592B are located in upper heat sink 1 504A and lower heat sink 1 504B, respectively.
  • Upper heat sink heater 1 590A and lower heat sink heater 1 590B (connected to electrical power via upper leads 1 591 A and lower leads 1 591 B, respectively) are used to transfer heat to the thermoelectric blocks 1 51 3, thereby allowing thermoelectric blocks 1 51 3 to operate at a higher temperature range.
  • Upper and lower sensors 1 570A and 1 570B are used to monitor the temperature of the adjacent reaction chamber 1 60. The speed with which the temperature of the reaction chamber 1 60 is increased or decreased is important for optimizing some nucleic acid amplification assays.
  • the temperature is raised to higher plateau temperature "H" by activating upper and lower heaters 440A and 440B until a temperature is reached that will lead to a temperature stabilization at temperature H.
  • Water flow through upper and lower conduits 430A and 430B can be activated to minimize temperature overshoots if needed.
  • Temperature H is maintained by intermittently operating upper and lower heaters 440A and 440B when the temperature of the reaction chamber 1 60 lowers beneath a temperature of H minus Y (where Y is a temperature differential) .
  • the controller activates the pump 451 (not illustrated) of console 450 to cause cooling water to flow through upper and lower conduits 430A and 430B.
  • the performance of such a heater device and cooling device can be simulated using a heat transfer simulation computer program using a finite element approximation of the heat flow equation.
  • the simulation is conducted with the following assumptions: the thickness of the reaction chamber 1 60 is 0.5 mm, the upper and lower films were 0.1 mm thick and the insulation between the heater and the auxiliary block was 0.025 mm thick.
  • Such a simulation has determined that a jump from 25°C to 75°C can be achieved within 3.2 seconds, where, after 3.2 seconds, the temperature in the 5 reaction chamber is substantially uniform.
  • the reciprocal cooling step can be achieved within about 3 seconds, resulting in a substantially uniform temperature in the reaction chamber.
  • the variation in temperature in the reaction chamber is no more than about 0.1 °C.
  • reaction chamber 1 60 temperatures between about -20°C and about 1 00°C can be maintained.
  • each such reaction flow-way when the parallel reaction device includes more than one reaction flow-way, each such reaction flow-way
  • reaction chamber 1 60 which will have at least one heating and cooling device made up of thermoelectric blocks 501 (such as the heating and cooling device described in the paragraph immediately above) capable of being aligned with a side of the reaction chamber. More preferably, each such reaction chamber 1 60 will have a heating and cooling device on
  • the cross-sectional area of upper or lower first end-plate 502A or 502B substantially matches the largest cross-sectional area of the reaction chamber 1 60 to which it is intended to transfer heat.
  • reaction chamber 1 60 heated and cooled with upper and lower auxiliary blocks 500A and 500B or upper and lower blocks 1 500A and 1 500B are the same as those outlined above for the upper and lower auxiliary blocks 400A and 400B of Figure 1 0.
  • the reaction chamber 1 60 is heated and cooled by passing a heated or cooled fluid, preferably a gas, either directly
  • the apparatus illustrated in Figure 1 0 can be modified to operate pursuant to this embodiment by (a) removing (or not using) the upper and lower heaters 440A and 440B and (b) adding a heater for
  • the parallel reaction device preferably has two fluid management systems, one for a hotter fluid and another for a cooler fluid, together with the valving required to inject the hotter or cooler fluid into the tubing leading to the reaction chamber 1 60 as appropriate for maintaining a given temperature in the reaction chamber.
  • the heating and cooling fluid is a gas
  • the temperature of the gas soon after it has passed by 5 the reaction chamber 1 60 will provide a useful indication of the temperature of the reaction chamber 1 60.
  • auxiliary blocks act as foot-pads or for other foot ⁇ pads
  • mechanical or electromechanical methods of drawing the foot-pads towards or away from the fluid chamber on which it acts are well known and 10 include solenoids, pneumatically activated plungers, screw mechanisms and the like.
  • Pumping action can also be achieved using, for instance, peristaltic pumps, mechanisms whereby a roller pushes down on the flexible
  • Such mechanisms include micro- electromechanical devices such as reported by Shoji et al., "Fabrication of a Pump for Integrated Chemical Analyzing Systems, " Electronics and
  • At least one reaction chamber 1 60 has a transparent retaining wall that is generally formed of upper film 1 1 0 or lower film 1 20 (or two retaining walls are transparent). Reaction chamber
  • 35 1 60 can be a chamber where a reaction occurs, such as one of lysing reaction chambers 340 or reaction chambers 380 (see Figure 3), it can be a supply chamber containing samples, controls or reagents, such as supply chambers 350, 360 and 390, or it can be a storage chamber, such as one of storage chambers 399A-E.
  • the parallel reaction device in this embodiment preferably includes a light source capable of directing light to the transparent upper or lower film 1 10 or 1 20 and a detection device for detecting (a) the light reflected from an illuminated reaction chamber 1 60, (b) the light transmitted through an illuminated chamber 1 60, or (c) the light emissions emanating from an excited molecule in a chamber 1 60.
  • a membrane is "transparent" if it is 80% transparent at a wavelength useful for detecting biological molecules.
  • the detection device can incorporate optical fibers, optical lenses, optical filters or other optical elements. Alternatively, where detection uses fluorescence, detection and quantitation can be done by photographing the detection channel 295 under appropriate excitation light. With fiber optics or other suitable optical devices, the size of the detection system that is adjacent to the parallel reaction device is minimized. This size minimization facilitates incorporating the detection system together with a temperature control device (described more fully below) into the parallel reaction device.
  • a particularly preferred light source is a solid state laser. The size of these light sources also facilitates incorporating a number of auxiliary components about the parallel reaction device.
  • the method used to detect amplified nucleic acid uses a dye that absorbs light at a wavelength higher than about 600 nm to indicate the presence of amplified nucleic acid, as described below.
  • dyes include Cy5TM, one of a series of proprietary cyanine class dyes. Cy5TM, and the related dyes, are products of Biological Systems, Inc. (Pittsburgh, PA) . This particular dye is relatively small, absorbs at about 650 nm and emits a fluorescent signal at about 667 nm.
  • a preferred solid state laser source is a Laser Max, Inc. (Rochester, NY) Model LAS 200-635.5, which emits a light with a wavelength at a maximum of 4 .
  • Other colorimetric detection methods for instance those utilizing biotin-avidin binding to associate horse radish peroxidase with a hybridized pair of polynucleotide sequences, can be used.
  • Signals from the detection device typically will be input into a controller 960, where they can be used to determine the presence, or absence, of material assayed for and the magnitude of the signal indicating the presence of the material. From these data, the amount of assay material can be calculated and the quality of the assay as indicated by the controls can be quantitated. This information is then stored for the assay report listing.
  • the cassette has one or more detection channels 295.
  • One such detection channel 295 is illustrated in Figures 1 5A and 1 5B. It is made up of a number of fibers 297, which together preferably transmit at least about 50% of light of a wavelength useful in the detection procedure, confined to the detection channel 295.
  • the fibers 297 can be bound in place for instance by cementing or crimping.
  • the fibers 297 can be fabricated of glass or suitably transparent plastics.
  • the fibers 297 are preferably between about 5 ⁇ m and about 50 ⁇ m in diameter, more preferably about 20 ⁇ m.
  • the detection channel typically has a width and depth of no more than about 3,000 ⁇ m, preferably between about 200 ⁇ m and about 1 ,000 ⁇ m.
  • Microchannels between the fibers 297 allow liquid to flow through the detection channel 295.
  • a detection-mediating molecule is bound to the fibers 297.
  • the detection-mediating molecule is an oligonucleotide that hybridizes with the nucleic acid to be amplified in a nucleic acid amplification reaction and the nucleic acid amplification reaction utilizes primers having a detectable moiety.
  • the detection-mediating molecules are bound to the fibers 297 by known methods.
  • first band 296A, second band 296B and third band 296C have separate detection-mediating molecules, which could be, for instance, designed to detect two separate species to be amplified in a nucleic acid amplification reaction and to provide a control for non-specific hybridizations.
  • detection-mediating molecules could be, for instance, designed to detect two separate species to be amplified in a nucleic acid amplification reaction and to provide a control for non-specific hybridizations.
  • oligonucleotide synthesis procedures that utilize photo-cleavable protecting groups and masks to protect certain bands 296 from photocleavage can be used. Such synthesis procedures are described in U.S. Patent No. 5,424, 1 86 (Fodor et al.).
  • the instrument 900 is preferably designed to provide heat control at the detection channels 295 for conducting hybridization reactions.
  • the sides 298 of the detection channel 295 are coated with a reflective coating so that light incident from above will reflect and twice pass through the detection channel 295.
  • a reflective coating is provided by metalizing, for instance using a sputtering or evaporation process.
  • the detection channels 295 contain membranes
  • hybridization probe 299 such as a nylon membrane, to which a hybridization probe has been bound. If two or more hybridization probes are used, they are each bound to a specific region of the membranes 299 using "dot blot" procedures such as are described in Bugawan et al., "A Method for Typing Polymorphism at the HLA-A Locus Using PCR Amplification and Immobilized Oligonucleotide Probes" Tissue Antigens 44: 1 37-147, 1 994 and Kawasaki et al., "Genetic Analysis Using Polymerase Chain Reaction-Amplified DNA and Immobilized Oligonucleotide Probes: Reverse Dot-Blot Typing" , Methods in Enzymology 21 8: 369-381 , 1 993. As described above, the amplification product hybridized with the bound probe or probes has attached -via the amplification primers - a detectable moiety.
  • the upper film 1 1 0 over the cavity is replaced with a cover 1 1 0' selected for its optical properties, such as, without limitation, a cover 1 1 0' made of optical quartz. Because pumping is effected elsewhere in the cassette, the cover 1 1 0' does not have to be flexible like an upper film 1 1 0. While in a preferred embodiment detection is done in situ in the cassette, in other embodiments the products of chemical reactions effected in the cassette are removed and detection methods or chemistries are done elsewhere, including in a different cassette.
  • Paramagnetic beads useful for facilitating chemical processes conducted in a cassette 1 00 are available from several sources including Bang Laboratories (Carmel, IN) for beads lacking conjugated biomolecules, Dynal (Lake Success, NY) for beads conjugated to various antibodies (for instance, antibodies that bind to the CD2 cell-surface receptor) and CPG (Lincoln Park, NJ) for beads with a glass matrix and a variety of surface bonded organics.
  • Bang Laboratories Carmel, IN
  • Dynal Lake Success, NY
  • CPG Longcoln Park, NJ
  • each bead will preferably have a diameter of less than about 1 mil, more preferably, less than about 0.5 mil, which diameter facilitates entry and exit through the channels by which material is inserted or evacuated from the reaction chamber 1 60.
  • the diameter is preferably sufficiently large to preclude their entry into these channels.
  • the entrances to such channels within a reaction chamber 1 60 are preferably positioned or designed so as to minimize the chance that a channel will be blocked by a bead that settles over the channel's entryway.
  • the beads are locked in place using magnetic fields.
  • the magnet used should preferably generate a sufficient magnetic field gradient within a reaction chamber 1 60.
  • Such magnets can be constructed by forming sharp edges on highly magnetic permanent magnets, such as those formed of rare earths, such as the neodymium-iron-boron class of permanent magnets.
  • Such a permanent magnet is available from, for example, Edmund Scientific (Barrington, NJ). Sharp edges of dimensions suitable for a particular reaction chamber 1 60 are, for example, formed by abrasive grinding of the magnetic material.
  • An example of such a shaped magnet 1 1 00 is shown in Figure 1 6, where the magnet has a roof-shape at one of the poles.
  • the illustration shows a preferred embodiment where there are two roof shapes and illustrates that the magnet can be brought adjacent to or can be removed from a cassette such as cassette 1 00 or cassette 200.
  • lower auxiliary block 1 600B has slots (not visible) that allow the magnet 1 1 00 to be placed adjacent to cassette 1 00 or 200.
  • This magnet suitably has dimensions such that the length of the peak of the roof-shape matches the cross-sectional size of a reaction chamber 1 60.
  • the peak 1 101 of the magnet 1 1 00 is placed adjacent to the reaction chamber or other structure in which the beads are located.
  • the paramagnetic beads are held in place by leaving the peak 1 1 01 adjacent to the beads.
  • the beads are impelled to move with the magnet.
  • Another way in which high magnetic field gradients can be achieved is to make uniform slices of a magnetic material and use an adhesive to join the slices in alternating N to S orientations. Such alternating slice magnets have high magnetic field gradients at the junctions of the slices.
  • the sharp-edged magnets described above are effective in adhering the paramagnetic beads in one place and in moving beads located, for instance, in a fluid exchange channel or in a reaction chamber, from one location to another. Such magnets thus can help retain the paramagnetic beads in one place, for instance when a fluid in a reaction chamber 1 60 is being removed from that chamber but it is desirable to leave the beads in the chamber. Magnets with locations having high magnetic field gradients that are particularly suitable for use in this context are described in U.S. Provisional Patent Application No. 60/006,202, filed November 3, 1 995, titled “Magnet, " Docket No. DSRC 1 1 904P, which is incorporated herein in its entirety by reference.
  • cell binding beads e.g., beads having bound antibodies specific for a certain subset of cells
  • the beads can be locked in place, for instance magnetically if the beads are paramagnetic, while non-adherent cells and fluids are washed away.
  • cell-binding beads can be used to concentrate small sub-populations of cells.
  • the beads suitably have attachment sites for coupling the building blocks of chemicals or polymers.
  • a septum 1 31 can be fixed in place in inlet 1 30 using heated die 1 200, as illustrated in Figures 1 7A and 1 7B.
  • the die 1 200 is heated sufficiently so that the angled, sharp edges 1 201 cut into body 1 05 and move melted material 1 32 such that it locks the septum 1 31 in place.
  • the controller 960 typically will be a microprocessor. However, it can also be a simpler device comprised of timers, switches, solenoids and the like.
  • the important feature of controller 460 is that it directs the activation of the means for impelling a fluid, the valves and the heating and cooling device, according to a pre-set or programmable schedule that results in the operation of an assay protocol, such as the protocol outlined below.
  • the controller 460 receives input indicating the temperature of the reaction chambers of the parallel reaction device and is capable of adjusting its control signals in response to this input.
  • nucleic acid purification step can increase the likelihood of getting a false positive result.
  • PCR reaction it is preferable to conduct parallel control PCR reactions when conducting PCR.
  • One control omits sample from the reaction or uses a sample previously characterized as negative.
  • Another control introduces a known amount of a purified nucleic acid that is known to contain the sequence or sequences that the PCR reaction is designed to amplify.
  • These types of controls can be accomplished on multiple parallel reaction devices or, more preferably, in separate reaction flow-ways on the same parallel reaction device whereby each reagent is distributed from a single source to all of the reaction flow-ways.
  • Another control technique used in PCR is to design the PCR reaction so that it will amplify multiple nucleic acid segments, each of which can be indicative of a disease or a genetic circumstance or marker. The different segments can be amplified in multiple reactions or in the same reaction chamber. If amplified in the same chamber, that binding competition between the various primers can necessitate extending the time, in each amplification cycle, spent at the replication temperature.
  • One method for removing cellular debris from a sample involves binding the cells in the sample to a bead that has attached thereto an antibody specific for a cell surface molecule found on the cells.
  • Beads that bind to the CD2 white blood cells or to E. coli bacteria (such as the 01 57E strain) are available from Dynal (Lake Success, NY).
  • Dynal Lake Success, NY
  • An ever-growing family of cell-surface molecules found on mammalian cells, bacterial cells, viruses and parasites has been characterized and antibodies against the majority of these molecules have been developed. See, e.g. , Adhesion Molecules, CD. Wegner, ed., Academic Press, New York, 1 994.
  • the cells can be adhered to the antibodies on the beads and lysed to release their nucleic acid content.
  • the lysis fluid together with the released nucleic acid can be moved to a separate compartment for further processing, leaving behind the beads and their adherent cellular debris.
  • the lysis fluid used to release nucleic acid from the sample cells can also interfere with the PCR reaction. Thus, in some protocols it is important to bind the nucleic acid to a substrate so that the lysis fluid can be washed away.
  • One such support is provided by beads that bind to DNA, such as glass beads that bind to DNA by ionic or other interactions such as Van der Waals interactions and hydrophobic interactions. Suitable beads, with surfaces chemically treated to maximize the number of interaction sites, are available from, for example, BioRad (Hercules, CA) . Paramagnetic beads with a number of DNA binding surfaces, such as nitrocellulose or nylon-coated surfaces, can be useful in operating the invention.
  • the beads it is desirable for the beads to be paramagnetic so that they can be manipulated using magnetic forces.
  • Paramagnetic glass beads are manufactured by CPG (Lincoln Park, NJ) . Once the nucleic acid is bound to the beads, the lysis fluid can be washed from the beads. The nucleic acid can be amplified with the beads present.
  • the lysis fluid used to release nucleic acid from the cells in a sample typically includes a detergent, preferably nonionic, and a buffer, usually the buffer used in the PCR amplification reaction.
  • the pH of the lysis fluid is preferably from about pH 7.8 (for protease K-containing lysis fluids, for example) to about pH 8.0 (for phenol-mediated lysis, for example), typically about pH 8.0.
  • Suitable detergents include, without limitation, Sarkosyl and
  • Nonidet P-40 Other components can includes salts, including MgCI 2 , chelators and proteases such as proteinase K. Proteinase K can be inactivated by heating, for instance, to about 1 00°C for about 1 0 minutes. Depending on the composition of the lysis buffer, it can be more or less important to wash the lysis buffer away from the nucleic acid prior to conducting the amplification assay.
  • the amplification buffer used to support the amplification reaction will typically include the four deoxynucleotide triphosphates (NTPs) (e.g., at a concentration of from about 0.2 mM each), a buffer (e.g., Tris-HCl, about 10 mM), potassium chloride (e.g., about 50 mM) and magnesium chloride (e.g., about 1 to 1 0 mM, usually optimized for a given PCR assay scheme).
  • the pH is preferably from about pH 8.0 to about pH 9.0, typically about pH 8.3. Other components such as gelatin (e.g., about 0.01 % w/v) can be added.
  • the individual primers are typically present in the reaction at a concentration of about 0.5 ⁇ M.
  • sample nucleic acid needed varies with the type of nucleic acid and the number of target nucleic acid segments in the nucleic acid sample.
  • a concentration of about 1 0 ⁇ g/ml is desirable.
  • the polymerase used in the procedure is a heat-resistant DNA polymerase such as Taq polymerase, recombinate Taq polymerase, Tfl DNA polymerase (Promega Corp., Madison, Wl), or 77/ DNA polymerase (Promega Corp., Madison, Wl) .
  • Heat stability allows the PCR reaction to proceed from cycle to cycle without the need for adding additional polymerase during the course of the reaction process to replace polymerase that is irreversibly denatured when the reaction vessel is brought to a DNA strand separation temperature.
  • the DNA polymerase used has the increased accuracy associated with the presence of a proofreading, 3' to 5' exonuclease activity, such as the proofreading activity of the TH DNA polymerase.
  • Blood provides one of the more convenient samples for diagnostic or genetic PCR testing. For most genetic testing, from about 1 0 to about 50 ⁇ l of blood is sufficient to provide enough sample DNA to allow for PCR amplification of specific target segments. For fetal cell analysis, however, as much as about 20 mis, which may contain as few as about 400 fetal cells, can be required. Such large sample volumes require concentration, for instance, using the methods described above. For testing for microbial diseases, the concentration of target nucleic acid in the sample can be quite low (e.g., no more than about 2-5 fg per bacterial genome). Thus, when using the parallel reaction device to test for such microbes, concentration methods may again be required.
  • RNA sample To specifically amplify RNA, it is necessary to first synthesize cDNA strands from the RNA in the sample using a reverse transcriptase (such as AMV reverse transcriptase available from Promega Corp., Madison, Wl).
  • a reverse transcriptase such as AMV reverse transcriptase available from Promega Corp., Madison, Wl.
  • Methods for conducting a PCR reaction from an RNA sample are described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York and PCR: A Practical Approach, IRL Press, 1 991 .
  • RNA for this purpose, a facile procedure uses a lysis buffer containing detergent (such as 0.5% Nonidet P-40), buffer (e.g., pH 8.3) and suitable salts that has been, immediately prior to use, mixed 1 : 1 000 with a 1 : 1 0 diethylpyrocarbonate solution in ethanol. After sample cells have been lysed with this solution, a supernate containing RNA is separated away from a pellet of nuclei by centrifugation. Primer, which is generally the same as one of the primers used in the subsequent PCR cycling reaction, is annealed to the RNA by heating (e.g., to about 65°C) and subsequently reducing the temperature to, generally, about 37°C.
  • detergent such as 0.5% Nonidet P-40
  • buffer e.g., pH 8.3
  • suitable salts that has been, immediately prior to use, mixed 1 : 1 000 with a 1 : 1 0 diethylpyrocarbonate solution in ethanol.
  • the reverse transcriptase, nucleotide triphosphates and suitable buffer are then added to initiate cDNA synthesis.
  • a small volume e.g., about 1 .0 to about 2.0 ⁇ l
  • a solution e.g. , about 50 to about 100 ⁇ l
  • the temperature cycling program is then initiated.
  • the advantages of the parallel reaction device as it relates to conducting PCR reactions also substantially apply to conducting hybridization procedures.
  • the ability of the valves of the parallel reaction device to accommodate elevated temperatures allows the system to be used in hybridization protocols. While hybridization reactions are not as sensitive to contamination as PCR reactions, these reactions are nonetheless very sensitive to contamination, the risk of which is substantially reduced with the disposable system of the invention.
  • Procedures for conducting hybridizations are well known in the art. See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Press, 1 989.
  • a nucleic acid such as (a) a sample source of nucleic acid containing a target sequence, or (b) a probe nucleic acid is bound to a solid support and, after this binding, the remaining binding sites on the support are inactivated. Then, the other species of nucleic acid, which has bound to it a detectable reporter molecule, is added under appropriate hybridization conditions. After washing, the amount of reporter molecule bound to (i.e. hybridized with) the nucleic acid on the solid support is measured.
  • a hybridization can be conducted in a reaction chamber in the parallel reaction device, where the reaction chamber contains a nitrocellulose membrane (or another membrane that binds nucleic acid) to which RNA has been bound (for instance, by electrophoretic or capillary blotting from a separation gel, followed by baking).
  • a Northern prehybridization solution can then be introduced into the reaction chamber from one of the fluid chambers.
  • the recipes for Northern prehybridization solution p. A1 -40
  • Northern hybridization solution p. A1 -39
  • SSC p. A1 -53, 20X recipe
  • Denhart's solution p.
  • incubation temperatures are in the range that is generally appropriate given the presence of 50% formamide in the prehybridization and hybridization solutions; for hybridizations conducted without formamide, incubation temperatures are typically higher, such as about 55°C to about 70°C.
  • the membrane is then exposed to Northern hybridization solution containing melted probe and incubated overnight at the same temperature used in the prehybridization.
  • the hybridization solution is pushed out of the reaction chamber, the reaction chamber is brought to about 25°C and a first wash solution ( 1 X SSC, 0.1 % w/v sodium dodecyl sulfate) is introduced. After 1 5 minutes, the wash is repeated. After an additional 1 5 minute wash, a third and final wash is conducted using 0.25X SSC, 0.1 % w/v sodium dodecyl sulfate.
  • 1 X SSC 0.1 % w/v sodium dodecyl sulfate
  • the antibody-antigen binding reactions are generally conducted at room temperature or at a reduced temperature, such as about 4°C.
  • positive results are generally indicated by an enzymic reaction, typically mediated by the enzyme alkaline phosphatase, which enzyme reaction is generally conducted at a temperature between about 20°C and about 40°C.
  • the parallel reaction device of the invention allows these assays to be automated in a system that allows fast and reliable temperature regulation in the temperature range between about 0°C and about 40°C.
  • an "antigen” which is a substance that when injected into an animal, often in the presence of "adjuvants” known to enhance antibody production, can cause the animal to manufacture antibodies specific for the antigen
  • an antibody is found on the surface of a cell, such as a bacteria or eukaryotic cell, and the cell can function as a solid support.
  • the antigen is bound to the support and a sample which may contain a first antibody specific for the antigen and produced by a first animal species is incubated with the bound antigen. After appropriate washing steps, a second antibody from a second animal species, which antibody is specific for antibodies of the first species and is attached to a detectable moiety (such as alkaline phosphatase), is incubated with the support. If the sample contained the first antibody, the second antibody will bind and be detectable using the detectable moiety.
  • a detectable moiety such as alkaline phosphatase
  • detectable moiety is alkaline phosphatase
  • detection can be conducted by adding a chemical, such as p-nitrophenyl phosphate, that develops a detectable characteristic (such as color or light emission) in the presence of a developing reagent such as a phosphatase enzyme.
  • This assay can, for instance, be used to test blood for the presence of antibodies to the AIDS virus.
  • a sample which may contain an antigen is incubated with the support together with a limiting amount of an antibody specific for the antigen, which antibody has an attached detectable moiety. Due to competition between the solution phase antigen and the support-bound antigen, the amount of antigen in the sample correlates with reduced amounts of antibody that bind to the support-bound antigen and a weaker signal produced by the detectable moiety.
  • antibody-sandwich ELISA uses a first antibody specific for an antigen, which antibody is bound to the support. A sample which may contain the antigen is then incubated with the support. Following this, a second antibody that binds to a second part of the antigen. and which has an attached detectable moiety, is incubated with the support. If the sample contained the antigen, the antigen will bind the support and then bind to the detectable second antibody. This is the basis for the home pregnancy test, where the antigen is the pregnancy-associated hormone chorionic gonadotropin.
  • a sample which may contain a first antibody from a first species is incubated with a support that has bound to it a second antibody from a second species that is specific for antibodies of the first species.
  • the antigen for the first antibody is then incubated with the support.
  • a third antibody specific for a portion of the antigen not bound by the first antibody is incubated with the support.
  • the third antibody has an attached detectable moiety. If the sample contained the first antibody, the detectable third antibody will bind to the support.
  • the following example illustrates fabrication methods used in constructing cassettes for a microfluidics device of the present invention.
  • Various cassettes have been fabricated containing components that are shown in Figure 1 .
  • Cassette bodies have been made from high-density polyethylene, both by machining and by molding.
  • the methods of fabrication and demonstrated performance include the following: Membrane embossing: The membrane covering the cassette body and forming the reaction chambers was embossed prior to sealing to the body. The membrane was stretched on a frame and embossed between positive and negative hot dies. For membranes of polyester/ polyethylene laminate, the dies were heated to a temperature of above 1 40°C.
  • a preferable material for embossing is a fluoropolymer/ polyethylene laminate which can be given a more permanent deformation at a lower temperature and which has a lower water permeability.
  • Heat sealing The membrane was sealed to the cassette body using a hot aluminum die with raised lands corresponding to the heat seal areas. A pressure, corresponding to approximately 1 50 to 300 psi over the actual seal area, was applied for 1 to 2 seconds. Following application of the pressure, the die was either rapidly quenched by water channels running through the die block, or the die was lifted. Superior results were obtained by quenching the die. With a 2 mil thick membrane of a polyester/low-density polyethylene laminate, sealed to a body of high-density polyethylene, a die temperature of 1 56°C was used. A blister 0.5 " in diameter sealed in this manner withstood internal pressures in excess of 50 psi.
  • the cassette regions at the seal were formed into a raised ridge, about 0.01 " high. Variation in the amount the die deforms the base material, originating from small variations in cassette thickness, can then occur with a minimum variation in the volume of base material displaced. This ridge structure was found to reduce the extruded material in regions such as the well surrounding a valve. BursapakTM structure: The outer seal of the BursapakTM was made as described above. The center seal was made using a die heated to temperature of about 1 56°C. This die contained small independently sprung steel pins which contacted the center seal. The lower conductivity of the steel and the air gap between the pins and the die were designed to restrict the amount of heat available for sealing.
  • Liquid fill Liquid fill of both Bursapaks and storage vessels similar to the "waste vessel" of Figure 1 was achieved.
  • the input needle was connected to a 2-way valve which could be switched between a vacuum pump and a syringe supplying the fill liquid. Following exhaustion of the vessel by the pump, for a few seconds, the valve was switched and the vessel filled by the syringe. The filled vessel then contained no air bubbles.
  • Both a septum, as shown in Figure 1 and a simple entry port were used for filling. Sealing of the entry channel was achieved by a hot rod, as indicated in Figure 2, which melted the channel closed but kept the polyester component of the membrane sufficiently intact.
  • Valve operation Valves, constructed as in Figures 1 , 5 and
  • the following example illustrates one embodiment of the present invention whereby a PCR amplification reaction is conducted in the context of a cassette in a microfluidics device.
  • a PCR assay is conducted using the cassette 200 illustrated in Figures 4A-4E, the device having alpha through delta first reaction chambers 262A-D, which are used for lysing the cells in the samples, and alpha through delta second reaction chambers 262A-D, with each first reaction chamber 261 - second reaction chamber 262 pair forming a separate reaction flow-way 265.
  • the parallel reaction device has a set of one upper auxiliary block, e.g. 1 500A and one lower auxiliary block, e.g. 1 500B (not shown), for each of first reaction chamber 261 and each second reaction chamber 262.
  • the cassette 200 has pumps for moving fluid from one chamber to another chamber.
  • the reaction protocol is as follows: 1 .
  • Each of the four first reaction chambers 261 receives from a connected first supply chamber 251 a suspension in 1 60 ⁇ l of paramagnetic DNA-binding beads having a diameter of 2-4 mils, that can be used in the cell lysis stage to bind the DNA released from the lysed cells (these beads are, e.g., Dynabeads ® DNA DirectTM, available from Dynal, Lake Success, NY).
  • Alpha first reaction chamber 261 A receives a fluid (40 ⁇ l) from alpha fifth supply chamber 255A containing purified DNA that includes the amplification sequence being tested for in an amount sufficient to generate a positive result, thereby creating a positive control.
  • Beta first reaction chamber 5 261 B receives from beta fifth supply chamber 255B buffer solution or a biological sample known to not contain the target sequence (40 ⁇ l) in place of the sample or positive control, and therefore serves as a negative control.
  • Blood sample (40 ⁇ l), stored in sixth supply chamber 256 is drawn into each of gamma and delta first supply chambers 261 C and 261 D.
  • a lysis solution ( 1 00 ⁇ l) that is drawn from alpha, gamma, epsilon and eta third supply chambers 253A, C, E and F, respectively.
  • the lysis solution is a solution of amplification buffer supplemented with 1 .0% v/w Tween 20 (Sigma Chemical Co., St. Louis, MO). (The lysis solution can be substituted with the solution provided by Dynal.)
  • the lysis solution is emptied into first waste chamber 271 .
  • the lysis solution which exits from alpha and beta first reaction chambers 261 A and 261 B, respectively, contains the cellular and serum residue of the blood sample.
  • wash solution 100 ⁇ l
  • amplification buffer 40 mM NaCl, 20 mM Tris-HCl, pH 8.3, 5 mM MgSO 4 , 0.01 % w/v gelatin, 0.1 % v/v Triton X-1 00, Sigma Chemical Co., St. Louis, MO
  • amplification buffer 40 mM NaCl, 20 mM Tris-HCl, pH 8.3, 5 mM MgSO 4 , 0.01 % w/v gelatin, 0.1 % v/v Triton X-1 00, Sigma Chemical Co., St. Louis, MO
  • Solutions (volume 30 ⁇ l) containing appropriate primers 30 for amplifying the target sequence are then drawn into first reaction chambers 261 from the connected beta, delta, zeta and theta third supply chambers 253B, 253D, 253F and 253H.
  • Solutions (volume 30 ⁇ l) containing the needed nucleotide triphosphates are introduced from the connected alpha, gamma, epsilon and eta fourth supply chambers 254A, 35 254C, 254E and 254F.
  • the controller then initiates a temperature program modelled on the protocol described by Wu et al., Proc. Natl. Acad. Sci. USA 86: 2752-2760, 1 989
  • the program first heats second reaction chambers 262 to a temperature of 55°C and maintains that temperature for 2 minutes
  • the controller cycles the temperature between a replication temperature of 72°C (maintained for 3 minutes) and a DNA strand separation temperature of 94°C (maintained for 1 minute)
  • the material in reaction chambers 262 is analyzed for the presence of the proper amplified sequence

Abstract

The invention provides a cassette (100) for conducting reactions therein comprising one or more fluid exchange channels (141), a supply chamber (150), a reaction chamber (160) and a waste chamber (170).

Description

PARALLEL REACTION CASSETTE AND ASSOCIATED DEVICES
This invention was made with U.S. Government support under Contract No. 70NANB5H 1037. The U.S. Government has certain rights in this invention.
The present invention relates to a disposable parallel reaction device for conducting reactions, which device can include a component containing all necessary supply and reaction chambers and connecting fluid exchange channels. The parallel reaction device is particularly adapted for conducting polymerase chain reaction ("PCR") assays, and other scientific, forensic and diagnostic assays. Synthetic reactions, including combinatorial chemistry, can also be conducted in the device.
The PCR assay has provided a powerful method of assaying for the presence of either defined segments of nucleic acids or nucleic acid segments that are highly homologous to such defined segments. The method can be used to assay body fluids for the presence of nucleic acid specific for particular pathogens, such as the mycobacterium causing Lyme disease, the HIV virus or other pathogenic microbes. The microbe diagnostic assay functions by adding, to a sample that may contain a target segment of nucleic acid from the microbe's genome, at least one pair of "primers" (i.e., relatively short nucleic acid segments or nucleic acid analogs) that specifically bind to (i.e., "hybridize" with) the target segment of nucleic acid. The first primer of a pair binds to a first strand of the two-stranded target nucleic acid segment and, when hybridized, can prime the enzymatic reproduction of a copy of the second strand of the target nucleic acid segment in a direction arbitrarily designated as the downstream direction. The second primer of a pair binds to the second strand of the target nucleic acid segment at a position downstream from the first primer hybridization site and can prime the enzymatic reproduction of a copy of the first strand of the target nucleic acid segment in the upstream direction. (In the case where the sample is made up of single-stranded target nucleic acids, the second primer will hybridize with the theoretical second strand determined with the Watson-Crick base-pairing rules.) To the sample are added the monomer building blocks of nucleic acid and an enzyme that specifically catalyzes nucleic acid reproduction from a single strand of nucleic acid to which the short primer is bound. The enzyme is preferably highly resistant to destruction by elevated temperatures. The sample is heated to a DNA melting temperature to separate the two strands of the sample nucleic acid and then cooled to a replication temperature. The replication temperature allows the primers to specifically bind to the separated strands and allows the reproductive enzyme to operate. After this cycle, the reaction mix contains two sets of the two stranded nucleic acid segment for each target nucleic acid segment that was originally present. Heating and replication temperature cycles are repeated until sufficient amounts of the nucleic acid segment are created through this exponential reproduction method. For instance, after 20 cycles the segment has been amplified as much as 220-fold, or roughly 1 ,000,000-fold. There are at least four critical problems associated with automating the PCR reaction. First, the degree of amplification achieved by the assay creates a large risk of contamination from foreign DNA from handling. Thus far, this risk has been dealt with in commercial, manual procedures by conducting the reactions in "clean" facilities that are extremely expensive to construct and maintain. For automation, this risk implies that all the reagents needed and the reaction chamber for the amplification should be contained in a disposable platform in which the sample can be inserted in a controlled, one-time operation. This risk also implies that sample preparation steps should be minimized and, to the extent possible, conducted within a disposable platform.
Second, the high temperatures needed to "melt" the nucleic acid so that the two strands separate imply that the reaction chamber must be well-sealed against vapor loss, even while allowing the insertion and removal of various reagent fluids. This goal is particularly hard to achieve on a suitable, disposable platform.
Third, the reactions should be conducted in relatively small volumes, generally volumes of no more than about 100 μl, to conserve expensive reagents and minimize the amount of sample, which could be a precious sample fluid or tissue that must be conserved to allow for other types of testing or is available only in a small amount.
Fourth, to provide assurance that a positive or negative result is meaningful, it is preferable to perform multiple, parallel reactions (for example, on positive and negative controls, in addition to the sample) using the same reagents for each reaction. Recently, there have been a number of publications on the mechanics of operating micro-scale reactors. These reactors are often described as constructed on silicon-based materials using the etching techniques developed by the semiconductor industry. This literature, however, does not present an effective solution to the problem of how to operate a disposable, high temperature microreactor. The present invention provides an economical, high temperature microreactor with effective valves suitable for use in conducting multiple, parallel PCR assays, each using the same reagents to assure meaningful results. The microreactor is also suitably adapted for conducting automated assays even when high temperature and considerably high vapor pressure are not a particular concern.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a device for conducting parallel reactions, comprising: (a) a cassette formed of a body having an upper surface, a lower surface, and an edge, and including an upper film or a lower film attached to the upper or lower surface, respectively, wherein the upper or lower film is formed of a flexible material; (b) two or more reaction flow-ways in the cassette, wherein each reaction flow-way comprises two or more fluid chambers which comprise a first supply chamber and a first reaction chamber having an upper wall and a lower wall, and wherein the fluid chambers are serially connected by first fluid exchange channels; (c) a valve for controlling the flow of fluid through a first fluid exchange channel; (d) a pump for moving fluids into or out of the fluid chambers; and (e) a first inlet port on the cassette connected to a first supply chamber in each reaction flow-way by a second fluid exchange channel. The first supply chamber is preferably a supply chamber having a releasable seal blocking the outlet into the first fluid exchange channel connecting the first supply chamber to its reaction flow-ways; more preferably, the first supply chamber is an internal-outlet supply chamber. The pump preferably comprises a foot-pad pump with foot-pads designed to push on the first supply chamber to open the sealed outlet and pump fluid into the connected first fluid exchange channel. Preferably, the first supply chamber is collapsible upon evacuation and fillable from a vacuum-collapsed state to a defined volume. In one aspect of the invention, the second fluid exchange channel is releasably sealed so as to block the flow of fluids through the second fluid exchange channel. Preferably, the second fluid exchange channel is heat-sealed; more preferable, the second fluid exchange channel is sealed at multiple locations to prevent fluid communication between the first supply chambers.
In another aspect, the valve used in the context of the present invention is a plunger-type valve that is controlled by a pressure control means for: (i) applying a positive pressure to the plunger-type valve such that the plunger-type valve presses against the upper or lower film so as to impede the flow of fluid in a first fluid exchange channel, and (ii) releasing the positive pressure to the plunger-type valve such that the plunger-type valve releases from the flexible film so as to permit the flow of fluid in the first fluid exchange channel. Preferably, the plunger of the plunger-type valve is affixed to an instrument from which the cassette is detachable.
The cassette can be formed of a body that comprises recesses in its upper or lower surface which, together with an associated upper or lower film, form the first and second fluid exchange channels, and a plurality of fluid chambers. In the invention, it is preferred that a fluid chamber is formed in the upper or lower surface and at least one first or second fluid exchange channel is formed on an upper or lower surface located above or below that fluid chamber. The cassette of the present invention further comprises: (f) at least one hole situated in the body so as to connect a first or second fluid exchange channel formed at the upper or lower surface of the body with a first or second fluid exchange channel formed at the other surface. Preferably, the portion of upper or lower film covering a said fluid chamber made up of a recess in the body is embossed to mirror the shape of the bottom of the fluid chamber such that when the chambers is evacuated the film portion will invert to match the shape of the bottom of the chamber. Preferably, one of the pumps is a foot-pad pump having a foot pad that fits against the surface of the inverted embossed film portion of said fluid chamber.
Preferably, the cassette further comprises: (g) one or more second supply chambers, wherein two or more fourth fluid exchange channels connect the second supply chamber to two or more reaction flow-ways, which fourth fluid exchange channels include two or more said valves so that fluid from the second supply chamber can be directed to any one of the connected reaction flow-ways to the exclusion of the other connected reaction flow-ways; and (h) one or more second inlet ports on the cassette each connected to one of the second supply chambers by a separate third fluid exchange channel. Preferably, the device further comprises (i) a metering chamber interposed between the second supply chamber and the connected reaction flow-way. The combination of elements (f), (g), (h), and optionally (i) forms a sample insertion device. Preferably, the cassette has more than one such sample insertion device and sufficient reaction flow-ways such that different experimental samples can be reacted in parallel.
Preferably, the upper and lower walls of each first reaction chamber are formed of an embossed portion of a said upper film and an embossed portion of a said lower film, wherein the embossing allows upper and lower walls of the first reaction chambers to be brought together to minimize the volume of the first reaction chambers. Preferably, at least one pump comprises a foot-pad pump with upper and lower foot-pads designed to push together the upper and lower walls of a first reaction chamber. Alternatively or in addition, at least one of the pumps comprises gas pressure conduits for applying a positive pressure to the flexible upper or lower walls of a first reaction chamber so as to cause the flexible upper or lower wall to press inward thereby decreasing the volume within the first reaction chamber and impelling the flow of fluids therefrom.
In a preferred embodiment, the cassette further comprises (j) one or more waste chambers; and (k) an exhaust port for evacuating one or more of the first reaction chambers or the waste chambers.
Each embodiment of the invention can further comprise (I) a heater for heating one or more of the fluid chambers; (m) a cooler for cooling one or more of the fluid chambers; and (n) a temperature monitor for monitoring the temperature of one or more of the fluid chambers. Preferably, a foot-pad for pumping fluid out of the fluid chamber is associated with a heater and cooler for the fluid chamber; more preferably, the heaters and the coolers comprise a thermoelectric heat pump attached to a heat sink having a heater element. Preferably, the heaters and the coolers can change the temperature of a fluid chamber at a rate of at least about 5°C per second. Additionally, each embodiment of the invention can further comprise (o) a permanent magnet that can be positioned adjacent to one or more of the fluid chambers, or removed therefrom, wherein further the invention comprises means for moving the magnet adjacent to or away from the cassette. Each embodiment of the invention can also comprise (p) a detection chamber or channel having a transparent wall. Further, each such - 6 - embodiment can include (q) a light source capable of directing light to the transparent wall of a chamber or channel; and also (r) a light detection device capable of detecting: ( 1 ) the light reflected from an illuminated chamber or channel having a transparent wall; (2) the light transmitted through an illuminated chamber or channel having a transparent wall; or (3) the light emissions emanating from an excited molecule in a chamber or channel having a transparent wall.
In a preferred embodiment, the invention includes at least one valve that comprises: ( 1 ) a shut-off means comprising a valve ball or pinch foot, and (2) switching means for positioning the valve ball or pinch foot so that the valve ball or pinch foot: (i) presses against the flexible film to cut off flow through a first or second fluid exchange channel, or (ii) releases away from the flexible film to allow flow through the first or second fluid exchange channel. The switching means preferably comprises spring loaded levers. Preferably, at least one valve comprises: ( D a spacer, (2) a spacer spring means for normally pressing the spacer against the flexible film so as to cut off the flow of fluids through a first fluid exchange channel, and (3) an electromagnet effective when activated to sufficiently release the pressure against the flexible film to allow the flow of fluids through the first or second fluid exchange channel.
In a preferred embodiment, the invention provides a device for conducting assays in parallel using fluids that are confined to a disposable cassette comprising the disposable assay cassette, which comprises (i) at least two reaction flow-ways, including a first reaction flow-way designed to receive and assay an experimental sample and a second reaction flow-way designed to receive and assay a negative control, (ii) for each reaction flow- way, at least one supply chamber connected thereto and containing fluids needed in the assay and at least one reaction chamber, (iii) a negative control supply chamber connected with the second reaction flow-way containing the negative control, and (iv) a test sample supply chamber connected with the first reaction flow-way designed to receive a test sample through an inlet connected with the test sample supply chamber, valves for controlling the flow of fluids in the cassette, and an instrument comprising a temperature control unit for controlling in parallel the temperature in a reaction chamber in each reaction flow-way, valve actuators for opening and closing the valves in the cassette, and one or more pumps for pushing fluid out of the various supply chambers and reaction chambers of the cassette. Preferably, the cassette further comprises (v) a third reaction flow-way designed to receive and assay a test sample and a positive control, (vi) connecting routes between the test sample supply chamber and both the first and third reaction flow- ways, wherein these connecting routes are controlled by valves that allow selective flow between the test sample supply chamber and either the first or third reaction flow-way, and (vii) a first positive control supply chamber connecting with the third reaction flow-way containing the positive control. Also preferably, the cassette further comprises (viii) a fourth reaction flow- way designed to receive and assay a positive control, and (ix) a second positive control supply chamber connecting with the fourth reaction flow-way containing the positive control. As well, the cassette preferably comprises (v) a third reaction flow-way designed to receive and assay a test sample and a positive control, (vi) connecting routes between the test sample supply chamber and both the first and third reaction flow-ways, wherein these connecting routes are controlled by valves that allow selective flow between the test sample supply chamber and either the first or third reaction flow-way, and (VIII) a first positive control supply chamber connecting with the third reaction flow-way containing the positive control. Preferably, the pumps comprise one or more foot-pad pumps. Further, the temperature control unit preferably comprises a thermoelectric heat pump; and the thermoelectric heat pump preferably is attached to a heat sink having a heater element. Preferably, the valves of this embodiment comprise plunger-type valves. The invention further provides a method of conducting assays, including chemical diagnostic assays, antibody-based assays and nucleic acid amplification-based assays, using one of the aforementioned devices, which method comprises (a) providing the device for conducting assays in parallel, wherein reagents and control materials are pre-loaded into the supply chambers; (b) inserting a test sample into the test sample supply chamber; and (c) reacting in parallel in separate reaction flow-ways ( 1 ) the test sample and (2) the negative control. Preferably, the reagents or control materials include binding domains derived from antibodies; alternatively, the reagents or control materials include fluids containing primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction. Preferably, the reacting comprises reacting in separate reaction flow-ways (1 ) test sample and (2) negative control with a suspension of nucleic acid-binding beads, wherein the suspension of nucleic acid-binding beads is provided by a separate supply chamber for each reaction flow-way; and replacing the fluid suspending the nucleic acid-binding beads with a fluid containing primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction. Preferably, the nucleic acid binding beads are paramagnetic beads and the replacing step comprises ( 1 ) magnetically locking the nucleic acid-binding beads in place while pushing the suspending fluid into a waste chamber, (2) resuspending the nucleic acid- binding beads in a wash fluid, wherein wash fluid is introduced from a separate supply chamber for each reaction flow-way, (3) magnetically locking the nucleic acid-binding beads in place while pushing the suspending fluid into a waste chamber, and (4) resuspending the nucleic acid-binding beads in the fluid containing primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction.
In a preferred embodiment, the invention relates to a method of conducting nucleic acid amplification reactions using the aforementioned device, which method comprises (a) providing the device for conducting assays in parallel, wherein reagents and control materials are pre-loaded into the supply chambers, wherein the reagents or control materials include primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction; (b) inserting a test sample into the test sample supply chamber; and (c) reacting in parallel in separate reaction flow- ways ( 1 ) the test sample, (2) a negative control and (3) a mixture of the test sample and a positive control. The present invention further preferably relates to a method of conducting nucleic acid amplification reactions using the aforementioned device, which method comprises: (a) providing the device for conducting assays in parallel, wherein reagents and control materials are pre-loaded into the supply chambers, wherein the reagents or control materials include primers, nucleotide triphosphates, and ions and buffers suitable for supporting a nucleic acid amplification reaction; (b) inserting a test sample into the test sample supply chamber; and (c) reacting in parallel in separate reaction flow-ways (1 ) the test sample, (2) a negative control, (3) a mixture of the test sample and a positive control and (4) a positive control.
The invention still further provides a device comprising a cassette suitable for conducting reactions therein, which cassette comprises a body having one or more recesses and one or more embossed films covering the recesses. Preferably, the cassette includes a hole extends through the body, further comprising a fluid exchange channel in communication with a valve, which valve is in communication with the hole, and a film having an embossed portion sealed to the body such that the hole and the fluid exchange channel are covered. The device further comprises preferably a pneumatically driven plunger for pressing the embossed film portion at or about the valve, and pressure control means for (i) applying a positive pressure to the pneumatically driven plunger such that the plunger presses against the flexible film so as to close the valve, and (ii) releasing the positive pressure to the pneumatically driven plunger such that the plunger releases from the flexible film so as to open the valve.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A, I B and 1 C show a top, side and bottom view of a cassette of the invention.
Figure 2A shows a side view of a Bursapak supply chamber. Figure 2B illustrates a method for sealing closed a fluid exchange channel.
Figure 2C illustrates how pressure can be used to open a Bursapak supply chamber.
Figures 2D and 2E illustrate a foot-pad that can be used to pressurize the fluid in the Bursapak supply chamber. Figure 3 schematically diagrams a parallel reaction device of the invention.
Figure 4A illustrates a cassette of the invention. Figures 4B-4E show the cassette of Figure 4A with various subsets of the features thereof illustrated and numbered. Figure 5A and 5B show a plunger-type valve mechanism for regulating fluid flow through a cassette.
Figure 6 shows in perspective view the part of a plunger-type valve located in the body of a cassette.
Figure 7 shows the parts of a plunger-type valve located outside the cassette (i.e., in the instrument).
Figures 8A, 8B and 8C show various configurations of valve mechanisms for regulating fluid flow through a cassette.
Figures 9A and 9B show a magnetic spring valve mechanism for regulating fluid flow through a cassette. Figure 10 shows a support device for rapidly heating and cooling a reaction chamber and providing a foot-pad for a foot-pad pump.
Figures 1 1 A and 1 1 B show the operation of a foot-pad pump on a reaction chamber.
Figure 12 shows a schematic of accessory support devices for rapidly heating or cooling a reaction chamber.
Figure 13 shows another mechanism for rapidly heating or cooling a reaction chamber.
Figure 14 shows yet another mechanism for rapidly heating or cooling a reaction chamber. Figures 15A and 15B show two side views of a detection channel.
Figure 16 shows an example of a magnet useful for locking paramagnetic beads at a certain location in a cassette.
Figures 17 A and B show a device for mounting a septum to the cassette.
DEFINITIONS
The following terms used in this disclosure shall have the meanings set forth below: • annealing temperature
PCR protocols and other nucleic acid amplification protocols often use an "annealing temperature" less than the replication temperature to accelerate the rate at which the primers bind to (i.e., hybridize with) the sample nucleic acid; this annealing temperature is typically between about 45°C and about 72°C, often about 55°C. Generally, the annealing temperature will be about 5°C below the lowest Tm for the interaction between (a) one of the primers used in reaction and (b) the target nucleic acid segment. * Bursapak chamber a chamber formed in a solid support and having a film formed of a flexible material that is sealed to the support at the edges of the chamber and has an outlet channel that is blocked by a portion of the film which is sealed over the outlet channel, wherein the seal over the outlet is broken or removed by pressurizing the fluid contents of the chamber at a pressure that does not affect the seal at the edges of the chamber; preferably, the film is on one face of the cassette body and the outlet is oriented toward the other. • cassette a disposable device for conducting reactions therein having a cassette body, one or more upper membranes or one or more lower membranes which individually or in combination define one or more supply chambers, one or more reaction chambers and fluid exchange channels connecting the supply chambers to reaction chambers.
• cassette body a solid portion having sufficient depth and sturdiness to allow cavities formed therein to provide the depth for fluid chambers and fluid exchange channels.
• collapsible upon evacuation some of the chambers described below will preferably be filled by first applying a vacuum to evacuate the chamber contents and then filling the evacuated chamber with fluid — preferably, these chambers are "collapsible" in that they have at least one flexible film that collapses to minimize chamber volume.
• connection (between fluid chambers, inlets or detection channels) two fluid chambers, inlets or detection channels are "connected" or have a "route of connection" therebetween if there is one or more fluid exchange channels joining the two such that fluid can move from one to the other.
• concentric Bursapak supply chamber an internal outlet Bursapak supply chamber wherein the outlet channel is located substantially in the center of the supply chamber; "substantially in the center" means that the distance between the center of the supply chamber and the geometric center of the supply chamber is no more than about 20% of the length of the supply chamber cross-section defined by the line joining the center of the outlet and the geometric center of the supply center.
• DNA strand separation temperature the temperature used in a nucleic acid amplification protocol to separate the complementary strands of nucleic acid that may be present in a sample; this temperature is typically between about 92°C and about 97°C, preferably about 94°C.
• elevated pressure a pressure more than ambient atmospheric pressure. • tillable from a vacuum-collapsed state to a defined volume these are chambers that unfold from the collapsed state to a first volume; preferably, the inserted fluid volume is within about 10% of the first volume, more preferably within about 3% of the first volume. The first volume is the maximum volume of fluid that can be inserted into the chamber without affecting the integrity of the chamber.
• fluid chamber the term "fluid chamber" encompasses reaction, supply, waste metering and sample storage chambers, and other fluid containing chambers. In those embodiments where contents of the chambers can be pumped out using a foot-pad having a shape that conforms to a covering film that is inverted to match the shape of the bottom of the chamber, the chamber can be closed by maintaining the foot-pad pressed against the inverted covering film. • fluid-tight a space or chamber is fluid-tight if it retains an aqueous fluid in the space at a temperature of 99°C for one hour; a seal between two materials is fluid- tight if the seal is substantially no more permeable to water than the most water-permeable such material. • foot-pad a plunger having a shape designed to conform to the inverted shape of the covering film of a supply chamber; when the plunger presses against the flexible film it pressurizes the fluid in the supply chamber and, if an exit is available, pushes the fluid out of the supply chamber. • foot-pad pump a mechanical, electromechanical or pneumatic device that uses a one or more, preferably two or more, foot-pads to press on one or more fluid chambers such as supply chambers or reaction chambers to pressurize the contents and push the contents out through an unobstructed connected fluid exchange channel.
• integral parts or elements of a valve are integral to a body layer or to a cassette if they cannot be facilely and reversibly detached from that body layer or cassette. • internal outlet Bursapak supply chamber a Bursapak supply chamber wherein the outlet channel is located away from the edges of the supply chamber such that fluid-containing space is interposed between the sealed outlet channel and the edges chamber.
• negative control a material designed to be comparable to a sample to be assayed but lacking the substance to be assayed for, such that a positive result upon assaying a negative control would indicate a problem with the assay protocol or assay reagents.
• nucleic acid melting temperature or Tm the transition temperature for two-stranded duplex of nucleic acid at which the equilibria shifts from favoring the base-paired duplex to favoring the separation of the two strands.
• positive control a material designed to generate, in the absence of a problem with the assay chemistry such as the presence of an interfering substance, a positive assay result.
• reaction flow-away a series of two or more serially connected fluid chambers through which fluids can move. • reduced pressure a pressure less than ambient atmospheric pressure.
• replication temperature the temperature used in a nucleic acid amplification protocol to allow the nucleic acid reproductive enzyme to reproduce the complementary strand of a nucleic acid to which a primer is bound (i.e., hybridized); this temperature is typically between about 69°C and about 78°C, preferably about 72°C, when using a heat stable polymerase such as Taq polymerase.
• serially connected two or more fluid chambers are serially connected if there are fluid exchange channels by which fluid from a first of the serially connected chambers can pass to a second of the serially connected chambers, and from there to a third of the serially connected chambers, and so on until the fluid passes to the last of the serially connected chambers.
• substantially uniform temperature in the reaction chamberwhere the temperature in a reaction chamber varies by no more than about ± 0.3°C. * target nucleic acid segment a segment of nucleic acid that is sought to be identified or measured in a sample, such as a sequence intended, if present, to be amplified in a nucleic acid amplification reaction such as a PCR reaction, strand displacement assay or ligase chain reaction; the target segment is typically part of a much larger nucleic acid molecule found in the sample.
* thermoelectric heat pump a device for heating and cooling fluid chambers that is made up of one or more thermoelectric blocks.
DETAILED DESCRIPTION
The cassette of the present invention includes at least one reaction chamber and at least one supply chamber in combination with interconnecting fluid exchange channels. The cassette comprises a body into which the aforementioned chambers and channels are formed such that when covered by a film and sealed, as described below, the formed body with film can hold fluids. The shape of the body can be any shape, although preferably it is a flat square, rectangular or circular structure of length and width or diameter substantially greater than its depth, such as, for example, 3 cm x 3 cm x 3 mm, inter alia, and the length and width or diameter can be further described with respect to a top or bottom surface, and the depth can be further described with respect to an edge. The chambers and channels prior to covering by the film can be open to any surface of the body, preferably is open to the top or bottom, more preferably is open to the top and bottom, although each chamber or channel preferably is open to one side only.
The present invention is described herein with respect to particular embodiments; however, these embodiments should not be construed as in any way limiting the scope of the present invention, which includes all modifications encompassed within the spirit and scope of the invention as described hereinbelow.
Exemplary Cassette
Figures 1 A, 1 B and 1 C show a top view, cross-sectional view and bottom view of a portion of one embodiment of a cassette 1 00 according to the invention. The cassette 1 00 has a body 1 05 in which are defined inlet 1 30, first fluid exchange channel 141 , supply chamber 1 50, second fluid exchange channel 1 42, reaction chamber 1 60, third fluid exchange channel 1 43 and waste chamber 1 70. The body 1 05 has first upper film 1 10A, second upper film 1 1 OB, third upper film 1 10C and lower film 1 20. In Figure 1 A, first seal portion 1 1 1 A (shaded area), second seal portion 1 1 1 B (shaded area) and third seal portion 1 1 1 C (shaded area) show where first upper film 1 1 0A, second upper film 1 1 0B and third upper film 1 1 0C, respectively, are sealed against body 105. In Figure 1 C, shading 1 21 shows where lower film 1 20 is sealed against body 1 05. Inlet 1 30 has a septum 1 31 . First, second and third upper films 1 10A-C are collectively referred to as "upper films 1 1 0. " Septum 1 31 can be, for instance a bilayer material formed of an outer layer of silicon or neoprene rubber and an inner layer of chemically inert material such as tetrafluoroethylene homopolymer (e.g. , Teflon, E.I. duPont de Nemours and Co., Wilmington, DE) facing the body 105. Second upper film 1 1 0B and lower film 1 20 are embossed or shaped at positions 1 61 and 1 62 to help form reaction chamber 1 60, as will be described in greater detail below with reference to Figures 1 1 A and 1 1 B. First upper film 1 1 0A is embossed or shaped at the location of supply chamber 1 50 so that first upper film protrudes above the upper surface of body 105, creating a greater volume for supply chamber 1 50 and facilitating the mechanism by which supply chamber 1 50 is emptied, as described further in the text below with reference to Figures 2A and 2B. Third upper film 1 1 0A is embossed or shaped at the location of waste chamber 1 70, which embossing facilitates the mechanism by which the waste chamber is filled. A valve 1 80 is formed in third fluid exchange channel 143. The outlet 1 51 of supply chamber 1 50 is sealed by a portion of first upper film 1 10A. Supply chamber 1 50 is a Bursapak supply chamber, which type of supply chamber is a particularly useful type of supply chamber for use in the cassette of the invention. Because many of the cassettes described below make use of this preferred type of supply chamber, Bursapak supply chambers are described in more detail in the following section.
Bursapak Supply Chambers
Figure 2A shows a side view of a Bursapak supply chamber 1 50 having supply cavity 1 55, which can contain a fluid. The Bursapak supply chamber 1 50 has an inlet first fluid exchange channel 141 , which is preferably sealed, for instance by heat sealing at sealing location 1 41 A, after the Bursapak supply chamber 1 50 has been filled with fluid, and an outlet second fluid exchange channel 1 42 which is initially sealed with a fourth seal portion 1 1 1 D of first upper film 1 1 0A. Figure 2B shows the use of die 1 300 to heat seal first fluid exchange channel 1 41 , at sealing location 1 41 A. Figure 2C illustrated how pressure -- indicated by the arrows -- applied to the fluid in Bursapak supply chamber 700 is effective to pull the seal portion 1 1 1 away from the outlet second fluid exchange channel 142. Figure 2D illustrates a foot-pad 210 that can be used to apply pressure to the fluid in Bursapak supply chamber 1 50 and pump it through outlet second fluid exchange channel 1 42 Foot-pads can be fabricated of any suitably sturdy material including, without limitation, aluminum, plastics, rubber, alumina, copper, sintered beryllia, and the like. Upper films 1 1 0 and lower films 1 20 are preferably constructed of a flexible film such as a polyethylene, polyvinylidene fluoride or polyethylene/polyethylene terephthalate bi-layer film Suitable films are available from Kapak Corporation, Minneapolis, MN or E.I duPont de Nemours and Co., Wilmington, DE. Polyethylene/polyethylene-terephthalate bi-layer film such as 3M No. 5 or 3M No 48 (3M Corp., MN) or Dupont M30 (DuPont de Nemours, Wilmington, DE) are particularly preferred. The polyethylene layer is preferably positioned against body 1 05. Figure 2E shows the foot-pad used to pump fluid out of Bursapak supply chamber 1 50
The first upper film 1 1 0A is embossed or shaped, for instance by applying suitably shaped, heated dies to the first upper film 1 10A, so that it can protrude away from the body 1 05 when the supply chamber 1 50 is filled and will rest, without substantial stretching, against the bottom of supply chamber 1 50 when the supply chamber 1 50 is evacuated.
It is believed that the application of force through a foot-pad results in the application of greater force per unit length at the edges of the fourth seal portion 1 1 1 D than at the edges of first seal portion 1 1 1 A, resulting in selective peeling of fourth seal portion 1 1 1 D. Whatever the mechanism, however, in operation Bursapak chambers operate as illustrated in Figures 2A- 2C. To assure proper functioning, in some embodiments it may be necessary to seal fourth portion 1 1 1 D relatively more weakly, for instance using a weaker adhesive or a lower temperature sealing die.
Materials. Dimensions for Cassette Components
Body 105 is preferably formed of a molded plastic, such as high density polyethylene, but other materials that are suitably resistant to the chemistries sought to be conducted on the parallel reaction device, such as glass and silicon-based materials, can be used. Where body 1 05 is plastic, it is preferably formed by a molding process that is used to form cavities and channels that will be sealed with upper and lower films 1 1 0 and 1 20 to form fluid chambers and fluid exchange channels. Such cavities and channels are formed in glass and silicon materials by chemical etching or laser ablation. Upper and lower films 1 1 0 and 1 20 typically have a thickness of from about 0.3 mils to about 5 mils, preferably from about 1 mil to about 3 mils. For fluid chambers having a diameter of about 1 cm or more, the film thickness is more preferably about 2 mils. Reaction chamber 1 30A typically has a thickness, between upper and lower films 1 10 and 1 20, of from about 0.1 mm to about 3 mm preferably of from about 0.5 to about 1 .0 mm and an area, defined by the inner surface of upper or lower films 1 1 0 or 1 20, of preferably from about 0.05 cm2 to about 2 cm2, more preferably from about 0.1 cm2 to about 1 cm2, yet more preferably about 0.5 cm2. The dimensions of reaction chamber are preferably sized small enough to permit rapid thermal cycling (on the order of about 10 seconds) .
Fluid exchange channels typically have a diameter between about 200 and about 500 μm. Supply chambers 1 50 typically have a volume between about 5 and about 500 μl, preferably from about 1 0 to about 200 μl, more preferably from about 30 to about 1 60 μl. Metering chambers preferably have a volume between about 5 and about 50 μl. Preferably, the total volume of each reaction chamber 1 60 is between about 5 μl and about 200 μl, more preferably, between about 10 μl and about 1 00 μl. Preferably, each reaction chamber has a thickness (i.e., distance between upper film 1 10 and lower film
120) of about 1 mm or less.
Upper and lower films 1 1 0 and 1 20 preferably are resistant to temperatures as high as about 1 20°C and are between about 1 and about 6 mils in thickness, more preferably, between about 2 and about 4. The thinness of the membranes facilitates rapid heat exchange between the reaction chamber and an adjacent heating or cooling device. Schematic of Parallel Reaction Device
Figure 3 illustrates schematically a parallel reaction device 301 according to the invention having five reaction flow-ways, each such flow-way, respectively, used for analyzing (A) a sample 300, (B) a positive control 31 0, (C) a negative control 320, (D) a positive control 330 combined with sample 300, and (E) a sample 300. Each of these samples and controls is introduced into one of first through fifth lysing chambers 340A-E (collectively, lysing chambers 340). Lysing reagents and washing buffer can be distributed from first supply chamber 350 and second supply chamber 360, respectively, to all five lysing chambers 340. Waste can be emptied from lysing chambers 340 into a single waste chamber 370. The remaining contents of each of lysing chambers 340 can then be transferred to one of first through fifth reaction chambers 380A-E, respectively (collectively, reaction chambers 380). Amplification reagents are added to each of reaction chambers 380 from a third supply chamber 390. Waste can be emptied from reaction chambers 380 into waste chamber 370. The remaining contents of each of reaction chambers 380A-E can then be transferred into one of first through fifth storage chambers 399A-E, respectively. Each valve which regulates the flow of fluids into and out of the various chambers is separately diagrammed in Figure 3 as an encircled letter "v. "
It should be noted that some of the arrows in Figure 3, which arrows represent fluid channels, apparently pass through a fluid chamber. These channels actually pass above or below the fluid chamber, as is described further in the text below. As is described further below, lysing chambers 340 and reaction chambers 380 preferably have flexible upper film 1 1 0 and lower film 1 20 that can be manipulated with a foot-pad pump or a gas pressure flow control means. If both upper and lower walls of a fluid chamber are formed with films 1 10 and 1 20, then channels passing through the region of the device occupied by the lysis chambers 340 or reaction chambers 380 must pass adjacent to such chambers rather than above or below the chambers.
Detailed Cassette - Structure
Another cassette 200 is illustrated in Figure 4A. The illustrated cassette 200 has planar dimensions of 31Λ inches by 5 5/1 6 inches, although other sizes are contemplated, including for instance in circumstances where the sizes of the fluid chambers and other components of the cassette differ from those illustrated. Because of the complexity of Figure 4A, Figures 4B - 4E show the body 205 of the cassette together with illustrations of various subsets of the components of body 205. In these illustrations the solid lines connecting inlets, valves or fluid chambers represent fluid exchange channels. Those fluid exchange channels represented by dark lines are formed in the upper surface of body 205, while those represented by lighter lines are formed in the lower surface of body 205. At the top of Figure 4B are illustrated the symbols used to represent an inlet 230 or a supply chambers 250 of various sizes (sizes recited for illustrative purposes only) .
In Figure 4B are illustrated: alpha first supply chamber 251 A, beta first supply chamber 251 B, and so on through delta first supply chamber 251 D, which are connected to first inlet 231 by alpha second fluid exchange channel 242A; alpha second supply chamber 252A, beta second supply chamber 252B, and so on through theta second supply chamber 253H, which are connected to second inlet 232 by beta second fluid exchange channel 242B; alpha third supply chamber 253A, beta third supply chamber 253B, and so on through theta third supply chamber 253H, of which alpha, gamma, epsilon and eta third supply chambers 253A, C, E and G are connected to third inlet 233 by gamma second fluid exchange channel 243B and beta, delta, zeta and theta third supply chambers 253B, 253D, 253F and 253H are connected to beta fourth inlet 234B, delta fourth inlet 234D, zeta fourth inlet 234F and theta fourth inlet 234H, respectively; and alpha fourth supply chamber 254A, beta fourth supply chamber 254B, and so on through theta fourth supply chamber 254H, of which alpha, gamma, epsilon and eta fourth supply chambers 254A, 254C, 254E and 254G are connected to fifth inlet 235 by delta second fluid exchange channel 242D and beta, delta, zeta and theta fourth supply chambers 254B, 254D, 254F and 254H are connected to sixth inlet 236 by epsilon second fluid exchange channel 242E. The connecting fluid exchange channels 21 5 between second fluid exchange channels 242 and supply chambers 250 are represented by solid circles.
Alpha first reaction chamber 261 A can receive fluid from any of seven supply chambers 250, which supply chambers 250 are alpha first supply chamber 251 A, alpha second supply chamber 252A, beta second supply chamber 252B, alpha third supply chamber 253A, beta third supply chamber 253B, alpha fourth supply chamber 254A and beta fourth supply chamber 254B. Beta first reaction chamber 261 B, gamma first reaction chamber 261 C and delta first reaction chamber 261 D each can receive fluid, in a manner parallel to the arrangement for alpha first reaction chamber 261 A, from seven supply chambers 250 as illustrated. Alpha first reaction chamber 261 A, beta first reaction chamber 261 B, gamma first reaction chamber 261 C 5 and delta first reaction chamber 261 D connect to alpha second reaction chamber 262A, beta second reaction chamber 262B, gamma second reaction chamber 262C and delta second reaction chamber 262D, respectively, via alpha first fluid exchange channel 241 A, beta first fluid exchange channel 241 B, gamma first fluid exchange channel 241 C and delta first fluid exchange
10 channel 241 D, respectively. Alpha second reaction chamber 262A, beta second reaction chamber 262B, gamma second reaction chamber 262C and delta second reaction chamber 262D connect to first waste chamber 271 under the control of alpha first valve 281 A, beta first valve 281 B, gamma first valve 281 C and delta first valve 281 D, respectively.
15 In Figure 4C are illustrated alpha seventh inlet 237A and beta seventh inlet 237B, which are connected to alpha fifth supply chamber 255A and beta fifth supply chamber 255B, respectively. Alpha fifth supply chamber 255A and beta fifth supply chamber 255B are connected to alpha second reaction chamber 262A and beta second reaction chamber 262B.
20 Exhaust port 275 allows the first reaction chambers 261 , second reaction chambers 262, first waste chamber 271 , second waste chamber 272, metering chamber 290 and detection channels 295 to be evacuated prior to use. This evacuation is possible because all of the first reaction chambers 261 , second reaction chambers 262, first waste chamber
25 271 , second waste chamber 272, metering chamber 290 and detection channels 295 communicate when the appropriate valves 280 are open. Alpha sealing position 276A and beta sealing position 276B can be heat sealed when the evacuation process is complete to lock the first reaction chambers 261 , second reaction chambers 262, first waste chamber 271 , second waste
30 chamber 272, metering chamber 290 and detection channels 295 in the evacuated state prior to operating the cassette.
In Figure 4D, sixth supply chamber 256 is filled using alpha eighth inlet 238A and is connected to metering chamber 290 under the control of alpha second valve 282A. Seventh supply chamber 257 is filled
35 using beta eighth inlet 238B and is connected to metering chamber 290 under the control of beta second valve 282B. From metering chamber 290 fluid can be directed to either gamma second reaction chamber 262C or delta second reaction chamber 262D under the control of gamma second valve 282C and delta second valve 282D, respectively.
In Figure 4E, fluid from alpha second reaction chamber 262A can be directed to alpha detection channel 295A under the control of alpha 5 third valve 283A. Corresponding connections from beta second reaction chamber 262B through delta second reaction chamber 262D to beta detection channel 295B through delta detection channel 295D, respectively, are controlled by beta third valve 283B through delta third valve 283D, respectively. Alpha eighth supply chamber 258A, beta eighth supply chamber 10 258B, and so on, are respectively connected to alpha detection channel 295A, beta detection channel 295B, and so on. Alpha eighth supply chamber 258A, beta eighth supply chamber 258B, and so on are filled through ninth inlet 239.
Detailed Cassette - Operational Features
15 This discussion of operational features of the cassette structure 200 shown in Figures 4A-4E assumes that the supply chambers of that structure are Bursapak supply chambers. The first supply chambers 251 can be used to store fluid having suspended paramagnetic beads used in preparing nucleic acid from biological samples, which paramagnetic beads are
20 described in greater detail below. A foot-pad pump operates propel in parallel the fluid and suspended beads from the first supply chambers 251 to the connected first reactions chambers 261 . To assure that the beads are suspended the foot-pad pump operating on the first supply chambers 251 and foot-pad pump operating on the first reaction chambers 261 can alternately be
25 operated to move the fluid back and forth between the first supply chambers 251 and first reaction chambers 261 , thereby agitating the fluid and re¬ suspending the beads.
The second supply chambers 252 can contain a buffer solution, such as a buffer solution used to wash the paramagnetic beads. The
30 associated foot-pad pump has four foot-pads designed to interact with either ( 1 ) alpha second supply chamber 252A, gamma second supply chamber 252C, epsilon second supply chamber 252E and eta second supply chamber 252G or (2) beta second supply chamber 252B, delta second supply chamber 252D, zeta second supply chamber 252F and theta second supply chamber
35 252H. Alternatively, the pump has two sets of four pads designed to interact with second supply chambers 252. The third supply chambers 253 alternate in size between supply chambers 253 having volumes of 1 00 μl and supply chambers 253 having volumes of 30 μl. The 1 00 μl supply chambers 253 can be used to store cell lysis solutions while the 30 μl supply chambers 253 can be used to 5 store solutions of primers.
Alpha, gamma, epsilon and eta fourth supply chambers 254A, 254C, 254E and 254G can be used to store a solution containing the appropriate nucleotide triphosphates for a nucleic acid amplification assay. Beta, delta, zeta and theta fourth supply chambers 254B, 254D, 254F and
10 254H can be used to store solutions containing the polymerase enzyme for the nucleic acid amplification assay.
A desirable feature for a cassette such as that illustrated in Figures 4A-4E is the ability to incorporate a positive control in one or more, but not all, of the reaction flow-ways 265 (not identified in Figures, first
15 reaction flow-way 265A includes alpha first and second reaction chambers 261 A and 262A, second reaction flow-way 265B includes beta first and second reaction chambers 261 B and 262B, and so on) . Thus, a material that should generate a positive assay result can be inserted into sample that otherwise may or may not produce a positive signal (i.e., experimental 0 samples) or in samples that should not produce a positive signal (i.e. , negative controls) . In this way, the source of any substances that interfere with the assay can be determined. Any failure of the reaction flow-ways containing a positive control to generate a positive signal or an appropriately strong positive signal would indicate that a standard solution used in the assay contains a 5 substance or has a property that interferes with the assay. Fluids expected to generate negative signals can also be incorporated into the cassette.
Controls, e.g., fluids that have a predetermined amount of a component to be tested for or that are known to lack the component, can be inserted into alpha and beta second reaction chambers 262A and 262B from
30 alpha and beta fifth supply chambers 255A and 255B. Note that this particular embodiment does not include a facile way to introduce both a positive control and a test sample into a reaction flow-way; however, modifications of the cassette 200 of Figures 2A-2E that would allow such a means of introduction are easily envisioned.
35 Not all Bursapak supply chambers 250 must be utilized. A
Bursapak supply chamber is avoided simply by not pumping its contents into the connected reaction chambers.
It is desirable to contain all waste fluids in the cassette 200. Thus, the illustrated cassette 200 has a first waste chamber 271 and a second waste chamber 272 (collectively waste chambers 270) of sufficient volume to accommodate all the fluids introduced into the cassette. Waste chambers 270 are prepared in an evacuated state such that the films forming the outer wall of the waste chambers 270 (see film 1 10C of Figure 1 ) rest against the inner surfaces of the waste chambers 270. As fluid is pumped into the waste chambers 270, the film will flex outwardly to provide room for the inserted fluid. It is desirable to confine the fluids to the cassette for instance to isolate biohazards or, in the case of nucleic acid amplification assays, to minimize the opportunity for aerosols to spread nucleic acid through the lab creating the potential for cross-contamination of other assays.
Supply chambers 250 are also evacuated in like manner prior to filling. Most supply chambers 250 will, in a preferred embodiment, be pre- filled prior to shipment to the laboratory where the assay will be conducted. Of course, the test sample will be inserted at the lab site. Fluid insertion is best described with reference to Figure 1 B. A needle can be inserted into septum 1 31 and used to evacuate supply chamber 1 50, causing film 1 1 0A to collapse onto the floor of supply chamber 1 50. Then, fluid can be inserted through the septum into supply chamber 1 50. The first fluid exchange channel is then blocked, for instance by heat sealing or by crimping.
Focusing on delta reaction flow-way 265D, note that experimental sample from sixth supply chamber 256 is first relayed to delta second reaction chamber 262D while flow to delta first reaction chamber 261 D is blocked by operating a foot-pad pump minimize the volume of delta first reaction chamber 261 D. Typically, the first reaction conducted on the experimental sample will occur in delta first reaction chamber 261 D. To move the experimental sample from delta second reaction chamber 262D to delta first reaction chamber 261 D, delta second valve 282D is closed, the foot-pad pump acting on delta first reaction chamber 261 D is released, and the foot¬ pad pump acting on delta second reaction chamber 262D is operated to pump the experimental sample into delta first reaction chamber 261 D.
Foot-pad pumps that operate to drain a supply chamber 1 50 can remain engaged with the supply chamber 1 50 to prevent back-flow into the supply chamber 1 50. Valves
Figures 5A, 5B, 6 and 7 illustrate yet another embodiment of the invention that utilizes plunger-type valves to control the flow of fluids in the cassette 1 00 or cassette 200. The operation of such a plunger-type valve in a cassette 1 00 or 200 is illustrated above with reference to Figures 5A and 5B. Plunger 810 has a plunger rod 81 1 and a piston 81 2. In the position illustrated in Figure 5A, plunger rod 81 1 is withdrawn away from such that third film 1 1 0C, which is embossed to protrude away from the seat 1 81 of valve 1 80, does not interfere with fluid flow from alpha third fluid exchange channel 143A, into valve 1 80, and out through beta third fluid exchange channel 1 43B. In Figure 5B, plunger rod 81 1 presses film 1 1 0C against valve seat 1 81 , blocking fluid flow. Figure 6 shows a three-dimensional view of valve 1 80, including valve seat 1 81 and valve trough 1 82. The plunger 81 0 can be constructed of numerous durable materials including without limitation a plastic such as polycarbonate or metal such as stainless steel or aluminum or the like. The diameter of plunger rod 81 1 is typically from about 20 to about 1 00 μm, preferably about 60 μm, while piston 81 2 typically has a diameter from about 100 to about 300 μm, preferably about 200 μm. Preferably, the ratio of the cross-sectional area of the piston 81 2 to that of the plunger rod 81 1 is at least about 1 0-fold, thereby providing a corresponding mechanical advantage.
A pneumatic mechanism for operating plunger 81 0 is illustrated in Figure 7. Instrument 900 (not shown) has a pneumatic device 800 formed of first portion 800A and second portion 800B which can be joined together, for instance, by bolts, rivets, adhesives or snap-fitting pieces. Interposed between the first and second portions 800A and 800B is flexible gasket 820, which can be formed of a suitable film such as poly (2-chloro-1 ,3- butadiene) (e.g., Neoprene, DuPont de Neumours, Wilmington, DE) or silicon rubber. Flexible gasket 820 can be held in place by the clamping action of first and second portions 800A and 800B, which adherent force can be supplemented using heat sealing or adhesive. Pneumatic cavity 830 is formed in both first and second portions 800A and 800B and has a cavity inlet 831 . Fluid, preferably a gas, is inserted through cavity inlet 831 to pressurize the part of pneumatic cavity 830 located above the gasket 820 and cause the gasket 820 to press against plunger 810, causing plunger 81 0 to press against valve seat 1 81 . In the absence of such fluid pressure in pneumatic cavity 830, pump induced pressure in third fluid exchange channel 1 43A is sufficient to displace (a) third upper cover into displacement cavity 840 and (b) plunger 81 0 from the valve seat 1 81 , thereby allowing flow. Pneumatic device 800 can be formed of numerous durable materials including without limitation a plastic such as polycarbonate or metal such as brass or aluminum or the like.
As an alternate to the above method of plunger actuation, other methods may be used which do not employ the piston. These include motor driven cam or screw, and external hydraulic or pneumatic cylinders.
In another valve embodiment of the invention shown in Figure 8A with reference to another cassette 600 (not shown) . Valve ball 620 is used to press lower film 1 20 flush against the lower surface of first body layer 601 so as to block fluid flow through hole 632. Valve ball 620 can be fabricated of any suitably material such as nylon, high density polyethylene, polycarbonate and the like. Lower film 1 20 is sealed to portions 601 A and 601 C of first body layer 601 , but typically is not sealed to portion 601 B. The sealing between lower film 1 20 and portions 601 A and 601 C can be done using, for instance, adhesives or by clamping the membrane between body layer 601 and second body layer 602. First body layer 601 , second body layer 602 and third body layer 603 can be joined together using, for instance, by bolts, rivets, adhesives or snap-fitting pieces. Pressure can be applied to valve ball 620 to press it against or release it from lower film 1 20 in a number of ways. Note that the valve is designed so that valve ball 620 will automatically center itself to properly seat itself against first layer 601 . Figure 8A shows a spring loaded lever 640 that allows a push motion to open the valve, where force is applied as indicated by arrow "B" . A push rod 643 (not illustrated) can be used to so engage spring loaded lever 640. Figure 8B illustrates another embodiment that uses pull rod 641 to open the valve. The function of both spring loaded level 640 and pull rod 641 depend on the spring 642 formed from third body layer 603. Both types of rods can be activated by a cam 650 that is driven by a shaft 652 (not illustrated). In operation, liquid flow is, for instance, in the direction indicated by arrow "A" and proceeds by first conduit 631 and second conduit 632. When valve ball 620 is seated against first body layer 601 , the valve is closed and flow is stopped. As the valve ball 620 is withdrawn, lower film 1 20 deforms in response to fluid pressure, into cavity 633 to form third conduit 633A (not shown) linking second conduit 632 with fourth conduit 634. Fourth conduit 634 connects with fifth conduit 635. Figure 8C illustrates the use of a cam 650 to activate a pull rod 641 that is spring loaded with pull rod spring 651 . All of the various pull rods 641 and pull rod springs 651 can be contained in a single base plate 604, such as that shown in Figure 8C, which can be attached to the instrument 900. The valve of Figure 8C also differs in employing a pinch foot 621 instead of a valve ball 620 and in seating the pinch foot 621 against portion 601 B instead of against the opening of second conduit 632. In the illustrated embodiments of Figures 8A-8C, the valves are normally in the closed position. The positioning of the valves can be programmed and activated by controller 960 (not shown) . To further ensure that fluid flow is blocked prior to attaching the cassette 600 to the base plate, temporary membranes or seals can be employed to maintain the various fluids in their chambers. These membranes could be broken by applying a light pressure. Alternatively, the fluids could be frozen prior during storage to attaching the parallel reaction device to the base plate.
Alternatively, second and third body layer 602 and 603, respectively, can be designed to be separable from first body layer 601 , which contains fluid exchange channels and fluid chambers. In this embodiment, prior to joining these separable parts, the valve locations are not strongly closed to fluid flow, although the lower film 1 20 can rest securely enough against portion 601 B to prevent inadvertent fluid flow. Where the valve includes a valve ball 620, a ball retention film 61 5 is usefully sealed to the upper side of second body layer 602 to assure that the value ball 620 does not fall out of the device. The advantage of separating these pieces is that the portions of the parallel reaction device containing mechanical elements can be re-used while the fluid-handling portion can be disposed of.
Figure 9B shows a closed electromagnetic valve 380 for use in controlling the flow of fluids in a cassette 300. Located in a portion 700 of instrument 900, the electromagnetic valve 380 has a spacer 730 that is pressed against a flexible upper film 1 1 0 by first spacer spring 731 and second spacer spring 732. The first and second spacer springs 731 and 732 or the spacer 730 are sufficiently magnetic or magnetically permeable that they can be drawn away from upper film 1 1 0 by activating electromagnetic coils 740. In Figure 9A, The electromagnetic valve 380 is shown in the open position with spacer 730 electromagnetically drawn away from valve seat 381 . Auxiliary Blocks
Figure 10 illustrates a part of instrument 900, reaction cell servicing device 400, having upper auxiliary block 400A for moving fluids into or out of a reaction chamber 1 60. Preferably, there will be a corresponding, upwardly oriented lower auxiliary block 400B located underneath reaction chamber 1 60. Upper auxiliary block 400A is honeycombed with upper conduit 430A. Upper conduit 430A has an upper inlet 431 A and an upper outlet 432A. First upper portion 401 A of upper auxiliary block 400A is fabricated of any suitably sturdy material, but is preferably constructed of the same material as third upper portion 403A. Second upper portion 402A is preferably fabricated of a heat-insulating material, such as, without limitation, nylon, polycarbonate and the like. Third and fourth upper portions 403A and 404A are preferably fabricated of a heat-conductive material, such as, without limitation, aluminum, copper, sintered beryllia, and the like. Upper portions 401 A-404A can be joined using, for instance, bolts, rivets, adhesives or snap- fitting pieces. Upper electrical heaters 440A are positioned adjacent to the reaction chamber 1 60.
The upper and lower heaters 440A and 440B are generally thin layers of conductive material that is separated from the heat-conductive upper and lower sections 402A and 402B of upper and lower auxiliary blocks 400A and 400B by a thin electrical insulation layer. Such an insulation layer is formed, for example, by direct deposition onto the substrate. For example, silicon nitride can be deposited from the gas phase or aluminum oxide can be deposited using a liquid carrier. The conducting layer forming upper and lower heaters 440A and 440B are, for example, deposited by vacuum evaporation (e.g., for a nichrome conducting layer) or by deposition from the vapor (e.g., for an indium tin oxide conducting layer) . Alternately, pre-formed heater sheets are cemented to the substrate, for instance using an epoxy cement or the adhesive recommended by the vendors. Appropriate heaters can be obtained from Elmwood Sensors Inc. (Pawtucket, Rl) or from Omega Engineering Inc. (Stamford, CT) . Typically, individual heater elements have planar dimensions appropriate, alone or in combination with electrically coupled heater elements, to match the size of the reaction chamber to be heated.
Fourth upper portion 404A constitutes an upper foot-pad 404A' for a foot-pad pump that operates to pump fluid out of a reaction chamber 1 60. In this context, where a foot-pad is associated with a heating and cooling device, it is preferably fabricated of a material with high thermal conductivity such as aluminum, copper, sintered beryllia, and the like. The operation of the foot-pad pump 460, which includes lower foot-pad 404B', is illustrated in Figures 1 1 A and 1 1 B. When the upper and lower foot-pads 404A' and 404B' are withdrawn away from reaction chamber 1 60, the reaction chamber 1 60 can be filled with fluid (see Figure 1 1 A) . When the upper and lower foot-pads 404A' and 404B' are brought towards each other (see Figure 1 1 B), fluid in reaction chamber 1 50 is pushed out either through second fluid exchange channel 1 42 or third fluid exchange channel 1 43. The embossing at location 1 61 (for the upper film 1 10B) or at location 1 62 (for lower film 1 20), allows the two films to be pushed together without substantial stretching. The embossing of upper film 1 1 0B and lower film 1 20 is done, for instance, by applying suitably shaped, heated dies.
Preferably, instrument 900 has a device for pumping and controlling reaction cell temperature, such as reaction cell servicing device 400, for each reaction chamber 1 60 in the cassette 1 00 or 200.
Figure 1 2 shows a schematic of the accessory support devices for the upper auxiliary block 400A of Figure 1 1 . Water is propelled through upper and lower conduits 430A and 430B, respectively, from pump and water cooler console 950. Pump and water cooler console 950 further includes fluid valves operating under the control of controller 960. Electrical current is supplied to upper and lower heaters 440A and 440B, respectively, by power supply 970, which is controlled by controller 960. Controller 960 receives input from upper and lower thermal sensors 450A and 450B, respectively.
In Figure 1 3, upper auxiliary block 500A includes a set of paired first and second upper thermoelectric blocks 51 1 A and 51 2A, respectively, while lower auxiliary block 500B has a set of paired first and second lower thermoelectric blocks 51 1 B and 51 2B, respectively. First upper and first lower thermoelectric blocks 51 1 A and 51 1 B, respectively, are made of p-type semiconductor material, while second upper and second lower thermoelectric blocks 51 2 A and 51 2B, respectively, are made of n-type semiconductor material. The thermoelectric blocks 51 1 and 51 2 are electrically connected in series by upper and lower connectors 51 3A and 51 3B as illustrated to form thermoelectric heat pumps. Such thermoelectric heat pumps are available for instance from Tellurex Corp. , Traverse City, Ml and Marlow Industries, Dallas, TX. Upper and lower gas inlet/outlets 510A and 51 OB are connected to upper and lower manifolds 520A and 520B, respectively, formed by the space between the upper and lower thermoelectric 5 blocks 501 A and 501 B. Upper and lower manifolds 520A and 520B (which are made up of the space between thermoelectric blocks) are connected, respectively, to an upper plurality of passageways 521 A or a lower plurality of passageways 521 B. The outer portions of upper and lower auxiliary blocks 500A and 500B are upper and lower heat sinks 504A and 504B, respectively,
10 which are preferably constructed of a heat-conductive material such as, without limitation, aluminum, copper, sintered beryllia, and the like. First upper air-tight collar 506A, second upper air-tight collar 507A, first lower air-tight collar 506B and second lower air-tight collar 507B help form upper and lower manifolds 520A and 520B. Upper and lower thermal sensors 570A
15 and 570B are connectable to a controller or a monitoring device by upper and lower leads 571 A and 571 B, respectively.
It will be recognized that upper end-plate 502A viewed from underneath or lower end-plate 502B viewed from above would have a series of holes which are the outlets of upper and lower passageways 521 A and
20 521 B. Another attribute of the auxiliary blocks 500A and 500B is that the thermoelectric blocks typically are arrayed in three dimensions rather than two.
Heating is achieved by applying voltage of the proper polarity to upper first and second leads 508A and 509A and to lower first and second
25 leads 508B and 509B. Cooling is achieved by reversing the polarity of the voltage. An important variable in the operation of these heating and cooling devices is temperature uniformity. To increase temperature uniformity, upper and lower first end-plates 502A and 502B are preferably constructed of a material of high thermal conductivity, such as sintered beryllia. Other suitable
30 materials include, without limitation, ceramics containing metallic aluminum. Preferably, the thermoconductivity of end-plates 502A and 502B is at least about 0.2 watt cm" 1 K~1 , more preferably at least about 2 watt-cm" 1-K"1. The upper and lower temperature sensors 570A and 570B can be, without limitation, thermocouples or resistive sensors. The upper and lower sensors 35 570A and 570B can, for example, be deposited on upper and lower first end-plates 502A and 502B as thin films or they can be in the form of thin wires embedded into holes in upper and lower first end-plates 502A and 502B.
Upper and lower auxiliary blocks 500A and 500B provide an alternate method of applying pressure to second upper film 1 1 0B and lower film 1 20 to push fluid out of reaction chamber 1 60. When gas pressure is applied through upper gas inlet/outlet 51 OA and corresponding lower gas inlet/outlet 51 OB (not shown) of lower auxiliary block 500B, the gas exiting upper and lower pressurized fluid channels 521 A and 521 B (not shown) forces upper and lower films 1 1 0 and 1 20 together, thereby forcing fluid from reaction chamber 1 60.
Upper or lower auxiliary block 500A or 500B can contain a plurality of upper or lower pressurized fluid channels 421 A or 421 B, respectively, which are used to operate a gas pressure flow control means. The fluid within these channels typically is a gas such as oxygen or nitrogen. Gas of higher than atmospheric pressure can be applied to the upper or lower pressurized fluid channels 421 A or 421 B from, for instance, a pressurized gas canister or a pump applied to upper or lower gas inlet/outlet 41 OA or 41 OB. A vacuum, usually a partial vacuum, can be applied to the upper or lower pressurized fluid channels 421 A or 421 B using, for instance, a vacuum pump. Numerous mechanisms for controlling the pressure of the pressurized fluid channels will be recognized by those of ordinary skill in the engineering arts. Figure 14 illustrate another upper auxiliary block 1 500A and lower auxiliary block 1 500B that use thermoelectric heat pumps but use a foot-pad pump instead of a gas-pressure mediated pumping device. Upper and lower foot-pads 1 505A and 1 505B are used to pump fluid out of reaction chamber 1 60. Thermoelectric blocks 1 51 3 are used to heat or cool as described above. Upper and lower heat sink thermal sensors 1 592A and 1 592B are located in upper heat sink 1 504A and lower heat sink 1 504B, respectively. Upper heat sink heater 1 590A and lower heat sink heater 1 590B (connected to electrical power via upper leads 1 591 A and lower leads 1 591 B, respectively) are used to transfer heat to the thermoelectric blocks 1 51 3, thereby allowing thermoelectric blocks 1 51 3 to operate at a higher temperature range. Upper and lower sensors 1 570A and 1 570B are used to monitor the temperature of the adjacent reaction chamber 1 60. The speed with which the temperature of the reaction chamber 1 60 is increased or decreased is important for optimizing some nucleic acid amplification assays. During the temperature cycling important for some nucleic acid amplification assays, it is important to operate at a relatively lower temperature where the nucleic acid sample is enzymatically reproduced and at a higher temperature where the nucleic acid sample is melted to separate the two strands of the nucleic acid. During the period when the assay apparatus cycles between the two preselected temperatures believed to be appropriate for a given nucleic acid amplification, various unwanted chemistries can be expected to occur. For instance, as the temperature increases from the lower temperature, the replication enzyme can be expected to continue to function, although not necessarily with the appropriate accuracy of replication achieved at the prescribed lower temperature. At the higher temperature set point, this unwanted enzymic activity is inhibited by the high temperature. Thus, it is important to rapidly change the reaction temperature between the two operating temperature plateaus. One mechanism by which the temperature can rapidly be changed in the reaction chamber is illustrated in Figure 1 0. Assume that the reaction chamber 1 60 is operating at lower plateau temperature "G". Under these conditions, cooling water does not flow through upper conduit 430A or corresponding lower conduit 430B (not shown) . The temperature is maintained by intermittently operating upper and lower heaters 440A and 440B when the temperature in reaction chamber 1 60 lowers beneath a temperature of G minus X (where X is a temperature differential) . At a pre-programmed time, the temperature is raised to higher plateau temperature "H" by activating upper and lower heaters 440A and 440B until a temperature is reached that will lead to a temperature stabilization at temperature H. Water flow through upper and lower conduits 430A and 430B can be activated to minimize temperature overshoots if needed. Temperature H is maintained by intermittently operating upper and lower heaters 440A and 440B when the temperature of the reaction chamber 1 60 lowers beneath a temperature of H minus Y (where Y is a temperature differential) . To cycle back to temperature G, the controller activates the pump 451 (not illustrated) of console 450 to cause cooling water to flow through upper and lower conduits 430A and 430B.
The performance of such a heater device and cooling device can be simulated using a heat transfer simulation computer program using a finite element approximation of the heat flow equation. The simulation is conducted with the following assumptions: the thickness of the reaction chamber 1 60 is 0.5 mm, the upper and lower films were 0.1 mm thick and the insulation between the heater and the auxiliary block was 0.025 mm thick. Such a simulation has determined that a jump from 25°C to 75°C can be achieved within 3.2 seconds, where, after 3.2 seconds, the temperature in the 5 reaction chamber is substantially uniform. The reciprocal cooling step can be achieved within about 3 seconds, resulting in a substantially uniform temperature in the reaction chamber. Preferably, after this cooling step the variation in temperature in the reaction chamber is no more than about 0.1 °C. Using the heating and cooling devices of the present
10 invention, including the device described in the immediately preceding paragraph, reaction chamber 1 60 temperatures between about -20°C and about 1 00°C can be maintained.
In one preferred embodiment, when the parallel reaction device includes more than one reaction flow-way, each such reaction flow-
15 way will include at least one reaction chamber 1 60 which will have at least one heating and cooling device made up of thermoelectric blocks 501 (such as the heating and cooling device described in the paragraph immediately above) capable of being aligned with a side of the reaction chamber. More preferably, each such reaction chamber 1 60 will have a heating and cooling device on
20 each of two opposing sides. In another preferred embodiment, the cross-sectional area of upper or lower first end-plate 502A or 502B substantially matches the largest cross-sectional area of the reaction chamber 1 60 to which it is intended to transfer heat.
The principles of temperature cycling for a reaction chamber
25 1 60 heated and cooled with upper and lower auxiliary blocks 500A and 500B or upper and lower blocks 1 500A and 1 500B are the same as those outlined above for the upper and lower auxiliary blocks 400A and 400B of Figure 1 0. In another embodiment, the reaction chamber 1 60 is heated and cooled by passing a heated or cooled fluid, preferably a gas, either directly
30 over one or more surfaces of the reaction chamber 1 60 or through a heat exchange apparatus that can be positioned adjacent to one or more surfaces of the reaction chamber 1 60. The apparatus illustrated in Figure 1 0 can be modified to operate pursuant to this embodiment by (a) removing (or not using) the upper and lower heaters 440A and 440B and (b) adding a heater for
35 heating the fluid. The parallel reaction device preferably has two fluid management systems, one for a hotter fluid and another for a cooler fluid, together with the valving required to inject the hotter or cooler fluid into the tubing leading to the reaction chamber 1 60 as appropriate for maintaining a given temperature in the reaction chamber. Particularly where the heating and cooling fluid is a gas, the temperature of the gas soon after it has passed by 5 the reaction chamber 1 60 will provide a useful indication of the temperature of the reaction chamber 1 60.
Where the auxiliary blocks act as foot-pads or for other foot¬ pads, mechanical or electromechanical methods of drawing the foot-pads towards or away from the fluid chamber on which it acts are well known and 10 include solenoids, pneumatically activated plungers, screw mechanisms and the like.
Auxiliary blocks and other features useful in conjunction with this invention are described in U.S. Patent Application No. < 1 1 772A > , filed October 31 , 1 996, titled "Assay System, " Docket No. DSRC 1 1 772A, which 15 is incorporated herein in its entirety by reference.
Miscellaneous Pumps
Pumping action can also be achieved using, for instance, peristaltic pumps, mechanisms whereby a roller pushes down on the flexible
20 film of a fluid chamber to reduce the volume of the chamber, plungers that press on the flexible film of a fluid chamber to reduce its volume, and other pumping schemes known to the art. Such mechanisms include micro- electromechanical devices such as reported by Shoji et al., "Fabrication of a Pump for Integrated Chemical Analyzing Systems, " Electronics and
25 Communications in Japan, Part 2, 70: 52-59, 1 989 or Esashi et al., "Normally closed microvalve and pump fabricated on a Silicon Wafer, " Sensors and Actuators, 20: 1 63-169, 1 989 or piezo-electric pumps such as described in Moroney et al., "Ultrasonically Induced Microtransport, " Proc. MEMS, 91 : 277-282, 1 991 .
30
Detection Devices
In a preferred embodiment, at least one reaction chamber 1 60 has a transparent retaining wall that is generally formed of upper film 1 1 0 or lower film 1 20 (or two retaining walls are transparent). Reaction chamber
35 1 60 can be a chamber where a reaction occurs, such as one of lysing reaction chambers 340 or reaction chambers 380 (see Figure 3), it can be a supply chamber containing samples, controls or reagents, such as supply chambers 350, 360 and 390, or it can be a storage chamber, such as one of storage chambers 399A-E. The parallel reaction device in this embodiment preferably includes a light source capable of directing light to the transparent upper or lower film 1 10 or 1 20 and a detection device for detecting (a) the light reflected from an illuminated reaction chamber 1 60, (b) the light transmitted through an illuminated chamber 1 60, or (c) the light emissions emanating from an excited molecule in a chamber 1 60. A membrane is "transparent" if it is 80% transparent at a wavelength useful for detecting biological molecules. The detection device can incorporate optical fibers, optical lenses, optical filters or other optical elements. Alternatively, where detection uses fluorescence, detection and quantitation can be done by photographing the detection channel 295 under appropriate excitation light. With fiber optics or other suitable optical devices, the size of the detection system that is adjacent to the parallel reaction device is minimized. This size minimization facilitates incorporating the detection system together with a temperature control device (described more fully below) into the parallel reaction device. A particularly preferred light source is a solid state laser. The size of these light sources also facilitates incorporating a number of auxiliary components about the parallel reaction device. When a nucleic acid amplification is conducted in an parallel reaction device that incorporates current technology solid state lasers, the method used to detect amplified nucleic acid uses a dye that absorbs light at a wavelength higher than about 600 nm to indicate the presence of amplified nucleic acid, as described below. Examples of such dyes include Cy5™, one of a series of proprietary cyanine class dyes. Cy5™, and the related dyes, are products of Biological Systems, Inc. (Pittsburgh, PA) . This particular dye is relatively small, absorbs at about 650 nm and emits a fluorescent signal at about 667 nm. Other, larger suitable dyes include structures derived from seaweed such as allophycocyanin and allophycocyanin-conjugated reagents (Sigma Chemical Co., St. Louis, MO). These dyes absorb in the 630-750 nm range. The relatively long wavelengths of the excitation light described above avoid much of the background fluorescence associated with biological materials, plastics or other possible components of the cassette 100. A preferred solid state laser source is a Laser Max, Inc. (Rochester, NY) Model LAS 200-635.5, which emits a light with a wavelength at a maximum of 4 . Other colorimetric detection methods, for instance those utilizing biotin-avidin binding to associate horse radish peroxidase with a hybridized pair of polynucleotide sequences, can be used.
Signals from the detection device typically will be input into a controller 960, where they can be used to determine the presence, or absence, of material assayed for and the magnitude of the signal indicating the presence of the material. From these data, the amount of assay material can be calculated and the quality of the assay as indicated by the controls can be quantitated. This information is then stored for the assay report listing.
In a preferred embodiment, the cassette has one or more detection channels 295. One such detection channel 295 is illustrated in Figures 1 5A and 1 5B. It is made up of a number of fibers 297, which together preferably transmit at least about 50% of light of a wavelength useful in the detection procedure, confined to the detection channel 295. The fibers 297 can be bound in place for instance by cementing or crimping. The fibers 297 can be fabricated of glass or suitably transparent plastics. The fibers 297 are preferably between about 5 μm and about 50 μm in diameter, more preferably about 20 μm. The detection channel typically has a width and depth of no more than about 3,000 μm, preferably between about 200 μm and about 1 ,000 μm. Microchannels between the fibers 297 allow liquid to flow through the detection channel 295. A detection-mediating molecule is bound to the fibers 297. Preferably the detection-mediating molecule is an oligonucleotide that hybridizes with the nucleic acid to be amplified in a nucleic acid amplification reaction and the nucleic acid amplification reaction utilizes primers having a detectable moiety. The detection-mediating molecules are bound to the fibers 297 by known methods. Preferably, discrete bands on the fibers such as first band 296A, second band 296B and third band 296C have separate detection-mediating molecules, which could be, for instance, designed to detect two separate species to be amplified in a nucleic acid amplification reaction and to provide a control for non-specific hybridizations. To manufacture the banding pattern of bound molecules, oligonucleotide synthesis procedures that utilize photo-cleavable protecting groups and masks to protect certain bands 296 from photocleavage can be used. Such synthesis procedures are described in U.S. Patent No. 5,424, 1 86 (Fodor et al.). The instrument 900 is preferably designed to provide heat control at the detection channels 295 for conducting hybridization reactions. In a preferred embodiment, the sides 298 of the detection channel 295 are coated with a reflective coating so that light incident from above will reflect and twice pass through the detection channel 295. Such a reflective coating is provided by metalizing, for instance using a sputtering or evaporation process. Alternatively, the detection channels 295 contain membranes
299 (not shown), such as a nylon membrane, to which a hybridization probe has been bound. If two or more hybridization probes are used, they are each bound to a specific region of the membranes 299 using "dot blot" procedures such as are described in Bugawan et al., "A Method for Typing Polymorphism at the HLA-A Locus Using PCR Amplification and Immobilized Oligonucleotide Probes" Tissue Antigens 44: 1 37-147, 1 994 and Kawasaki et al., "Genetic Analysis Using Polymerase Chain Reaction-Amplified DNA and Immobilized Oligonucleotide Probes: Reverse Dot-Blot Typing" , Methods in Enzymology 21 8: 369-381 , 1 993. As described above, the amplification product hybridized with the bound probe or probes has attached -via the amplification primers - a detectable moiety.
Note that for cavities in the cassette 200 intended for use in detection, such as detection channels 295, in a preferred embodiment of the invention the upper film 1 1 0 over the cavity is replaced with a cover 1 1 0' selected for its optical properties, such as, without limitation, a cover 1 1 0' made of optical quartz. Because pumping is effected elsewhere in the cassette, the cover 1 1 0' does not have to be flexible like an upper film 1 1 0. While in a preferred embodiment detection is done in situ in the cassette, in other embodiments the products of chemical reactions effected in the cassette are removed and detection methods or chemistries are done elsewhere, including in a different cassette.
Paramagnetic Beads and High Field Gradient Magnet
Paramagnetic beads useful for facilitating chemical processes conducted in a cassette 1 00 are available from several sources including Bang Laboratories (Carmel, IN) for beads lacking conjugated biomolecules, Dynal (Lake Success, NY) for beads conjugated to various antibodies (for instance, antibodies that bind to the CD2 cell-surface receptor) and CPG (Lincoln Park, NJ) for beads with a glass matrix and a variety of surface bonded organics. For applications where it is anticipated that the beads will be washed into and out of reaction chambers, each bead will preferably have a diameter of less than about 1 mil, more preferably, less than about 0.5 mil, which diameter facilitates entry and exit through the channels by which material is inserted or evacuated from the reaction chamber 1 60. For applications where the beads are anticipated to remain in the reaction chamber 1 60, in one embodiment of the invention, the diameter is preferably sufficiently large to preclude their entry into these channels. The entrances to such channels within a reaction chamber 1 60 are preferably positioned or designed so as to minimize the chance that a channel will be blocked by a bead that settles over the channel's entryway. In a preferred embodiment, the beads are locked in place using magnetic fields. To generate sufficient movement among the beads, it has been determined that the magnet used should preferably generate a sufficient magnetic field gradient within a reaction chamber 1 60. Such magnets can be constructed by forming sharp edges on highly magnetic permanent magnets, such as those formed of rare earths, such as the neodymium-iron-boron class of permanent magnets. Such a permanent magnet is available from, for example, Edmund Scientific (Barrington, NJ). Sharp edges of dimensions suitable for a particular reaction chamber 1 60 are, for example, formed by abrasive grinding of the magnetic material. An example of such a shaped magnet 1 1 00 is shown in Figure 1 6, where the magnet has a roof-shape at one of the poles. The illustration shows a preferred embodiment where there are two roof shapes and illustrates that the magnet can be brought adjacent to or can be removed from a cassette such as cassette 1 00 or cassette 200. In the illustration, lower auxiliary block 1 600B has slots (not visible) that allow the magnet 1 1 00 to be placed adjacent to cassette 1 00 or 200. This magnet suitably has dimensions such that the length of the peak of the roof-shape matches the cross-sectional size of a reaction chamber 1 60. To maximize the field gradient acting on the paramagnetic beads, the peak 1 101 of the magnet 1 1 00 is placed adjacent to the reaction chamber or other structure in which the beads are located. The paramagnetic beads are held in place by leaving the peak 1 1 01 adjacent to the beads. By moving the magnet with its peak 1 1 01 adjacent to the beads, the beads are impelled to move with the magnet. Another way in which high magnetic field gradients can be achieved is to make uniform slices of a magnetic material and use an adhesive to join the slices in alternating N to S orientations. Such alternating slice magnets have high magnetic field gradients at the junctions of the slices. The sharp-edged magnets described above are effective in adhering the paramagnetic beads in one place and in moving beads located, for instance, in a fluid exchange channel or in a reaction chamber, from one location to another. Such magnets thus can help retain the paramagnetic beads in one place, for instance when a fluid in a reaction chamber 1 60 is being removed from that chamber but it is desirable to leave the beads in the chamber. Magnets with locations having high magnetic field gradients that are particularly suitable for use in this context are described in U.S. Provisional Patent Application No. 60/006,202, filed November 3, 1 995, titled "Magnet, " Docket No. DSRC 1 1 904P, which is incorporated herein in its entirety by reference.
Various cell binding beads (e.g., beads having bound antibodies specific for a certain subset of cells) can be used to adhere selected cells from a population of cells. The beads can be locked in place, for instance magnetically if the beads are paramagnetic, while non-adherent cells and fluids are washed away. Thus, cell-binding beads can be used to concentrate small sub-populations of cells.
In synthetic chemistry applications, the beads suitably have attachment sites for coupling the building blocks of chemicals or polymers.
Septum Manufacture
A septum 1 31 can be fixed in place in inlet 1 30 using heated die 1 200, as illustrated in Figures 1 7A and 1 7B. The die 1 200 is heated sufficiently so that the angled, sharp edges 1 201 cut into body 1 05 and move melted material 1 32 such that it locks the septum 1 31 in place.
Controller
The controller 960 typically will be a microprocessor. However, it can also be a simpler device comprised of timers, switches, solenoids and the like. The important feature of controller 460 is that it directs the activation of the means for impelling a fluid, the valves and the heating and cooling device, according to a pre-set or programmable schedule that results in the operation of an assay protocol, such as the protocol outlined below. Preferably, the controller 460 receives input indicating the temperature of the reaction chambers of the parallel reaction device and is capable of adjusting its control signals in response to this input. PCR Procedures Using the Assay System of the Invention
Often an important variable in PCR reactions is the amount of interfering cellular debris, including membrane fragments and cellular chemicals such as enzymes, fats and non-target nucleic acid, present in the sample to be assayed. Ideally, only highly purified nucleic acid is used as the sample subjected to a PCR amplification. However, such purification is not practical with the small amounts of tissue or fluid available for a diagnostic assay. Further, given the sensitivity of the assay to contamination by environmental sources of nucleic acid, a nucleic acid purification step can increase the likelihood of getting a false positive result. In some areas of diagnostic or forensic PCR this concern about interference by cellular debris has been eased somewhat by improvements in the characterization of PCR reaction conditions, such that often much cruder nucleic acid samples can be used without adverse effect. See Rolfs et al., PCR: Clinical Diagnostics and Research, Springer Lab, 1 992 (particularly Chapter 4 et seq.). See, also, the literature available with such commercial products as GeneReleaser (BioVentures, Inc., Murphreesboro, TN), Pall Leukosorb™ media (Pall, East Hills, NY) and Dynbeads® DNA Direct™ (Dynal, Lake Success, NY). (On PCR procedures, see generally, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York and PCR: A Practical Approach, IRL Press, 1 991 .) Nonetheless, it is desirable to have the capability of at least removing the cellular debris associated with the cell membranes of the cells that may be present in the sample. Such a technique for use in conjunction with the parallel reaction device of the invention is described below. Such a cleanup step can be applied when needed to achieve the needed level of sensitivity or accuracy, or omitted if not needed.
It is preferable to conduct parallel control PCR reactions when conducting PCR. One control omits sample from the reaction or uses a sample previously characterized as negative. Another control introduces a known amount of a purified nucleic acid that is known to contain the sequence or sequences that the PCR reaction is designed to amplify. These types of controls can be accomplished on multiple parallel reaction devices or, more preferably, in separate reaction flow-ways on the same parallel reaction device whereby each reagent is distributed from a single source to all of the reaction flow-ways. Another control technique used in PCR is to design the PCR reaction so that it will amplify multiple nucleic acid segments, each of which can be indicative of a disease or a genetic circumstance or marker. The different segments can be amplified in multiple reactions or in the same reaction chamber. If amplified in the same chamber, that binding competition between the various primers can necessitate extending the time, in each amplification cycle, spent at the replication temperature.
One method for removing cellular debris from a sample involves binding the cells in the sample to a bead that has attached thereto an antibody specific for a cell surface molecule found on the cells. Beads that bind to the CD2 white blood cells or to E. coli bacteria (such as the 01 57E strain) are available from Dynal (Lake Success, NY). An ever-growing family of cell-surface molecules found on mammalian cells, bacterial cells, viruses and parasites has been characterized and antibodies against the majority of these molecules have been developed. See, e.g. , Adhesion Molecules, CD. Wegner, ed., Academic Press, New York, 1 994. Many of these antibodies are available for use in fabricating other types of cell-affinity beads (for instance, from Sigma Chemical Co., St. Louis, MO). The cells can be adhered to the antibodies on the beads and lysed to release their nucleic acid content. The lysis fluid together with the released nucleic acid can be moved to a separate compartment for further processing, leaving behind the beads and their adherent cellular debris.
The lysis fluid used to release nucleic acid from the sample cells can also interfere with the PCR reaction. Thus, in some protocols it is important to bind the nucleic acid to a substrate so that the lysis fluid can be washed away. One such support is provided by beads that bind to DNA, such as glass beads that bind to DNA by ionic or other interactions such as Van der Waals interactions and hydrophobic interactions. Suitable beads, with surfaces chemically treated to maximize the number of interaction sites, are available from, for example, BioRad (Hercules, CA) . Paramagnetic beads with a number of DNA binding surfaces, such as nitrocellulose or nylon-coated surfaces, can be useful in operating the invention. In some embodiments, it is desirable for the beads to be paramagnetic so that they can be manipulated using magnetic forces. Paramagnetic glass beads are manufactured by CPG (Lincoln Park, NJ) . Once the nucleic acid is bound to the beads, the lysis fluid can be washed from the beads. The nucleic acid can be amplified with the beads present.
The lysis fluid used to release nucleic acid from the cells in a sample typically includes a detergent, preferably nonionic, and a buffer, usually the buffer used in the PCR amplification reaction. The pH of the lysis fluid is preferably from about pH 7.8 (for protease K-containing lysis fluids, for example) to about pH 8.0 (for phenol-mediated lysis, for example), typically about pH 8.0. Suitable detergents include, without limitation, Sarkosyl and
Nonidet P-40. Other components can includes salts, including MgCI2, chelators and proteases such as proteinase K. Proteinase K can be inactivated by heating, for instance, to about 1 00°C for about 1 0 minutes. Depending on the composition of the lysis buffer, it can be more or less important to wash the lysis buffer away from the nucleic acid prior to conducting the amplification assay.
The amplification buffer used to support the amplification reaction will typically include the four deoxynucleotide triphosphates (NTPs) (e.g., at a concentration of from about 0.2 mM each), a buffer (e.g., Tris-HCl, about 10 mM), potassium chloride (e.g., about 50 mM) and magnesium chloride (e.g., about 1 to 1 0 mM, usually optimized for a given PCR assay scheme). The pH is preferably from about pH 8.0 to about pH 9.0, typically about pH 8.3. Other components such as gelatin (e.g., about 0.01 % w/v) can be added. The individual primers are typically present in the reaction at a concentration of about 0.5 μM. The amount of sample nucleic acid needed varies with the type of nucleic acid and the number of target nucleic acid segments in the nucleic acid sample. For genomic DNA, where each cell in the sample has about 2 copies of target nucleic acid, a concentration of about 1 0 μg/ml is desirable.
For simplicity, the polymerase used in the procedure is a heat-resistant DNA polymerase such as Taq polymerase, recombinate Taq polymerase, Tfl DNA polymerase (Promega Corp., Madison, Wl), or 77/ DNA polymerase (Promega Corp., Madison, Wl) . Heat stability allows the PCR reaction to proceed from cycle to cycle without the need for adding additional polymerase during the course of the reaction process to replace polymerase that is irreversibly denatured when the reaction vessel is brought to a DNA strand separation temperature. Preferably, the DNA polymerase used has the increased accuracy associated with the presence of a proofreading, 3' to 5' exonuclease activity, such as the proofreading activity of the TH DNA polymerase.
Blood provides one of the more convenient samples for diagnostic or genetic PCR testing. For most genetic testing, from about 1 0 to about 50 μl of blood is sufficient to provide enough sample DNA to allow for PCR amplification of specific target segments. For fetal cell analysis, however, as much as about 20 mis, which may contain as few as about 400 fetal cells, can be required. Such large sample volumes require concentration, for instance, using the methods described above. For testing for microbial diseases, the concentration of target nucleic acid in the sample can be quite low (e.g., no more than about 2-5 fg per bacterial genome). Thus, when using the parallel reaction device to test for such microbes, concentration methods may again be required.
To specifically amplify RNA, it is necessary to first synthesize cDNA strands from the RNA in the sample using a reverse transcriptase (such as AMV reverse transcriptase available from Promega Corp., Madison, Wl). Methods for conducting a PCR reaction from an RNA sample are described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York and PCR: A Practical Approach, IRL Press, 1 991 . To prepare RNA for this purpose, a facile procedure uses a lysis buffer containing detergent (such as 0.5% Nonidet P-40), buffer (e.g., pH 8.3) and suitable salts that has been, immediately prior to use, mixed 1 : 1 000 with a 1 : 1 0 diethylpyrocarbonate solution in ethanol. After sample cells have been lysed with this solution, a supernate containing RNA is separated away from a pellet of nuclei by centrifugation. Primer, which is generally the same as one of the primers used in the subsequent PCR cycling reaction, is annealed to the RNA by heating (e.g., to about 65°C) and subsequently reducing the temperature to, generally, about 37°C. The reverse transcriptase, nucleotide triphosphates and suitable buffer (if not already present) are then added to initiate cDNA synthesis. Generally, a small volume (e.g., about 1 .0 to about 2.0 μl) of material from the cDNA synthesis is added to a solution (e.g. , about 50 to about 100 μl) containing the buffer, DNA polymerase, nucleotide triphosphates and primers needed for the PCR amplification. The temperature cycling program is then initiated. Hybridization Procedures
The advantages of the parallel reaction device as it relates to conducting PCR reactions also substantially apply to conducting hybridization procedures. The ability of the valves of the parallel reaction device to accommodate elevated temperatures allows the system to be used in hybridization protocols. While hybridization reactions are not as sensitive to contamination as PCR reactions, these reactions are nonetheless very sensitive to contamination, the risk of which is substantially reduced with the disposable system of the invention. Procedures for conducting hybridizations are well known in the art. See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Press, 1 989. In these procedures, a nucleic acid such as (a) a sample source of nucleic acid containing a target sequence, or (b) a probe nucleic acid is bound to a solid support and, after this binding, the remaining binding sites on the support are inactivated. Then, the other species of nucleic acid, which has bound to it a detectable reporter molecule, is added under appropriate hybridization conditions. After washing, the amount of reporter molecule bound to (i.e. hybridized with) the nucleic acid on the solid support is measured.
For instance, a hybridization can be conducted in a reaction chamber in the parallel reaction device, where the reaction chamber contains a nitrocellulose membrane (or another membrane that binds nucleic acid) to which RNA has been bound (for instance, by electrophoretic or capillary blotting from a separation gel, followed by baking). A Northern prehybridization solution can then be introduced into the reaction chamber from one of the fluid chambers. (The recipes for Northern prehybridization solution (p. A1 -40), Northern hybridization solution (p. A1 -39), SSC (p. A1 -53, 20X recipe) and Denhart's solution (p. A1 - 14, 100X recipe) of Ausubel et al., Short Protocols in Molecular Biology, 2nd Edition, John Wiley & Sons, 1992 are incorporated herein by reference to more fully exemplify the hybridization methods that can be conducted in the parallel reaction device; note that the salmon sperm DNA recited in two of these recipes, which DNA serves as a competitor to reduce nonspecific hybridizations, is typically sheared prior to use.) The membrane and prehybridization solution are incubated overnight at a temperature between about 37°C and about 42°C, depending on the melting temperature for the interaction between target sequence and the probe sequence. Note that these incubation temperatures are in the range that is generally appropriate given the presence of 50% formamide in the prehybridization and hybridization solutions; for hybridizations conducted without formamide, incubation temperatures are typically higher, such as about 55°C to about 70°C. The membrane is then exposed to Northern hybridization solution containing melted probe and incubated overnight at the same temperature used in the prehybridization. Following hybridization, the hybridization solution is pushed out of the reaction chamber, the reaction chamber is brought to about 25°C and a first wash solution ( 1 X SSC, 0.1 % w/v sodium dodecyl sulfate) is introduced. After 1 5 minutes, the wash is repeated. After an additional 1 5 minute wash, a third and final wash is conducted using 0.25X SSC, 0.1 % w/v sodium dodecyl sulfate. The above outlined hybridization method is exemplary only.
Numerous other hybridization methods can be conducted in the assay system, including those described in the following sections of Ausubel et al., Short Protocols in Molecular Biology, which are incorporated herein by reference: Unit 2.9, pp. 2-24 to 2-30 and the recipes of Appendix 1 referred to therein; Unit 6.3, pp. 6-6 to 6-7 and the recipes of Appendix 1 referred to therein; and Unit 1 3.1 2, p. 1 3-44 and the recipes of Appendix 1 referred to therein. Using the parallel reaction device of the invention, the elevated temperatures required for hybridization reactions can be handled in an automated apparatus. For instance, hybridizations can be conducted at a temperature defined by the melting temperature Tm. Tm values for any hybridization probe can be calculated using commercially available software such as Oligo TM v4.0 from National Biosciences, Inc. , Plymouth, MN.
Immunological Procedures Using the System of the Invention In immunoassay procedures, the antibody-antigen binding reactions are generally conducted at room temperature or at a reduced temperature, such as about 4°C. After the binding reactions, positive results are generally indicated by an enzymic reaction, typically mediated by the enzyme alkaline phosphatase, which enzyme reaction is generally conducted at a temperature between about 20°C and about 40°C. The parallel reaction device of the invention allows these assays to be automated in a system that allows fast and reliable temperature regulation in the temperature range between about 0°C and about 40°C.
Typically, modern antibody-based screening procedures use a solid support to which an "antigen" (which is a substance that when injected into an animal, often in the presence of "adjuvants" known to enhance antibody production, can cause the animal to manufacture antibodies specific for the antigen) or an antibody has been attached. Alternatively, the antigen is found on the surface of a cell, such as a bacteria or eukaryotic cell, and the cell can function as a solid support.
In one assay (indirect ELISA), the antigen is bound to the support and a sample which may contain a first antibody specific for the antigen and produced by a first animal species is incubated with the bound antigen. After appropriate washing steps, a second antibody from a second animal species, which antibody is specific for antibodies of the first species and is attached to a detectable moiety (such as alkaline phosphatase), is incubated with the support. If the sample contained the first antibody, the second antibody will bind and be detectable using the detectable moiety. For instance, if the detectable moiety is alkaline phosphatase, detection can be conducted by adding a chemical, such as p-nitrophenyl phosphate, that develops a detectable characteristic (such as color or light emission) in the presence of a developing reagent such as a phosphatase enzyme. This assay can, for instance, be used to test blood for the presence of antibodies to the AIDS virus. In another assay (direct competitive ELISA) that uses a support with bound antigen, a sample which may contain an antigen is incubated with the support together with a limiting amount of an antibody specific for the antigen, which antibody has an attached detectable moiety. Due to competition between the solution phase antigen and the support-bound antigen, the amount of antigen in the sample correlates with reduced amounts of antibody that bind to the support-bound antigen and a weaker signal produced by the detectable moiety.
Another assay (antibody-sandwich ELISA) uses a first antibody specific for an antigen, which antibody is bound to the support. A sample which may contain the antigen is then incubated with the support. Following this, a second antibody that binds to a second part of the antigen. and which has an attached detectable moiety, is incubated with the support. If the sample contained the antigen, the antigen will bind the support and then bind to the detectable second antibody. This is the basis for the home pregnancy test, where the antigen is the pregnancy-associated hormone chorionic gonadotropin.
In another assay (double antibody-sandwich ELISA) that uses a support with bound antibody, a sample which may contain a first antibody from a first species is incubated with a support that has bound to it a second antibody from a second species that is specific for antibodies of the first species. The antigen for the first antibody is then incubated with the support. Finally, a third antibody specific for a portion of the antigen not bound by the first antibody is incubated with the support. The third antibody has an attached detectable moiety. If the sample contained the first antibody, the detectable third antibody will bind to the support. These and other immunoassays are described in Units 1 1 . 1 and 1 1 .2 of Ausubel et al., Short Protocols in Molecular Biology (pp. 1 1 -1 to 1 1 -1 7), which text and the recipes of Appendix 1 cited therein, are incorporated herein by reference.
The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope.
Example 1 - Cassette Fabrication
The following example illustrates fabrication methods used in constructing cassettes for a microfluidics device of the present invention. Various cassettes have been fabricated containing components that are shown in Figure 1 . Cassette bodies have been made from high-density polyethylene, both by machining and by molding. The methods of fabrication and demonstrated performance include the following: Membrane embossing: The membrane covering the cassette body and forming the reaction chambers was embossed prior to sealing to the body. The membrane was stretched on a frame and embossed between positive and negative hot dies. For membranes of polyester/ polyethylene laminate, the dies were heated to a temperature of above 1 40°C. Since this is above the melting point of the polyethylene, the die in contact with the polyethylene was made of polytetrafluoroethylene, to prevent adhesion. A preferable material for embossing is a fluoropolymer/ polyethylene laminate which can be given a more permanent deformation at a lower temperature and which has a lower water permeability.
Heat sealing: The membrane was sealed to the cassette body using a hot aluminum die with raised lands corresponding to the heat seal areas. A pressure, corresponding to approximately 1 50 to 300 psi over the actual seal area, was applied for 1 to 2 seconds. Following application of the pressure, the die was either rapidly quenched by water channels running through the die block, or the die was lifted. Superior results were obtained by quenching the die. With a 2 mil thick membrane of a polyester/low-density polyethylene laminate, sealed to a body of high-density polyethylene, a die temperature of 1 56°C was used. A blister 0.5 " in diameter sealed in this manner withstood internal pressures in excess of 50 psi.
To preserve uniformity of seal over a cassette of extended size, the cassette regions at the seal were formed into a raised ridge, about 0.01 " high. Variation in the amount the die deforms the base material, originating from small variations in cassette thickness, can then occur with a minimum variation in the volume of base material displaced. This ridge structure was found to reduce the extruded material in regions such as the well surrounding a valve. Bursapak™ structure: The outer seal of the Bursapak™ was made as described above. The center seal was made using a die heated to temperature of about 1 56°C. This die contained small independently sprung steel pins which contacted the center seal. The lower conductivity of the steel and the air gap between the pins and the die were designed to restrict the amount of heat available for sealing. When the seal was formed at the center in this way, melting of the cassette base material was minimal, although the low-density polyethylene of the membrane was above its melting point. This seal was demonstrated to withstand an internal excess pressure of about 1 6 psi. Above this pressure, it ruptured as required by the design and released the contents of the Bursapak™ through the central port.
Liquid fill: Liquid fill of both Bursapaks and storage vessels similar to the "waste vessel" of Figure 1 was achieved. The input needle was connected to a 2-way valve which could be switched between a vacuum pump and a syringe supplying the fill liquid. Following exhaustion of the vessel by the pump, for a few seconds, the valve was switched and the vessel filled by the syringe. The filled vessel then contained no air bubbles. Both a septum, as shown in Figure 1 , and a simple entry port were used for filling. Sealing of the entry channel was achieved by a hot rod, as indicated in Figure 2, which melted the channel closed but kept the polyester component of the membrane sufficiently intact. Valve operation: Valves, constructed as in Figures 1 , 5 and
6, were fabricated according to the above descriptions. A molded polyethylene body and polyester/polyethylene membrane was used. Functioning was tested using pneumatically operated steel plungers. With a plunger force of approximately 0.8 lb. and a water pressure of 20 psi, the leakage rate was less than 0.1 microliter per minute.
Example 2 - PCR Amplification Reaction
The following example illustrates one embodiment of the present invention whereby a PCR amplification reaction is conducted in the context of a cassette in a microfluidics device.
A PCR assay is conducted using the cassette 200 illustrated in Figures 4A-4E, the device having alpha through delta first reaction chambers 262A-D, which are used for lysing the cells in the samples, and alpha through delta second reaction chambers 262A-D, with each first reaction chamber 261 - second reaction chamber 262 pair forming a separate reaction flow-way 265. The parallel reaction device has a set of one upper auxiliary block, e.g. 1 500A and one lower auxiliary block, e.g. 1 500B (not shown), for each of first reaction chamber 261 and each second reaction chamber 262. The cassette 200 has pumps for moving fluid from one chamber to another chamber. For instance, the gas pressure flow control means or the foot-pad pumps described above can be used to empty chambers and push the fluid therefrom into another chamber. Valves located between the various chambers contained in the device regulate this flow of fluids between and among the chambers. The reaction protocol is as follows: 1 . Each of the four first reaction chambers 261 receives from a connected first supply chamber 251 a suspension in 1 60 μl of paramagnetic DNA-binding beads having a diameter of 2-4 mils, that can be used in the cell lysis stage to bind the DNA released from the lysed cells (these beads are, e.g., Dynabeads® DNA Direct™, available from Dynal, Lake Success, NY). The beads are locked in place in the lysing reaction chambers 261 using the magnet 1 1 00 and suspending liquid is drained into first waste chamber 271 . Alpha first reaction chamber 261 A receives a fluid (40 μl) from alpha fifth supply chamber 255A containing purified DNA that includes the amplification sequence being tested for in an amount sufficient to generate a positive result, thereby creating a positive control. Beta first reaction chamber 5 261 B receives from beta fifth supply chamber 255B buffer solution or a biological sample known to not contain the target sequence (40 μl) in place of the sample or positive control, and therefore serves as a negative control. Blood sample (40 μl), stored in sixth supply chamber 256, is drawn into each of gamma and delta first supply chambers 261 C and 261 D. The first reaction
10 chambers 261 are then filled with a lysis solution ( 1 00 μl) that is drawn from alpha, gamma, epsilon and eta third supply chambers 253A, C, E and F, respectively. The lysis solution is a solution of amplification buffer supplemented with 1 .0% v/w Tween 20 (Sigma Chemical Co., St. Louis, MO). (The lysis solution can be substituted with the solution provided by Dynal.)
15 The temperature of first reaction chambers 261 is now maintained at 56°C.
2. After 10 minutes, the lysis solution is emptied into first waste chamber 271 . The lysis solution which exits from alpha and beta first reaction chambers 261 A and 261 B, respectively, contains the cellular and serum residue of the blood sample. The DNA-binding beads, to which the
20 cellular DNA is bound, remain in first reaction chambers 261 .
3. Wash solution ( 100 μl) composed of amplification buffer (40 mM NaCl, 20 mM Tris-HCl, pH 8.3, 5 mM MgSO4, 0.01 % w/v gelatin, 0.1 % v/v Triton X-1 00, Sigma Chemical Co., St. Louis, MO) is now introduced into first reaction chambers 261 from the connected second supply 25 chambers 252.
4. After 10 minutes, the wash solution is transferred out of first reaction chambers 261 into first waste chamber 271 .
5. Steps 3 and 4 are repeated.
6. Solutions (volume 30 μl) containing appropriate primers 30 for amplifying the target sequence (0.5μM) are then drawn into first reaction chambers 261 from the connected beta, delta, zeta and theta third supply chambers 253B, 253D, 253F and 253H. Solutions (volume 30 μl) containing the needed nucleotide triphosphates (0.2 mM each), are introduced from the connected alpha, gamma, epsilon and eta fourth supply chambers 254A, 35 254C, 254E and 254F. Solutions (volume 30 μl) containing Taq polymerase (2 Units, available from Promega Corp , Madison, Wl) are introduced from the connected beta, delta, zeta and theta fourth supply chambers 254B, 254D, 254F and 254H. The contents of each of first reaction chambers 261 are then transferred to the corresponding one of alpha through delta second reaction chambers 262A-D.
7. The controller then initiates a temperature program modelled on the protocol described by Wu et al., Proc. Natl. Acad. Sci. USA 86: 2752-2760, 1 989 The program first heats second reaction chambers 262 to a temperature of 55°C and maintains that temperature for 2 minutes Next, the controller cycles the temperature between a replication temperature of 72°C (maintained for 3 minutes) and a DNA strand separation temperature of 94°C (maintained for 1 minute) After the replication temperature incubation has been conducted 25 times, the material in reaction chambers 262 is analyzed for the presence of the proper amplified sequence
While this invention has been described with an emphasis upon a preferred embodiment, it will be obvious to those of ordinary skill in the art that variations in the preferred composition and method may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims

Claims

WHAT IS CLAIMED:
1 . A device for conducting parallel reactions, comprising:
(a) a cassette formed of a body having an upper surface, a lower surface, and an edge, and including an upper film or a lower film attached to the upper or lower surface, respectively, wherein the upper or lower film is formed of a flexible material;
(b) two or more reaction flow-ways in the cassette, wherein each reaction flow-way comprises two or more fluid chambers which comprise a first supply chamber and a first reaction chamber having an upper wall and a lower wall, and wherein the fluid chambers are serially connected by first fluid exchange channels;
(c) a valve for controlling the flow of fluid through a first fluid exchange channel;
(d) a pump for moving fluids into or out of the fluid chambers; and
(e) a first inlet port on the cassette connected to a first supply chamber in each reaction flow-way by a second fluid exchange channel.
2. The device of claim 1 , wherein the body comprises recesses in its upper or lower surface which, together with an associated upper or lower film, form the first and second fluid exchange channels, further comprising:
(f) at least one hole situated in the body so as to connect a first or second fluid exchange channel formed at the upper or lower surface of the body with a first or second fluid exchange channel formed at the other surface.
3. The device of claim 1 , further comprising: (g) one or more second supply chambers, wherein two or more fourth fluid exchange channels connect the second supply chamber to two or more reaction flow-ways, which fourth fluid exchange channels include two or more said valves so that fluid from the second supply chamber can be directed to any one of the connected reaction flow-ways to the exclusion of the other connected reaction flow-ways; and
(h) one or more second inlet ports on the cassette each connected to one of the second supply chambers by a separate third fluid exchange channel.
4. The device of claim 5, further comprising: (i) a metering chamber interposed between the second supply chamber and the connected reaction flow-way.
5. The device of claim 1 , wherein the upper and lower walls of each first reaction chamber are formed of an embossed portion of a said upper film and an embossed portion of a said lower film, wherein the embossing allows upper and lower walls of the first reaction chambers to be brought together to minimize the volume of the first reaction chambers.
6. The device of claim 1 , further comprising: (j) one or more waste chambers; and
(k) an exhaust port for evacuating one or more of the first reaction chambers or the waste chambers.
7. The device of claim 1 , further comprising: (I) a heater for heating one or more of the fluid chambers;
(m) a cooler for cooling one or more of the fluid chambers; and
(n) a temperature monitor for monitoring the temperature of one or more of the fluid chambers.
8. The device of claim 1 , further comprising:
(o) a permanent magnet that can be positioned adjacent to one or more of the fluid chambers.
9. The device of claim 1 , further comprising (p) a detection chamber or channel having a transparent wall.
1 0. The device of claim 9, further comprising:
(q) a light source capable of directing light to the transparent wall of a chamber or channel.
1 1 . The device of claim 1 0, further comprising:
(r) a light detection device capable of detecting:
(1 ) the light reflected from an illuminated chamber or channel having a transparent wall,
(2) the light transmitted through an illuminated chamber or channel having a transparent wall, or
(3) the light emissions emanating from an excited molecule in a chamber or channel having a transparent wall.
1 2. A device for conducting assays in parallel using fluids that are confined to a disposable cassette comprising: the disposable assay cassette, which comprises (i) at least two reaction flow-ways, including a first reaction flow-way designed to receive and assay an experimental sample and a second reaction flow-way designed to receive and assay a negative control, (ii) for each reaction flow- way, at least one supply chamber connected thereto and containing fluids needed in the assay and at least one reaction chamber, (iii) a negative control supply chamber connected with the second reaction flow-way containing the negative control, and (iv) a test sample supply chamber connected with the first reaction flow-way designed to receive a test sample through an inlet connected with the test sample supply chamber, valves for controlling the flow of fluids in the cassette, and an instrument comprising a temperature control unit for controlling in parallel the temperature in a reaction chamber in each reaction flow-way, valve actuators for opening and closing the valves in the cassette, and one or more pumps for pushing fluid out of the various supply chambers and reaction chambers of the cassette.
1 3. The device of claim 1 2, wherein the cassette further comprises (v) a third reaction flow-way designed to receive and assay a test sample and a positive control, (vi) connecting routes between the test sample supply chamber and both the first and third reaction flow-ways, wherein these connecting routes are controlled by valves that allow selective flow between the test sample supply chamber and either the first or third reaction flow-way, and (vii) a first positive control supply chamber connecting with the third reaction flow-way containing the positive control.
14. The device of claim 1 2, wherein the cassette further comprises (viii) a fourth reaction flow-way designed to receive and assay a positive control, and (ix) a second positive control supply chamber connecting with the fourth reaction flow-way containing the positive control.
1 5. The device of claim 1 4, wherein the cassette further comprises (v) a third reaction flow-way designed to receive and assay a test sample and a positive control, (vi) connecting routes between the test sample supply chamber and both the first and third reaction flow-ways, wherein these connecting routes are controlled by valves that allow selective flow between the test sample supply chamber and either the first or third reaction flow-way, and (viii) a first positive control supply chamber connecting with the third reaction flow-way containing the positive control.
1 6. A method of conducting assays using the device of claim 1 6, which method comprises:
(a) providing the device for conducting assays in parallel, wherein reagents and control materials are pre-loaded into the supply chambers;
(b) inserting a test sample into the test sample supply chamber; and
(c) reacting in parallel in separate reaction flow-ways ( 1 ) the test sample and (2) the negative control.
1 7. A device comprising a cassette suitable for conducting reactions therein, which cassette comprises a body having one or more recesses and one or more embossed films covering the recesses.
PCT/US1997/000298 1996-01-24 1997-01-24 Parallel reaction cassette and associated devices WO1997027324A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU18251/97A AU1825197A (en) 1996-01-24 1997-01-24 Parallel reaction cassette and associated devices

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1051396P 1996-01-24 1996-01-24
US60/010,513 1996-01-24

Publications (1)

Publication Number Publication Date
WO1997027324A1 true WO1997027324A1 (en) 1997-07-31

Family

ID=21746101

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/000298 WO1997027324A1 (en) 1996-01-24 1997-01-24 Parallel reaction cassette and associated devices

Country Status (3)

Country Link
US (1) US5863502A (en)
AU (1) AU1825197A (en)
WO (1) WO1997027324A1 (en)

Cited By (82)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL1006813C1 (en) * 1997-08-20 1998-01-21 Sipke Wadman Packaging containing solid reaction carrier for chemical synthesis
WO1999023492A1 (en) * 1997-10-31 1999-05-14 Sarnoff Corporation Method for enhancing fluorescence
WO1999045141A1 (en) * 1998-03-05 1999-09-10 Thuraiayah Vinayagamoorthy Multi-zone polymerase/ligase chain reaction
US6007690A (en) * 1996-07-30 1999-12-28 Aclara Biosciences, Inc. Integrated microfluidic devices
FR2782934A1 (en) * 1998-09-08 2000-03-10 Bio Merieux Analysis card for medical diagnostics with built in valve isolating analysis chambers
WO2000013795A1 (en) * 1998-09-08 2000-03-16 Bio Merieux Microfluid system for reactions and transfers
WO2000040334A1 (en) * 1999-01-08 2000-07-13 Pe Corporation (Ny) Fiber array for contacting chemical species and methods for using and making same
WO2000053320A1 (en) * 1999-03-09 2000-09-14 Biomerieux S.A. Pumping device for transferring at least a fluid into a consumable
WO2000078453A1 (en) * 1999-06-22 2000-12-28 Biomerieux S.A. Valves enabling a liquid to be directed in a diagnostic chart, diagnostic charts and diagnostic device comprising several charts
FR2798867A1 (en) * 1999-09-23 2001-03-30 Commissariat Energie Atomique Device for injecting fluids simultaneously into microfluidic system for biochemical reactions has layer with deformable small diameter, open ended pipes and device for pressurizing fluids in pipes
EP1110084A1 (en) * 1998-08-03 2001-06-27 Qualisys Diagnostics Inc. Methods and apparatus for conducting tests
US6344326B1 (en) 1996-07-30 2002-02-05 Aclara Bio Sciences, Inc. Microfluidic method for nucleic acid purification and processing
FR2813207A1 (en) * 2000-08-28 2002-03-01 Bio Merieux REACTIONAL CARD AND USE OF SUCH A CARD
WO2002044566A1 (en) 2000-12-01 2002-06-06 Biomerieux S.A. Valves activated by electrically active polymers or by shape-memory materials, device containing same and method for using same
JP2003094395A (en) * 2001-09-26 2003-04-03 Olympus Optical Co Ltd Arrayed micro fluid control device
JP2003166910A (en) * 2001-11-30 2003-06-13 Asahi Kasei Corp Liquid-feeding mechanism and analyzer provided with the same
EP1327474A1 (en) * 2000-09-22 2003-07-16 Kawamura Institute Of Chemical Research Very small chemical device and flow rate adjusting method therefor
EP1343973A1 (en) * 2000-11-16 2003-09-17 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
JP2003287479A (en) * 2002-03-28 2003-10-10 Asahi Kasei Corp Valve mechanism
WO2003022435A3 (en) * 2001-09-11 2003-12-04 Iquum Inc Sample vessels
US6748332B2 (en) 1998-06-24 2004-06-08 Chen & Chen, Llc Fluid sample testing system
JP2004212361A (en) * 2003-01-09 2004-07-29 Yokogawa Electric Corp Cartridge for biochip
JP2004226207A (en) * 2003-01-22 2004-08-12 Asahi Kasei Corp Liquid-feeding mechanism and analyzer provided with the same
US6780617B2 (en) 2000-12-29 2004-08-24 Chen & Chen, Llc Sample processing device and method
WO2005009617A1 (en) * 2003-05-28 2005-02-03 Smiths Detection Inc. Device for polymerase chain reactions
JP2005037368A (en) * 2003-05-12 2005-02-10 Yokogawa Electric Corp Cartridge for chemical reaction, its manufacturing method, and driving system for cartridge for chemical reaction
US6875619B2 (en) 1999-11-12 2005-04-05 Motorola, Inc. Microfluidic devices comprising biochannels
EP1531936A2 (en) * 2002-07-26 2005-05-25 Applera Corporation Actuator for deformable valves in a microfluidic device, and method
EP1625888A2 (en) * 2004-08-13 2006-02-15 Alps Electric Co., Ltd. Test plate and test method using the same
WO2006045619A1 (en) * 2004-10-28 2006-05-04 Directif Gmbh Process and device for processing biopolymers in parallel
US7169353B1 (en) 1999-03-09 2007-01-30 Biomerieux S.A. Apparatus enabling liquid transfer by capillary action therein
US7259021B2 (en) 2000-03-07 2007-08-21 Bio Merieux Method for using a test card
WO2009002447A1 (en) * 2007-06-21 2008-12-31 Gen-Probe Incorporated Instrument and receptacles for use in performing processes
US7473551B2 (en) 2004-05-21 2009-01-06 Atonomics A/S Nano-mechanic microsensors and methods for detecting target analytes
US7491497B2 (en) 1999-06-22 2009-02-17 Biomerieux S.A. Device for implementing an analysis pack, analysis pack and method using same
US7595189B2 (en) 1999-01-08 2009-09-29 Applied Biosystems, Llc Integrated optics fiber array
US7648835B2 (en) 2003-06-06 2010-01-19 Micronics, Inc. System and method for heating, cooling and heat cycling on microfluidic device
US7655129B2 (en) 1998-06-23 2010-02-02 Osmetech Technology Inc. Binding acceleration techniques for the detection of analytes
US7815868B1 (en) 2006-02-28 2010-10-19 Fluidigm Corporation Microfluidic reaction apparatus for high throughput screening
US7820427B2 (en) 2001-11-30 2010-10-26 Fluidigm Corporation Microfluidic device and methods of using same
US7833708B2 (en) 2001-04-06 2010-11-16 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US7854897B2 (en) 2003-05-12 2010-12-21 Yokogawa Electric Corporation Chemical reaction cartridge, its fabrication method, and a chemical reaction cartridge drive system
WO2011124688A1 (en) * 2010-04-08 2011-10-13 Aj Innuscreen Gmbh Device for detecting nucleic acids
EP2409767A1 (en) 2005-06-23 2012-01-25 Biocartis SA Modular cartridge, system and method for automated medical diagnosis
US8216832B2 (en) 2007-07-31 2012-07-10 Micronics, Inc. Sanitary swab collection system, microfluidic assay device, and methods for diagnostic assays
EP2520368A1 (en) * 2005-10-03 2012-11-07 Rheonix, Inc. Microfluidic membrane pump and valve
CN102929309A (en) * 2005-01-25 2013-02-13 欧西里其有限责任公司 Temperature controller for small fluid samples having different heat capacities
US8435462B2 (en) 2000-06-28 2013-05-07 3M Innovative Properties Company Sample processing devices
DE102011056273A1 (en) * 2011-12-12 2013-06-13 sense2care GmbH Fluid reservoir for a device for analyzing patient samples
US8486247B2 (en) 1999-04-21 2013-07-16 Osmetch Technology, Inc. Use of microfluidic systems in the electrochemical detection of target analytes
US8658418B2 (en) 2002-04-01 2014-02-25 Fluidigm Corporation Microfluidic particle-analysis systems
US8871446B2 (en) 2002-10-02 2014-10-28 California Institute Of Technology Microfluidic nucleic acid analysis
US8883424B2 (en) 1999-05-21 2014-11-11 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
WO2014191519A1 (en) * 2013-05-30 2014-12-04 Commissariat à l'énergie atomique et aux énergies alternatives Fluidic card comprising a fluidic channel provided with an opening resealable by means of a flexible film
US8936933B2 (en) 2003-02-05 2015-01-20 IQumm, Inc. Sample processing methods
US20150024436A1 (en) * 2011-10-21 2015-01-22 Integenx Inc, Sample preparation, processing and analysis systems
JP2016052254A (en) * 2014-09-02 2016-04-14 株式会社東芝 Nucleic acid detection cassette
JP2016052253A (en) * 2014-09-02 2016-04-14 株式会社東芝 Nucleic acid detection cassette
WO2016124908A1 (en) * 2015-02-02 2016-08-11 Atlas Genetics Limited Instrument for performing a diagnostic test on a fluidic cartridge
WO2016128570A1 (en) * 2015-02-13 2016-08-18 Espci Paper device for genetic diagnosis
US9714443B2 (en) 2002-09-25 2017-07-25 California Institute Of Technology Microfabricated structure having parallel and orthogonal flow channels controlled by row and column multiplexors
US9915613B2 (en) 2011-02-24 2018-03-13 Gen-Probe Incorporated Systems and methods for distinguishing optical signals of different modulation frequencies in an optical signal detector
WO2018052768A1 (en) * 2016-09-16 2018-03-22 General Electric Company Compact valve array with actuator system
WO2018153950A1 (en) * 2017-02-22 2018-08-30 Selfdiagnostics Deutschland Gmbh Microfluidic test device
US10799862B2 (en) 2006-03-24 2020-10-13 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using same
US10821446B1 (en) 2006-03-24 2020-11-03 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
US10821435B2 (en) 2014-09-02 2020-11-03 Canon Medical Systems Corporation Nucleic acid detection cassette
US10844368B2 (en) 2007-07-13 2020-11-24 Handylab, Inc. Diagnostic apparatus to extract nucleic acids including a magnetic assembly and a heater assembly
US10865437B2 (en) 2003-07-31 2020-12-15 Handylab, Inc. Processing particle-containing samples
US10875022B2 (en) 2007-07-13 2020-12-29 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US10900066B2 (en) 2006-03-24 2021-01-26 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US11060082B2 (en) 2007-07-13 2021-07-13 Handy Lab, Inc. Polynucleotide capture materials, and systems using same
US11142785B2 (en) 2006-03-24 2021-10-12 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US11266987B2 (en) 2007-07-13 2022-03-08 Handylab, Inc. Microfluidic cartridge
US11441171B2 (en) 2004-05-03 2022-09-13 Handylab, Inc. Method for processing polynucleotide-containing samples
US11453906B2 (en) 2011-11-04 2022-09-27 Handylab, Inc. Multiplexed diagnostic detection apparatus and methods
US11549959B2 (en) 2007-07-13 2023-01-10 Handylab, Inc. Automated pipetting apparatus having a combined liquid pump and pipette head system
US11666919B2 (en) 2015-02-02 2023-06-06 Binx Health Limited Instrument for performing a diagnostic test on a fluidic cartridge
US11684918B2 (en) 2011-10-21 2023-06-27 IntegenX, Inc. Sample preparation, processing and analysis systems
US11788127B2 (en) 2011-04-15 2023-10-17 Becton, Dickinson And Company Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection
DE102022203778A1 (en) 2022-04-14 2023-10-19 Robert Bosch Gesellschaft mit beschränkter Haftung Microfluidic cartridge with a trench-shaped depression to prevent heat conduction in the outer wall
US11806718B2 (en) 2006-03-24 2023-11-07 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system

Families Citing this family (384)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6319472B1 (en) * 1993-11-01 2001-11-20 Nanogen, Inc. System including functionally separated regions in electrophoretic system
US20040077074A1 (en) * 1993-11-01 2004-04-22 Nanogen, Inc. Multi-chambered analysis device
US7857957B2 (en) * 1994-07-07 2010-12-28 Gamida For Life B.V. Integrated portable biological detection system
US6403367B1 (en) * 1994-07-07 2002-06-11 Nanogen, Inc. Integrated portable biological detection system
US20020022261A1 (en) * 1995-06-29 2002-02-21 Anderson Rolfe C. Miniaturized genetic analysis systems and methods
US6048734A (en) 1995-09-15 2000-04-11 The Regents Of The University Of Michigan Thermal microvalves in a fluid flow method
US6399023B1 (en) 1996-04-16 2002-06-04 Caliper Technologies Corp. Analytical system and method
DE19648695C2 (en) * 1996-11-25 1999-07-22 Abb Patent Gmbh Device for the automatic and continuous analysis of liquid samples
US6235471B1 (en) 1997-04-04 2001-05-22 Caliper Technologies Corp. Closed-loop biochemical analyzers
US6391622B1 (en) 1997-04-04 2002-05-21 Caliper Technologies Corp. Closed-loop biochemical analyzers
ATE347615T1 (en) * 1997-04-16 2006-12-15 Applera Corp NUCLEIC ACID COLLECTION
US6872527B2 (en) * 1997-04-16 2005-03-29 Xtrana, Inc. Nucleic acid archiving
US6143496A (en) 1997-04-17 2000-11-07 Cytonix Corporation Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly
US6004752A (en) * 1997-07-29 1999-12-21 Sarnoff Corporation Solid support with attached molecules
US7214298B2 (en) * 1997-09-23 2007-05-08 California Institute Of Technology Microfabricated cell sorter
US6833242B2 (en) * 1997-09-23 2004-12-21 California Institute Of Technology Methods for detecting and sorting polynucleotides based on size
US5961930A (en) * 1997-10-15 1999-10-05 Eastman Kodak Company Integrated micro-ceramic chemical plant with insertable reaction chambers and micro-filters
US5965092A (en) * 1997-10-15 1999-10-12 Eastman Kodak Company Integrated micro-ceramic chemical plant with insertable micro-filters
US5976472A (en) * 1997-10-15 1999-11-02 Eastman Kodak Company Integrated micro-ceramic chemical plant with insertable catalytic reaction chambers
EP1179585B1 (en) 1997-12-24 2008-07-09 Cepheid Device and method for lysis
US6723290B1 (en) * 1998-03-07 2004-04-20 Levine Robert A Container for holding biologic fluid for analysis
US6818437B1 (en) * 1998-05-16 2004-11-16 Applera Corporation Instrument for monitoring polymerase chain reaction of DNA
US7498164B2 (en) * 1998-05-16 2009-03-03 Applied Biosystems, Llc Instrument for monitoring nucleic acid sequence amplification reaction
EP1078245B1 (en) * 1998-05-16 2008-08-06 Applera Corporation Instrument for monitoring polymerase chain reaction of dna
WO1999060397A1 (en) * 1998-05-18 1999-11-25 University Of Washington Liquid analysis cartridge
US6830729B1 (en) 1998-05-18 2004-12-14 University Of Washington Sample analysis instrument
US7799521B2 (en) * 1998-06-24 2010-09-21 Chen & Chen, Llc Thermal cycling
US6306658B1 (en) * 1998-08-13 2001-10-23 Symyx Technologies Parallel reactor with internal sensing
US6601613B2 (en) * 1998-10-13 2003-08-05 Biomicro Systems, Inc. Fluid circuit components based upon passive fluid dynamics
AU2180200A (en) * 1998-12-14 2000-07-03 Li-Cor Inc. A heterogeneous assay for pyrophosphate detection
US6431476B1 (en) 1999-12-21 2002-08-13 Cepheid Apparatus and method for rapid ultrasonic disruption of cells or viruses
US7914994B2 (en) 1998-12-24 2011-03-29 Cepheid Method for separating an analyte from a sample
US20050026209A1 (en) * 1999-01-08 2005-02-03 Vann Charles S. Optical fiber bundle for detecting binding of chemical species
US6463649B1 (en) * 1999-01-22 2002-10-15 Industrial Technology Research Institute Method of manufacturing disposable reaction module
US6284195B1 (en) * 1999-01-25 2001-09-04 Industrial Technology Research Institute Disposable reaction module
AU3372800A (en) 1999-02-23 2000-09-14 Caliper Technologies Corporation Manipulation of microparticles in microfluidic systems
US7214544B2 (en) * 1999-03-02 2007-05-08 Qualigen, Inc. Semi-continuous blood separation using magnetic beads
US7150994B2 (en) * 1999-03-03 2006-12-19 Symyx Technologies, Inc. Parallel flow process optimization reactor
US6303343B1 (en) * 1999-04-06 2001-10-16 Caliper Technologies Corp. Inefficient fast PCR
US7312087B2 (en) * 2000-01-11 2007-12-25 Clinical Micro Sensors, Inc. Devices and methods for biochip multiplexing
US20020177135A1 (en) * 1999-07-27 2002-11-28 Doung Hau H. Devices and methods for biochip multiplexing
US20040053290A1 (en) * 2000-01-11 2004-03-18 Terbrueggen Robert Henry Devices and methods for biochip multiplexing
US7410793B2 (en) 1999-05-17 2008-08-12 Applera Corporation Optical instrument including excitation source
US7423750B2 (en) * 2001-11-29 2008-09-09 Applera Corporation Configurations, systems, and methods for optical scanning with at least one first relative angular motion and at least one second angular motion or at least one linear motion
US20050279949A1 (en) * 1999-05-17 2005-12-22 Applera Corporation Temperature control for light-emitting diode stabilization
US7387891B2 (en) * 1999-05-17 2008-06-17 Applera Corporation Optical instrument including excitation source
US6485690B1 (en) 1999-05-27 2002-11-26 Orchid Biosciences, Inc. Multiple fluid sample processor and system
AU782343B2 (en) 1999-05-28 2005-07-21 Cepheid Apparatus and method for analyzing a fluid sample
US8815521B2 (en) 2000-05-30 2014-08-26 Cepheid Apparatus and method for cell disruption
US20040200909A1 (en) * 1999-05-28 2004-10-14 Cepheid Apparatus and method for cell disruption
US6818185B1 (en) * 1999-05-28 2004-11-16 Cepheid Cartridge for conducting a chemical reaction
US9073053B2 (en) 1999-05-28 2015-07-07 Cepheid Apparatus and method for cell disruption
US6664104B2 (en) 1999-06-25 2003-12-16 Cepheid Device incorporating a microfluidic chip for separating analyte from a sample
DE19935433A1 (en) * 1999-08-01 2001-03-01 Febit Ferrarius Biotech Gmbh Microfluidic reaction carrier
US6932951B1 (en) 1999-10-29 2005-08-23 Massachusetts Institute Of Technology Microfabricated chemical reactor
US6692952B1 (en) * 1999-11-10 2004-02-17 Massachusetts Institute Of Technology Cell analysis and sorting apparatus for manipulation of cells
US6361958B1 (en) * 1999-11-12 2002-03-26 Motorola, Inc. Biochannel assay for hybridization with biomaterial
US6432290B1 (en) 1999-11-26 2002-08-13 The Governors Of The University Of Alberta Apparatus and method for trapping bead based reagents within microfluidic analysis systems
CA2290731A1 (en) * 1999-11-26 2001-05-26 D. Jed Harrison Apparatus and method for trapping bead based reagents within microfluidic analysis system
EP1111281A1 (en) * 1999-12-23 2001-06-27 Scitec Research SA Analyzer comprising a miniaturised flow plate
AU2001240040A1 (en) * 2000-03-03 2001-09-17 California Institute Of Technology Combinatorial array for nucleic acid analysis
US8071051B2 (en) 2004-05-14 2011-12-06 Honeywell International Inc. Portable sample analyzer cartridge
US6597438B1 (en) * 2000-08-02 2003-07-22 Honeywell International Inc. Portable flow cytometry
US7242474B2 (en) * 2004-07-27 2007-07-10 Cox James A Cytometer having fluid core stream position control
US7420659B1 (en) * 2000-06-02 2008-09-02 Honeywell Interantional Inc. Flow control system of a cartridge
US7262838B2 (en) * 2001-06-29 2007-08-28 Honeywell International Inc. Optical detection system for flow cytometry
US6970245B2 (en) * 2000-08-02 2005-11-29 Honeywell International Inc. Optical alignment detection system
US7641856B2 (en) * 2004-05-14 2010-01-05 Honeywell International Inc. Portable sample analyzer with removable cartridge
US7978329B2 (en) * 2000-08-02 2011-07-12 Honeywell International Inc. Portable scattering and fluorescence cytometer
US20060263888A1 (en) * 2000-06-02 2006-11-23 Honeywell International Inc. Differential white blood count on a disposable card
US7630063B2 (en) * 2000-08-02 2009-12-08 Honeywell International Inc. Miniaturized cytometer for detecting multiple species in a sample
US7215425B2 (en) * 2000-08-02 2007-05-08 Honeywell International Inc. Optical alignment for flow cytometry
US8329118B2 (en) * 2004-09-02 2012-12-11 Honeywell International Inc. Method and apparatus for determining one or more operating parameters for a microfluidic circuit
US7283223B2 (en) * 2002-08-21 2007-10-16 Honeywell International Inc. Cytometer having telecentric optics
US7130046B2 (en) * 2004-09-27 2006-10-31 Honeywell International Inc. Data frame selection for cytometer analysis
US7016022B2 (en) * 2000-08-02 2006-03-21 Honeywell International Inc. Dual use detectors for flow cytometry
US7471394B2 (en) * 2000-08-02 2008-12-30 Honeywell International Inc. Optical detection system with polarizing beamsplitter
US20020015959A1 (en) * 2000-06-23 2002-02-07 Bardell Ronald L. Fluid mixing in microfluidic structures
AU2001273057A1 (en) * 2000-06-27 2002-01-08 Fluidigm Corporation A microfluidic design automation method and system
AU7024801A (en) * 2000-06-28 2002-01-08 3M Innovative Properties Co Enhanced sample processing devices, systems and methods
US6720187B2 (en) * 2000-06-28 2004-04-13 3M Innovative Properties Company Multi-format sample processing devices
US6734401B2 (en) * 2000-06-28 2004-05-11 3M Innovative Properties Company Enhanced sample processing devices, systems and methods
US7277166B2 (en) * 2000-08-02 2007-10-02 Honeywell International Inc. Cytometer analysis cartridge optical configuration
US7061595B2 (en) * 2000-08-02 2006-06-13 Honeywell International Inc. Miniaturized flow controller with closed loop regulation
US7000330B2 (en) * 2002-08-21 2006-02-21 Honeywell International Inc. Method and apparatus for receiving a removable media member
AU2001288249A1 (en) * 2000-08-14 2002-02-25 The Regents Of The University Of California Biosensors and methods for their use
US7027683B2 (en) 2000-08-15 2006-04-11 Nanostream, Inc. Optical devices with fluidic systems
ATE448875T1 (en) * 2000-09-14 2009-12-15 Caliper Life Sciences Inc MICROFLUIDIC DEVICES AND METHODS FOR CARRYING OUT TEMPERATURE-MEDIATED REACTIONS
DE60103415D1 (en) 2000-09-29 2004-06-24 Nanostream Inc MICROFLUIDIC DEVICE FOR HEAT TRANSFER
EP1336097A4 (en) * 2000-10-13 2006-02-01 Fluidigm Corp Microfluidic device based sample injection system for analytical devices
US8097471B2 (en) * 2000-11-10 2012-01-17 3M Innovative Properties Company Sample processing devices
GB0028647D0 (en) * 2000-11-24 2001-01-10 Nextgen Sciences Ltd Apparatus for chemical assays
FR2817343B1 (en) * 2000-11-29 2003-05-09 Commissariat Energie Atomique METHOD AND DEVICES FOR TRANSPORTING AND CONCENTRATING AN ANALYTE PRESENT IN A SAMPLE
JP2004533670A (en) * 2001-02-13 2004-11-04 コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ Processing means for copy protection signal
US6692700B2 (en) 2001-02-14 2004-02-17 Handylab, Inc. Heat-reduction methods and systems related to microfluidic devices
ITMI20010434A1 (en) * 2001-03-02 2002-09-02 Oglio Stefano Dall DISPOSABLE STERILIZABLE SAMPLING UNIT FOR DETERMINATIONS IN MICROBIOLOGY AND CHEMICAL-CLINICS
US20050196785A1 (en) * 2001-03-05 2005-09-08 California Institute Of Technology Combinational array for nucleic acid analysis
US6742544B2 (en) * 2001-03-07 2004-06-01 Symyx Technologies, Inc. Injection valve array
WO2002072264A1 (en) * 2001-03-09 2002-09-19 Biomicro Systems, Inc. Method and system for microfluidic interfacing to arrays
US7323140B2 (en) * 2001-03-28 2008-01-29 Handylab, Inc. Moving microdroplets in a microfluidic device
US7192557B2 (en) * 2001-03-28 2007-03-20 Handylab, Inc. Methods and systems for releasing intracellular material from cells within microfluidic samples of fluids
US7829025B2 (en) * 2001-03-28 2010-11-09 Venture Lending & Leasing Iv, Inc. Systems and methods for thermal actuation of microfluidic devices
US7270786B2 (en) 2001-03-28 2007-09-18 Handylab, Inc. Methods and systems for processing microfluidic samples of particle containing fluids
US6852287B2 (en) 2001-09-12 2005-02-08 Handylab, Inc. Microfluidic devices having a reduced number of input and output connections
US7010391B2 (en) 2001-03-28 2006-03-07 Handylab, Inc. Methods and systems for control of microfluidic devices
US8895311B1 (en) 2001-03-28 2014-11-25 Handylab, Inc. Methods and systems for control of general purpose microfluidic devices
US6575188B2 (en) 2001-07-26 2003-06-10 Handylab, Inc. Methods and systems for fluid control in microfluidic devices
US20030118804A1 (en) * 2001-05-02 2003-06-26 3M Innovative Properties Company Sample processing device with resealable process chamber
US20050009101A1 (en) * 2001-05-17 2005-01-13 Motorola, Inc. Microfluidic devices comprising biochannels
US7118907B2 (en) * 2001-06-06 2006-10-10 Li-Cor, Inc. Single molecule detection systems and methods
US6981522B2 (en) * 2001-06-07 2006-01-03 Nanostream, Inc. Microfluidic devices with distributing inputs
US6729352B2 (en) 2001-06-07 2004-05-04 Nanostream, Inc. Microfluidic synthesis devices and methods
US20020186263A1 (en) * 2001-06-07 2002-12-12 Nanostream, Inc. Microfluidic fraction collectors
US7318912B2 (en) * 2001-06-07 2008-01-15 Nanostream, Inc. Microfluidic systems and methods for combining discrete fluid volumes
JP4679818B2 (en) * 2001-07-16 2011-05-11 アイダホ テクノロジー インコーポレーテッド Thermal cycle system and method of use thereof
US6825127B2 (en) 2001-07-24 2004-11-30 Zarlink Semiconductor Inc. Micro-fluidic devices
EP1438567B1 (en) * 2001-07-26 2018-07-25 Handylab, Inc. Methods and systems for microfluidic processing
DE10137565B4 (en) * 2001-07-30 2004-07-15 Filt Lungen- Und Thoraxdiagnostik Gmbh Method for determining parameters of a breath condensate
US6939632B2 (en) * 2001-08-06 2005-09-06 Massachusetts Institute Of Technology Thermally efficient micromachined device
GB0121340D0 (en) * 2001-09-04 2001-10-24 Provalis Diagnostics Ltd Device fo9r use in fluid array
US20030108664A1 (en) * 2001-10-05 2003-06-12 Kodas Toivo T. Methods and compositions for the formation of recessed electrical features on a substrate
US8440093B1 (en) 2001-10-26 2013-05-14 Fuidigm Corporation Methods and devices for electronic and magnetic sensing of the contents of microfluidic flow channels
US20030175947A1 (en) * 2001-11-05 2003-09-18 Liu Robin Hui Enhanced mixing in microfluidic devices
US7635588B2 (en) * 2001-11-29 2009-12-22 Applied Biosystems, Llc Apparatus and method for differentiating multiple fluorescence signals by excitation wavelength
US7691333B2 (en) 2001-11-30 2010-04-06 Fluidigm Corporation Microfluidic device and methods of using same
US6739576B2 (en) 2001-12-20 2004-05-25 Nanostream, Inc. Microfluidic flow control device with floating element
US6889468B2 (en) * 2001-12-28 2005-05-10 3M Innovative Properties Company Modular systems and methods for using sample processing devices
US20040109793A1 (en) * 2002-02-07 2004-06-10 Mcneely Michael R Three-dimensional microfluidics incorporating passive fluid control structures
AU2003215340A1 (en) * 2002-02-22 2003-09-09 Nanostream, Inc. Ratiometric dilution devices and methods
US7312085B2 (en) * 2002-04-01 2007-12-25 Fluidigm Corporation Microfluidic particle-analysis systems
US9943847B2 (en) 2002-04-17 2018-04-17 Cytonome/St, Llc Microfluidic system including a bubble valve for regulating fluid flow through a microchannel
US8409508B2 (en) * 2002-04-23 2013-04-02 Biofire Diagnostics, Inc. Sample withdrawal and dispensing device
JP2005526253A (en) * 2002-05-17 2005-09-02 アプレラ コーポレイション Apparatus and method for differentiating multiple fluorescent signals by excitation wavelength
WO2003098279A2 (en) 2002-05-17 2003-11-27 Applera Corporation Optical instrument includung excitation source
US20030217923A1 (en) * 2002-05-24 2003-11-27 Harrison D. Jed Apparatus and method for trapping bead based reagents within microfluidic analysis systems
WO2004000721A2 (en) * 2002-06-24 2003-12-31 Fluidigm Corporation Recirculating fluidic network and methods for using the same
US20040005247A1 (en) * 2002-07-03 2004-01-08 Nanostream, Inc. Microfluidic closed-end metering systems and methods
US20040101444A1 (en) * 2002-07-15 2004-05-27 Xeotron Corporation Apparatus and method for fluid delivery to a hybridization station
US20040011650A1 (en) * 2002-07-22 2004-01-22 Frederic Zenhausern Method and apparatus for manipulating polarizable analytes via dielectrophoresis
WO2004011666A2 (en) * 2002-07-26 2004-02-05 Applera Corporation Mg-mediated hot start biochemical reactions
US20040018115A1 (en) * 2002-07-29 2004-01-29 Nanostream, Inc. Fault tolerant detection regions in microfluidic systems
WO2004016728A1 (en) * 2002-08-19 2004-02-26 Olympus Corporation Incubator and culture device
TW590982B (en) * 2002-09-27 2004-06-11 Agnitio Science & Technology I Micro-fluid driving device
AU2003277153A1 (en) * 2002-09-27 2004-04-19 The General Hospital Corporation Microfluidic device for cell separation and uses thereof
US20040092033A1 (en) * 2002-10-18 2004-05-13 Nanostream, Inc. Systems and methods for preparing microfluidic devices for operation
US7010964B2 (en) * 2002-10-31 2006-03-14 Nanostream, Inc. Pressurized microfluidic devices with optical detection regions
SE524730C2 (en) * 2002-11-20 2004-09-21 Boule Medical Ab Blood Unit
US7507376B2 (en) * 2002-12-19 2009-03-24 3M Innovative Properties Company Integrated sample processing devices
JP4395133B2 (en) * 2002-12-20 2010-01-06 カリパー・ライフ・サイエンシズ・インク. Single molecule amplification and detection of DNA
AU2003303594A1 (en) * 2002-12-30 2004-07-29 The Regents Of The University Of California Methods and apparatus for pathogen detection and analysis
WO2004065930A2 (en) * 2003-01-14 2004-08-05 Micronics Inc. Microfluidic devices for fluid manipulation and analysis
US7419638B2 (en) * 2003-01-14 2008-09-02 Micronics, Inc. Microfluidic devices for fluid manipulation and analysis
US8641987B2 (en) * 2003-01-24 2014-02-04 Applied Biosystems, Llc Sample chamber array and method for processing a biological sample
US20050129580A1 (en) * 2003-02-26 2005-06-16 Swinehart Philip R. Microfluidic chemical reactor for the manufacture of chemically-produced nanoparticles
WO2004085668A2 (en) * 2003-03-20 2004-10-07 Northeastern Ohio Universities College Of Medecine Self-contained assay device for rapid detection of biohazardous agents
EP1473084B1 (en) * 2003-03-31 2015-07-29 Canon Kabushiki Kaisha Biochemical reaction cartridge
EP1473085B1 (en) * 2003-03-31 2015-07-22 Canon Kabushiki Kaisha Biochemical reaction cartridge
JP5419248B2 (en) * 2003-04-03 2014-02-19 フルイディグム コーポレイション Microfluidic device and method of use thereof
US8828663B2 (en) * 2005-03-18 2014-09-09 Fluidigm Corporation Thermal reaction device and method for using the same
US7476363B2 (en) * 2003-04-03 2009-01-13 Fluidigm Corporation Microfluidic devices and methods of using same
US7604965B2 (en) * 2003-04-03 2009-10-20 Fluidigm Corporation Thermal reaction device and method for using the same
US20050145496A1 (en) 2003-04-03 2005-07-07 Federico Goodsaid Thermal reaction device and method for using the same
WO2004101151A1 (en) * 2003-05-08 2004-11-25 Nanostream, Inc. Sample preparation for parallel chromatography
JP3711988B2 (en) * 2003-05-12 2005-11-02 株式会社日立製作所 Fine particle array analysis system, fine particle array kit, and chemical analysis method
EP1636564A1 (en) * 2003-06-13 2006-03-22 The General Hospital Corporation Microfluidic systems for size based removal of red blood cells and platelets from blood
US20040253143A1 (en) * 2003-06-16 2004-12-16 Yokogawa Electric Corporation Method for processing waste liquid in cartridges and a chemical reaction cartridge applying the method
DE10336850B4 (en) * 2003-08-11 2006-10-26 Thinxxs Gmbh micro storage
US7413712B2 (en) * 2003-08-11 2008-08-19 California Institute Of Technology Microfluidic rotary flow reactor matrix
US20050047967A1 (en) * 2003-09-03 2005-03-03 Industrial Technology Research Institute Microfluidic component providing multi-directional fluid movement
DE10344229A1 (en) * 2003-09-24 2005-05-19 Steag Microparts Gmbh A microstructured device for removably storing small amounts of liquid and method for withdrawing the liquid stored in said device
US7718133B2 (en) * 2003-10-09 2010-05-18 3M Innovative Properties Company Multilayer processing devices and methods
JP4459718B2 (en) * 2003-10-31 2010-04-28 セイコーインスツル株式会社 Micro valve mechanism
US20050170401A1 (en) * 2004-01-29 2005-08-04 Canon Kabushiki Kaisha Hybridization apparatus and method
CA2557819A1 (en) * 2004-03-03 2005-09-15 The General Hospital Corporation Magnetic device for isolation of cells and biomolecules in a microfluidic environment
US8961900B2 (en) * 2004-04-28 2015-02-24 Yokogawa Electric Corporation Chemical reaction cartridge, method of producing chemical reaction cartridge, and mechanism for driving chemical reaction cartridge
AU2005241080B2 (en) * 2004-05-03 2011-08-11 Handylab, Inc. Processing polynucleotide-containing samples
DE102004022263A1 (en) * 2004-05-06 2005-12-15 Clondiag Chip Technologies Gmbh Apparatus and method for detecting molecular interactions
US8323564B2 (en) 2004-05-14 2012-12-04 Honeywell International Inc. Portable sample analyzer system
WO2005114186A1 (en) * 2004-05-20 2005-12-01 Wako Pure Chemical Industries, Ltd. Method of assaying hyaluronic acid by using hyaluronic acid-binding protein
US7799553B2 (en) * 2004-06-01 2010-09-21 The Regents Of The University Of California Microfabricated integrated DNA analysis system
DE102004033317A1 (en) * 2004-07-09 2006-02-09 Roche Diagnostics Gmbh Analytical test element
US7932090B2 (en) * 2004-08-05 2011-04-26 3M Innovative Properties Company Sample processing device positioning apparatus and methods
US7612871B2 (en) * 2004-09-01 2009-11-03 Honeywell International Inc Frequency-multiplexed detection of multiple wavelength light for flow cytometry
CN101073002B (en) * 2004-09-15 2012-08-08 英特基因有限公司 Microfluidic devices
US7630075B2 (en) 2004-09-27 2009-12-08 Honeywell International Inc. Circular polarization illumination based analyzer system
US8445265B2 (en) * 2004-10-06 2013-05-21 Universal Bio Research Co., Ltd. Reaction vessel and reaction controller
EP1650297B1 (en) * 2004-10-19 2011-04-13 Samsung Electronics Co., Ltd. Method and apparatus for the rapid disruption of cells or viruses using micro magnetic beads and laser
DE102004051573B4 (en) * 2004-10-22 2007-03-15 Yokogawa Electric Corporation, Musashino Process for treating a waste liquid in chemical reaction cartridges and chemical reaction cartridge in which the process is used
KR100601972B1 (en) * 2004-11-03 2006-07-18 삼성전자주식회사 Apparatus and method for the purification of nucleic acids by phase separation using Laser and beads
US20060094028A1 (en) * 2004-11-04 2006-05-04 Welch Allyn, Inc. Rapid diagnostic assay
CN100462710C (en) * 2004-11-09 2009-02-18 横河电机株式会社 Process for processing waste liquid in box and chemical reaction box using the same process
WO2006053588A1 (en) * 2004-11-17 2006-05-26 Agilent Technologies, Inc. Supply arrangement with supply reservoir element and fluidic device
US9260693B2 (en) 2004-12-03 2016-02-16 Cytonome/St, Llc Actuation of parallel microfluidic arrays
KR20070104347A (en) 2004-12-03 2007-10-25 사이토놈, 인크. Unitary cartridge for particle processing
US20060203236A1 (en) * 2005-03-08 2006-09-14 Zhenghua Ji Sample cell
EP1707267A1 (en) * 2005-03-30 2006-10-04 F. Hoffman-la Roche AG Device having a self sealing fluid port
US20060246493A1 (en) * 2005-04-04 2006-11-02 Caliper Life Sciences, Inc. Method and apparatus for use in temperature controlled processing of microfluidic samples
US20070026413A1 (en) * 2005-07-29 2007-02-01 Mehmet Toner Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US20070196820A1 (en) * 2005-04-05 2007-08-23 Ravi Kapur Devices and methods for enrichment and alteration of cells and other particles
US20070026417A1 (en) * 2005-07-29 2007-02-01 Martin Fuchs Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US20070026414A1 (en) * 2005-07-29 2007-02-01 Martin Fuchs Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US20070026415A1 (en) * 2005-07-29 2007-02-01 Martin Fuchs Devices and methods for enrichment and alteration of circulating tumor cells and other particles
WO2006119106A1 (en) * 2005-04-29 2006-11-09 Honeywell International Inc. Cytometer cell counting and size measurement method
EP1920070B1 (en) * 2005-05-09 2019-08-07 BioFire Diagnostics, LLC Self-contained biological analysis
ES2820430T3 (en) 2005-05-09 2021-04-21 Labrador Diagnostics Llc Fluid systems for care centers and their uses
WO2007005974A2 (en) 2005-07-01 2007-01-11 Honeywell International, Inc. A flow metered analyzer
EP1901846B1 (en) 2005-07-01 2015-01-14 Honeywell International Inc. A microfluidic card for rbc analysis
JP4995197B2 (en) 2005-07-01 2012-08-08 ハネウェル・インターナショナル・インコーポレーテッド Molded cartridge with 3D hydrodynamic focusing
US7323660B2 (en) * 2005-07-05 2008-01-29 3M Innovative Properties Company Modular sample processing apparatus kits and modules
US7763210B2 (en) * 2005-07-05 2010-07-27 3M Innovative Properties Company Compliant microfluidic sample processing disks
US7754474B2 (en) 2005-07-05 2010-07-13 3M Innovative Properties Company Sample processing device compression systems and methods
US20070026416A1 (en) * 2005-07-29 2007-02-01 Martin Fuchs Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US20070059680A1 (en) * 2005-09-15 2007-03-15 Ravi Kapur System for cell enrichment
US20090181421A1 (en) * 2005-07-29 2009-07-16 Ravi Kapur Diagnosis of fetal abnormalities using nucleated red blood cells
US8921102B2 (en) 2005-07-29 2014-12-30 Gpb Scientific, Llc Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US7843563B2 (en) * 2005-08-16 2010-11-30 Honeywell International Inc. Light scattering and imaging optical system
JP2009507193A (en) * 2005-09-02 2009-02-19 カリフォルニア インスティチュート オブ テクノロジー Method and apparatus for mechanical actuation of valves in fluidic devices
US20070059716A1 (en) * 2005-09-15 2007-03-15 Ulysses Balis Methods for detecting fetal abnormality
US20070059718A1 (en) * 2005-09-15 2007-03-15 Mehmet Toner Systems and methods for enrichment of analytes
US20070059774A1 (en) * 2005-09-15 2007-03-15 Michael Grisham Kits for Prenatal Testing
US20070059719A1 (en) * 2005-09-15 2007-03-15 Michael Grisham Business methods for prenatal Diagnosis
US20070059781A1 (en) * 2005-09-15 2007-03-15 Ravi Kapur System for size based separation and analysis
US20070059683A1 (en) * 2005-09-15 2007-03-15 Tom Barber Veterinary diagnostic system
JP2007090138A (en) * 2005-09-27 2007-04-12 Yokogawa Electric Corp Cartridge for chemical treatments, and its using method
WO2007041486A2 (en) * 2005-09-29 2007-04-12 Siemens Medical Solutions Usa, Inc. Microfluidic chip for synthesizing radioactively labeled molecules suitable for human imaging with positron emission tomography
JP4830432B2 (en) * 2005-09-30 2011-12-07 横河電機株式会社 Chemical reaction cartridge and method of use thereof
JP4692200B2 (en) * 2005-10-06 2011-06-01 横河電機株式会社 Chemical treatment cartridge and method of use thereof
JP2009511059A (en) * 2005-10-11 2009-03-19 ハンディーラブ インコーポレイテッド Polynucleotide sample preparation device
EP1963817A2 (en) 2005-12-22 2008-09-03 Honeywell International Inc. Portable sample analyzer cartridge
EP1963866B1 (en) * 2005-12-22 2018-05-16 Honeywell International Inc. Hematological analyzer system with removable cartridge
JP5431732B2 (en) 2005-12-29 2014-03-05 ハネウェル・インターナショナル・インコーポレーテッド Assay implementation in microfluidic format
JP4906362B2 (en) * 2006-01-30 2012-03-28 株式会社日立ハイテクノロジーズ Chemical analysis pretreatment equipment
US7749365B2 (en) * 2006-02-01 2010-07-06 IntegenX, Inc. Optimized sample injection structures in microfluidic separations
US7862000B2 (en) * 2006-02-03 2011-01-04 California Institute Of Technology Microfluidic method and structure with an elastomeric gas-permeable gasket
WO2008030631A2 (en) * 2006-02-03 2008-03-13 Microchip Biotechnologies, Inc. Microfluidic devices
KR20090006053A (en) * 2006-02-03 2009-01-14 아르카디츠 엘리자로프 A microfluidic method and structure with an elastomeric gas-permeable gasket
US20080003564A1 (en) * 2006-02-14 2008-01-03 Iquum, Inc. Sample processing
AU2007225038B2 (en) * 2006-03-15 2013-08-29 Perkinelmer Health Sciences, Inc. Integrated nucleic acid assays
US7766033B2 (en) * 2006-03-22 2010-08-03 The Regents Of The University Of California Multiplexed latching valves for microfluidic devices and processors
US8741230B2 (en) 2006-03-24 2014-06-03 Theranos, Inc. Systems and methods of sample processing and fluid control in a fluidic system
US11287421B2 (en) 2006-03-24 2022-03-29 Labrador Diagnostics Llc Systems and methods of sample processing and fluid control in a fluidic system
US8088616B2 (en) 2006-03-24 2012-01-03 Handylab, Inc. Heater unit for microfluidic diagnostic system
US8007999B2 (en) 2006-05-10 2011-08-30 Theranos, Inc. Real-time detection of influenza virus
US20100216657A1 (en) * 2006-05-16 2010-08-26 Arcxis Biotechnologies, Inc. Pcr-free sample preparation and detection systems for high speed biologic analysis and identification
WO2007136715A2 (en) * 2006-05-16 2007-11-29 Arcxis Biotechnologies Pcr-free sample preparation and detection systems for high speed biologic analysis and identification
US20080050739A1 (en) 2006-06-14 2008-02-28 Roland Stoughton Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
EP2589668A1 (en) 2006-06-14 2013-05-08 Verinata Health, Inc Rare cell analysis using sample splitting and DNA tags
US20080124721A1 (en) * 2006-06-14 2008-05-29 Martin Fuchs Analysis of rare cell-enriched samples
EP1886727A1 (en) * 2006-07-14 2008-02-13 Roche Diagnostics GmbH Analytical device
WO2008012550A2 (en) 2006-07-28 2008-01-31 Diagnostics For The Real World, Ltd. Device, system and method for processing a sample
GB0618966D0 (en) * 2006-09-26 2006-11-08 Iti Scotland Ltd Cartridge system
WO2008147382A1 (en) * 2006-09-27 2008-12-04 Micronics, Inc. Integrated microfluidic assay devices and methods
US20080131327A1 (en) * 2006-09-28 2008-06-05 California Institute Of Technology System and method for interfacing with a microfluidic chip
JP5382347B2 (en) * 2006-10-11 2014-01-08 フルイディウム コーポレーション Disposable micro purification card, method and system
US8012744B2 (en) 2006-10-13 2011-09-06 Theranos, Inc. Reducing optical interference in a fluidic device
FR2907228B1 (en) * 2006-10-13 2009-07-24 Rhodia Recherches & Tech FLUID FLOW DEVICE, ASSEMBLY FOR DETERMINING AT LEAST ONE CHARACTERISTIC OF A PHYSICO-CHEMICAL SYSTEM COMPRISING SUCH A DEVICE, DETERMINING METHOD AND CORRESPONDING SCREENING METHOD
US8841116B2 (en) * 2006-10-25 2014-09-23 The Regents Of The University Of California Inline-injection microdevice and microfabricated integrated DNA analysis system using same
WO2008061165A2 (en) * 2006-11-14 2008-05-22 Handylab, Inc. Microfluidic cartridge and method of making same
US20080113391A1 (en) 2006-11-14 2008-05-15 Ian Gibbons Detection and quantification of analytes in bodily fluids
US9102911B2 (en) 2009-05-15 2015-08-11 Biofire Diagnostics, Llc High density self-contained biological analysis
CN101563562B (en) * 2006-12-19 2013-09-11 皇家飞利浦电子股份有限公司 Micro fluidic device
EP1935496A1 (en) * 2006-12-22 2008-06-25 Eppendorf Array Technologies SA Device and/or method for the detection of amplified nucleotides sequences on micro-arrays
TW200844420A (en) * 2006-12-22 2008-11-16 3M Innovative Properties Co Enhanced sample processing devices, systems and methods
EP1946841A1 (en) * 2006-12-22 2008-07-23 Eppendorf Array Technologies SA Device and/or method for the detection of amplified nucleotides sequences on micro-arrays
EP2117713B1 (en) 2006-12-22 2019-08-07 DiaSorin S.p.A. Thermal transfer methods for microfluidic systems
US20080163946A1 (en) * 2006-12-28 2008-07-10 The Trustees Of California State University Magnetically controlled valve for flow manipulation in polymer microfluidic devices
JP2008164566A (en) * 2007-01-05 2008-07-17 Yokogawa Electric Corp Cartridge for chemical reaction and method for using the same
US7829032B2 (en) * 2007-01-23 2010-11-09 Siemens Medical Solutions Usa, Inc. Fully-automated microfluidic system for the synthesis of radiolabeled biomarkers for positron emission tomography
US20080245740A1 (en) * 2007-01-29 2008-10-09 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Fluidic methods
EP2109666A4 (en) 2007-02-05 2011-09-14 Integenx Inc Microfluidic and nanofluidic devices, systems, and applications
US7799656B2 (en) * 2007-03-15 2010-09-21 Dalsa Semiconductor Inc. Microchannels for BioMEMS devices
WO2008116941A1 (en) * 2007-03-26 2008-10-02 Fundación Gaiker Method and device for detecting genetic material by means of polymerase chain reaction
US8071035B2 (en) * 2007-04-12 2011-12-06 Siemens Medical Solutions Usa, Inc. Microfluidic radiosynthesis system for positron emission tomography biomarkers
US9618139B2 (en) 2007-07-13 2017-04-11 Handylab, Inc. Integrated heater and magnetic separator
US20090136385A1 (en) * 2007-07-13 2009-05-28 Handylab, Inc. Reagent Tube
USD621060S1 (en) 2008-07-14 2010-08-03 Handylab, Inc. Microfluidic cartridge
US8133671B2 (en) * 2007-07-13 2012-03-13 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US8158430B1 (en) 2007-08-06 2012-04-17 Theranos, Inc. Systems and methods of fluidic sample processing
WO2009024773A1 (en) * 2007-08-17 2009-02-26 Diagnostics For The Real World, Ltd Device, system and method for processing a sample
JP5641184B2 (en) * 2007-09-10 2014-12-17 日本電気株式会社 Microchip sample processing equipment
JP2009083382A (en) * 2007-10-01 2009-04-23 Brother Ind Ltd Image forming device and image processing program
NZ584963A (en) 2007-10-02 2012-11-30 Theranos Inc Modular Point-of-care devices as addressible assay units with tips of assay units having interior to immobilize reagents by capillary action
JP5523327B2 (en) * 2007-10-12 2014-06-18 レオニックス,インコーポレイテッド Integrated microfluidic device and method
US8381169B2 (en) * 2007-10-30 2013-02-19 International Business Machines Corporation Extending unified process and method content to include dynamic and collaborative content
WO2009068025A1 (en) * 2007-11-26 2009-06-04 Atonomics A/S Integrated separation, activation, purification and detection cartridge
WO2009068862A1 (en) * 2007-11-26 2009-06-04 The Secretary Of State For Innovation, Universities And Skills Of Her Majesty's Britannic Government Electrochemical detection using silver nanoparticle labelled antibodies
GB0812679D0 (en) 2008-07-10 2008-08-20 Sec Dep For Innovation Universities Sample carrier for effecting chemical assays
WO2009068585A1 (en) * 2007-11-26 2009-06-04 Atonomics A/S Integrated separation and detection cartridge using magnetic particles with bimodal size distribution
US20090253181A1 (en) * 2008-01-22 2009-10-08 Microchip Biotechnologies, Inc. Universal sample preparation system and use in an integrated analysis system
US8961902B2 (en) * 2008-04-23 2015-02-24 Bioscale, Inc. Method and apparatus for analyte processing
KR100960066B1 (en) 2008-05-14 2010-05-31 삼성전자주식회사 Microfluidic device containing lyophilized reagent therein and analysing method using the same
GB0812681D0 (en) * 2008-07-10 2008-08-20 Sec Dep For Innovation Universities Apparatus and methods for effecting chemical assays
US20100009351A1 (en) * 2008-07-11 2010-01-14 Handylab, Inc. Polynucleotide Capture Materials, and Method of Using Same
USD618820S1 (en) 2008-07-11 2010-06-29 Handylab, Inc. Reagent holder
USD787087S1 (en) 2008-07-14 2017-05-16 Handylab, Inc. Housing
US20100034704A1 (en) * 2008-08-06 2010-02-11 Honeywell International Inc. Microfluidic cartridge channel with reduced bubble formation
US7947492B2 (en) * 2008-08-20 2011-05-24 Northeastern Ohio Universities College Of Medicine Device improving the detection of a ligand
US8037354B2 (en) 2008-09-18 2011-10-11 Honeywell International Inc. Apparatus and method for operating a computing platform without a battery pack
EP2349566B1 (en) 2008-10-03 2016-01-06 Micronics, Inc. Microfluidic apparatus and methods for performing blood typing and crossmatching
US20100093098A1 (en) * 2008-10-14 2010-04-15 Siemens Medical Solutions Nonflow-through appratus and mehod using enhanced flow mechanisms
US8247191B2 (en) * 2008-11-13 2012-08-21 Ritzen Kalle Disposable cassette and method of use for blood analysis on blood analyzer
CN102341691A (en) 2008-12-31 2012-02-01 尹特根埃克斯有限公司 Instrument with microfluidic chip
US7927904B2 (en) 2009-01-05 2011-04-19 Dalsa Semiconductor Inc. Method of making BIOMEMS devices
JP5175778B2 (en) * 2009-03-11 2013-04-03 株式会社東芝 Liquid feeding device
DE102009015395B4 (en) 2009-03-23 2022-11-24 Thinxxs Microtechnology Gmbh Flow cell for treating and/or examining a fluid
US8388908B2 (en) 2009-06-02 2013-03-05 Integenx Inc. Fluidic devices with diaphragm valves
CA2764464A1 (en) 2009-06-05 2010-12-09 Integenx Inc. Universal sample preparation system and use in an integrated analysis system
KR101274113B1 (en) * 2009-09-01 2013-06-13 한국전자통신연구원 Magnetic microvalve using metal ball and manufacturing method thereof
KR101875858B1 (en) 2009-10-19 2018-07-06 테라노스, 인코포레이티드 Integrated health data capture and analysis system
US20110091873A1 (en) * 2009-10-21 2011-04-21 Microfluidic Systems, Inc. Integrated sample preparation and amplification for nucleic acid detection from biological samples
US20110117607A1 (en) * 2009-11-13 2011-05-19 3M Innovative Properties Company Annular compression systems and methods for sample processing devices
USD667561S1 (en) 2009-11-13 2012-09-18 3M Innovative Properties Company Sample processing disk cover
USD638951S1 (en) 2009-11-13 2011-05-31 3M Innovative Properties Company Sample processing disk cover
US8834792B2 (en) 2009-11-13 2014-09-16 3M Innovative Properties Company Systems for processing sample processing devices
USD638550S1 (en) 2009-11-13 2011-05-24 3M Innovative Properties Company Sample processing disk cover
US8584703B2 (en) 2009-12-01 2013-11-19 Integenx Inc. Device with diaphragm valve
CN102740976B (en) 2010-01-29 2016-04-20 精密公司 Sampling-response microfluidic cartridge
US8512538B2 (en) 2010-05-28 2013-08-20 Integenx Inc. Capillary electrophoresis device
CN103589627B (en) * 2010-07-23 2015-11-18 贝克曼考尔特公司 For carrying out heat circulator module and the system of PCR in real time in PCR reaction vessel
US8763642B2 (en) 2010-08-20 2014-07-01 Integenx Inc. Microfluidic devices with mechanically-sealed diaphragm valves
EP2606154B1 (en) 2010-08-20 2019-09-25 Integenx Inc. Integrated analysis system
US8747747B2 (en) 2010-12-29 2014-06-10 Abbott Point Of Care Inc. Reader devices for manipulating multi-fluidic cartridges for sample analysis
CN103282122B (en) 2010-12-29 2016-03-16 雅培医护站股份有限公司 For multi-fluid cylinder tank and their using method of sample analysis
EP2666008B1 (en) 2011-01-21 2021-08-11 Labrador Diagnostics LLC Systems and methods for sample use maximization
WO2012120506A2 (en) 2011-03-09 2012-09-13 Pixcell Medical Technologies Ltd. Disposable cartridge for preparing a sample fluid containing cells for analysis
DE102011005811A1 (en) * 2011-03-18 2012-09-20 Robert Bosch Gmbh Microfluidic valve and microfluidic platform
US8709353B2 (en) * 2011-03-24 2014-04-29 Boehringer Ingelheim Microparts Gmbh Device and method for producing a fluidic connection between cavities
USD672467S1 (en) 2011-05-18 2012-12-11 3M Innovative Properties Company Rotatable sample processing disk
JP2014517291A (en) 2011-05-18 2014-07-17 スリーエム イノベイティブ プロパティズ カンパニー System and method for valve operation of a sample processing apparatus
MX337943B (en) 2011-05-18 2016-03-29 Focus Diagnostics Inc Systems and methods for detecting the presence of a selected volume of material in a sample processing device.
AU2012255144B2 (en) 2011-05-18 2015-01-29 Diasorin Italia S.P.A. Systems and methods for volumetric metering on a sample processing device
WO2013008442A1 (en) * 2011-07-14 2013-01-17 株式会社エンプラス Fluid handling device, fluid handling method, and fluid handling system
US9365418B2 (en) * 2011-09-02 2016-06-14 The Regents Of The University Of California Universal hardware platform and toolset for operating and fabricating microfluidic devices
AU2012315595B2 (en) 2011-09-30 2015-10-22 Becton, Dickinson And Company Unitized reagent strip
USD692162S1 (en) 2011-09-30 2013-10-22 Becton, Dickinson And Company Single piece reagent holder
US8852919B2 (en) * 2011-11-17 2014-10-07 Rheonix, Inc. Microfluidic apparatus, method, and applications
US8663583B2 (en) 2011-12-27 2014-03-04 Honeywell International Inc. Disposable cartridge for fluid analysis
US8741233B2 (en) 2011-12-27 2014-06-03 Honeywell International Inc. Disposable cartridge for fluid analysis
US8741234B2 (en) 2011-12-27 2014-06-03 Honeywell International Inc. Disposable cartridge for fluid analysis
US8741235B2 (en) 2011-12-27 2014-06-03 Honeywell International Inc. Two step sample loading of a fluid analysis cartridge
BR112014018995B1 (en) 2012-02-03 2021-01-19 Becton, Dickson And Company systems to perform automated testing
US11485968B2 (en) 2012-02-13 2022-11-01 Neumodx Molecular, Inc. Microfluidic cartridge for processing and detecting nucleic acids
US9637775B2 (en) * 2012-02-13 2017-05-02 Neumodx Molecular, Inc. System and method for processing biological samples
US11648561B2 (en) 2012-02-13 2023-05-16 Neumodx Molecular, Inc. System and method for processing and detecting nucleic acids
US9101930B2 (en) 2012-02-13 2015-08-11 Neumodx Molecular, Inc. Microfluidic cartridge for processing and detecting nucleic acids
US9675973B2 (en) * 2012-06-14 2017-06-13 Paratus Diagnostics, LLC Specimen delivery apparatus
EP2684609A1 (en) * 2012-07-09 2014-01-15 Biocartis SA Heater for a disposable dignostics cartridge
EP2872892B1 (en) 2012-07-10 2017-12-20 Lexogen GmbH Flexible dna sensor carrier and method
GB201217390D0 (en) 2012-09-28 2012-11-14 Agplus Diagnostics Ltd Test device and sample carrier
JP6935167B2 (en) 2012-12-21 2021-09-15 ペルキネルマー ヘルス サイエンシーズ, インコーポレイテッド Low elasticity film for microfluidic use
EP2935908B1 (en) 2012-12-21 2019-08-14 PerkinElmer Health Sciences, Inc. Fluidic circuits and related manufacturing methods
KR20150097764A (en) 2012-12-21 2015-08-26 마이크로닉스 인코포레이티드. Portable fluorescence detection system and microassay cartridge
JP6202713B2 (en) * 2013-02-22 2017-09-27 株式会社日立ハイテクノロジーズ Biochemical cartridge and biochemical feed system
EP2994750B1 (en) 2013-05-07 2020-08-12 PerkinElmer Health Sciences, Inc. Microfluidic devices and methods for performing serum separation and blood cross-matching
CA2911303C (en) 2013-05-07 2021-02-16 Micronics, Inc. Methods for preparation of nucleic acid-containing samples using clay minerals and alkaline solutions
US10087440B2 (en) 2013-05-07 2018-10-02 Micronics, Inc. Device for preparation and analysis of nucleic acids
GB2514614A (en) * 2013-05-31 2014-12-03 Simon Johnson Chemical process apparatus and methods
CN104329484B (en) * 2013-06-24 2018-11-30 浙江盾安禾田金属有限公司 The miniature valve of pollution resistance with enhancing
US10376880B2 (en) * 2013-07-30 2019-08-13 Carehealth America Corporation Lateral flow devices and methods of manufacture and use
WO2015073999A1 (en) 2013-11-18 2015-05-21 Integenx Inc. Cartridges and instruments for sample analysis
EP2878375A1 (en) * 2013-11-29 2015-06-03 Genewave Microfluidic cartridge for molecular diagnosis, docking station using such a microfluidic cartridge, and process for analyzing a biological sample
WO2015086863A1 (en) * 2013-12-11 2015-06-18 Ikerlan, S. Coop. Multiplexed valve for microfluidic devices
JP6548356B2 (en) * 2014-03-20 2019-07-24 キヤノンメディカルシステムズ株式会社 Liquid transfer device
WO2015179098A1 (en) 2014-05-21 2015-11-26 Integenx Inc. Fluidic cartridge with valve mechanism
EP3151965B1 (en) 2014-06-04 2021-02-24 Edan Instruments, Inc. Sample collection and analysis devices
CN107106983B (en) 2014-10-22 2021-04-16 尹特根埃克斯有限公司 Systems and methods for sample preparation, processing, and analysis
JP6619429B2 (en) * 2014-12-03 2019-12-11 ベンド リサーチ, インコーポレイテッド Disposable cell removal system
US10094490B2 (en) 2015-06-16 2018-10-09 Dunan Microstaq, Inc. Microvalve having contamination resistant features
US10040069B2 (en) * 2015-07-23 2018-08-07 General Electric Company Amplification and detection of nucleic acids
US9682378B1 (en) 2015-12-08 2017-06-20 Paratus Diagnostics, LLC Mating adaptor for coupling a point-of-care diagnostic cartridge to a computing device
US10436781B2 (en) 2016-01-27 2019-10-08 Paratus Diagnostics, LLC Point-of-care diagnostic cartridge having a digital micro-fluidic testing substrate
US9901014B2 (en) * 2016-04-15 2018-02-20 Ford Global Technologies, Llc Peristaltic pump for power electronics assembly
AU2017330438A1 (en) * 2016-09-23 2019-05-16 ArcherDX, Inc. Fluidic system and related methods
US11305278B2 (en) 2016-10-07 2022-04-19 Boehringer Ingelheim Vetmedica Gmbh Cartridge for testing a biological sample
US20200016590A1 (en) * 2016-10-07 2020-01-16 Boehringer Ingelheim Vetmedica Gmbh Analysis device, cartridge and method for testing a sample
KR20190104041A (en) * 2016-12-29 2019-09-05 아도르 디아그노스틱스 에스.알.엘. Electrophoresis chip for electrophoretic applications
US9719892B1 (en) 2017-01-30 2017-08-01 Paratus Diagnostic, Llc Processing device for processing a highly viscous sample
BR112021006315A2 (en) 2018-10-01 2021-07-06 Boehringer Ingelheim Vetmedica Gmbh peristaltic pump and analyzer to test a sample
US20210001333A1 (en) * 2019-07-03 2021-01-07 King Abdulaziz University Microfluidic device for measuring the enzymatic activity of thiopurin s-methyltransferase
US20210115368A1 (en) * 2019-10-17 2021-04-22 Roman Serheyevich Voronov Automated addressable microfluidic technology for minimally disruptive manipulation of cells and fluids within living cultures
WO2021100189A1 (en) * 2019-11-22 2021-05-27 株式会社日立ハイテク Pcr vessel, pcr vessel support device, thermal cycler, and genetic testing device
JP2022049382A (en) * 2020-09-16 2022-03-29 株式会社エンプラス Fluid handling device and manufacturing method of fluid handling device
WO2022098747A1 (en) * 2020-11-03 2022-05-12 Single Helix Genomics, Inc. Nucleic acid synthesis device and methods of use
JP2023063027A (en) * 2021-10-22 2023-05-09 株式会社エンプラス Fluid handling device and fluid handling system including the same

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4849340A (en) * 1987-04-03 1989-07-18 Cardiovascular Diagnostics, Inc. Reaction system element and method for performing prothrombin time assay
US5587128A (en) * 1992-05-01 1996-12-24 The Trustees Of The University Of Pennsylvania Mesoscale polynucleotide amplification devices

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1617732C2 (en) * 1966-03-01 1972-12-21 Promoveo-Sobioda & Cie, Seyssinet (Frankreich) Device for examining living cells of microorganisms
US4761381A (en) * 1985-09-18 1988-08-02 Miles Inc. Volume metering capillary gap device for applying a liquid sample onto a reactive surface
DE4022655A1 (en) * 1990-07-17 1992-01-23 Boehringer Mannheim Gmbh TEST KIT FOR DETERMINING ANALYTES IN A PASTOESE SAMPLE, ESPECIALLY IN CHAIR
US5208163A (en) * 1990-08-06 1993-05-04 Miles Inc. Self-metering fluid analysis device
US5154888A (en) * 1990-10-25 1992-10-13 Eastman Kodak Company Automatic sealing closure means for closing off a passage in a flexible cuvette
US5605662A (en) * 1993-11-01 1997-02-25 Nanogen, Inc. Active programmable electronic devices for molecular biological analysis and diagnostics
US5288463A (en) * 1992-10-23 1994-02-22 Eastman Kodak Company Positive flow control in an unvented container
US5500187A (en) * 1992-12-08 1996-03-19 Westinghouse Electric Corporation Disposable optical agglutination assay device and method for use
US5302348A (en) * 1992-12-10 1994-04-12 Itc Corporation Blood coagulation time test apparatus and method
US5627041A (en) * 1994-09-02 1997-05-06 Biometric Imaging, Inc. Disposable cartridge for an assay of a biological sample
US5631166A (en) * 1995-03-21 1997-05-20 Jewell; Charles R. Specimen disk for blood analyses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4849340A (en) * 1987-04-03 1989-07-18 Cardiovascular Diagnostics, Inc. Reaction system element and method for performing prothrombin time assay
US5587128A (en) * 1992-05-01 1996-12-24 The Trustees Of The University Of Pennsylvania Mesoscale polynucleotide amplification devices

Cited By (178)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6007690A (en) * 1996-07-30 1999-12-28 Aclara Biosciences, Inc. Integrated microfluidic devices
US6344326B1 (en) 1996-07-30 2002-02-05 Aclara Bio Sciences, Inc. Microfluidic method for nucleic acid purification and processing
NL1006813C1 (en) * 1997-08-20 1998-01-21 Sipke Wadman Packaging containing solid reaction carrier for chemical synthesis
WO1999023492A1 (en) * 1997-10-31 1999-05-14 Sarnoff Corporation Method for enhancing fluorescence
US6118126A (en) * 1997-10-31 2000-09-12 Sarnoff Corporation Method for enhancing fluorescence
US6140110A (en) * 1998-03-05 2000-10-31 Vinayagamoorthy; Thuraiayah Apparatus for multi-zone polymerase chain reaction
WO1999045141A1 (en) * 1998-03-05 1999-09-10 Thuraiayah Vinayagamoorthy Multi-zone polymerase/ligase chain reaction
US7655129B2 (en) 1998-06-23 2010-02-02 Osmetech Technology Inc. Binding acceleration techniques for the detection of analytes
US7833489B2 (en) 1998-06-24 2010-11-16 Chen & Chen, Llc Fluid sample testing system
US9005551B2 (en) 1998-06-24 2015-04-14 Roche Molecular Systems, Inc. Sample vessels
US6748332B2 (en) 1998-06-24 2004-06-08 Chen & Chen, Llc Fluid sample testing system
US7337072B2 (en) 1998-06-24 2008-02-26 Chen & Chen, Llc Fluid sample testing system
US10022722B2 (en) 1998-06-24 2018-07-17 Roche Molecular Systems, Inc. Sample vessels
EP1110084A4 (en) * 1998-08-03 2001-06-27 Qualisys Diagnostics Inc Methods and apparatus for conducting tests
EP1110084A1 (en) * 1998-08-03 2001-06-27 Qualisys Diagnostics Inc. Methods and apparatus for conducting tests
AU747591B2 (en) * 1998-09-08 2002-05-16 Bio Merieux Microfluid system for reactions and transfers
FR2782934A1 (en) * 1998-09-08 2000-03-10 Bio Merieux Analysis card for medical diagnostics with built in valve isolating analysis chambers
EP1201305A3 (en) * 1998-09-08 2002-11-13 Bio Merieux Microfluidic reaction and transfer system
WO2000013795A1 (en) * 1998-09-08 2000-03-16 Bio Merieux Microfluid system for reactions and transfers
US6929239B1 (en) 1998-09-08 2005-08-16 Bio Merieux Microfluid system for reactions and transfers
US6649404B1 (en) 1999-01-08 2003-11-18 Applera Corporation Method for using and making a fiber array
US7595189B2 (en) 1999-01-08 2009-09-29 Applied Biosystems, Llc Integrated optics fiber array
US6573089B1 (en) 1999-01-08 2003-06-03 Applera Corporation Method for using and making a fiber array
WO2000040334A1 (en) * 1999-01-08 2000-07-13 Pe Corporation (Ny) Fiber array for contacting chemical species and methods for using and making same
US6982149B2 (en) 1999-01-08 2006-01-03 Applera Corporation Fiber array and methods for using and making same
AU772719B2 (en) * 1999-01-08 2004-05-06 Applera Corporation Fiber array for contacting chemical species and methods for using and making same
US6635470B1 (en) 1999-01-08 2003-10-21 Applera Corporation Fiber array and methods for using and making same
FR2790681A1 (en) * 1999-03-09 2000-09-15 Biomerieux Sa PUMPING DEVICE FOR TRANSFERRING AT LEAST ONE FLUID INTO A CONSUMABLE
US7169353B1 (en) 1999-03-09 2007-01-30 Biomerieux S.A. Apparatus enabling liquid transfer by capillary action therein
WO2000053320A1 (en) * 1999-03-09 2000-09-14 Biomerieux S.A. Pumping device for transferring at least a fluid into a consumable
US9557295B2 (en) 1999-04-21 2017-01-31 Osmetech Technology, Inc. Use of microfluidic systems in the electrochemical detection of target analytes
US9151746B2 (en) 1999-04-21 2015-10-06 Osmetech Technology, Inc. Use of microfluidic systems in the electrochemical detection of target analytes
US8486247B2 (en) 1999-04-21 2013-07-16 Osmetch Technology, Inc. Use of microfluidic systems in the electrochemical detection of target analytes
US9289766B2 (en) 1999-05-21 2016-03-22 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
US8883424B2 (en) 1999-05-21 2014-11-11 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
US6902706B1 (en) * 1999-06-22 2005-06-07 Biomerieux S.A. Valves enabling a liquid to be directed in a diagnostic chart diagnostic charts and diagnostic device comprising several charts
WO2000078453A1 (en) * 1999-06-22 2000-12-28 Biomerieux S.A. Valves enabling a liquid to be directed in a diagnostic chart, diagnostic charts and diagnostic device comprising several charts
FR2795476A1 (en) 1999-06-22 2000-12-29 Biomerieux Sa Valve for directing fluid to a diagnostic card, used in micro-assay devices, has thin and/or deformable film partly attached to surface of diagnostic card, and device for compressing the film
US7491497B2 (en) 1999-06-22 2009-02-17 Biomerieux S.A. Device for implementing an analysis pack, analysis pack and method using same
FR2798867A1 (en) * 1999-09-23 2001-03-30 Commissariat Energie Atomique Device for injecting fluids simultaneously into microfluidic system for biochemical reactions has layer with deformable small diameter, open ended pipes and device for pressurizing fluids in pipes
US6875619B2 (en) 1999-11-12 2005-04-05 Motorola, Inc. Microfluidic devices comprising biochannels
US7259021B2 (en) 2000-03-07 2007-08-21 Bio Merieux Method for using a test card
US9926521B2 (en) 2000-06-27 2018-03-27 Fluidigm Corporation Microfluidic particle-analysis systems
US8435462B2 (en) 2000-06-28 2013-05-07 3M Innovative Properties Company Sample processing devices
WO2002018823A1 (en) * 2000-08-28 2002-03-07 Biomerieux S.A. Reaction card and use of same
US7537730B2 (en) 2000-08-28 2009-05-26 Biomerieux S.A. Reaction card and use of same
FR2813207A1 (en) * 2000-08-28 2002-03-01 Bio Merieux REACTIONAL CARD AND USE OF SUCH A CARD
EP1327474A1 (en) * 2000-09-22 2003-07-16 Kawamura Institute Of Chemical Research Very small chemical device and flow rate adjusting method therefor
EP1327474A4 (en) * 2000-09-22 2004-12-29 Kawamura Inst Chem Res Very small chemical device and flow rate adjusting method therefor
US7238325B2 (en) 2000-09-22 2007-07-03 Kawamura Institute Of Chemical Research Very small chemical device and flow rate adjusting method therefor
US8455258B2 (en) 2000-11-16 2013-06-04 California Insitute Of Technology Apparatus and methods for conducting assays and high throughput screening
US8673645B2 (en) 2000-11-16 2014-03-18 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US8273574B2 (en) 2000-11-16 2012-09-25 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
EP1343973A1 (en) * 2000-11-16 2003-09-17 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US10509018B2 (en) 2000-11-16 2019-12-17 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US9176137B2 (en) 2000-11-16 2015-11-03 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
EP2381116A1 (en) * 2000-11-16 2011-10-26 California Institute of Technology Apparatus and methods for conducting assays and high throughput screening
US7887753B2 (en) 2000-11-16 2011-02-15 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
EP1343973A4 (en) * 2000-11-16 2004-12-22 California Inst Of Techn Apparatus and methods for conducting assays and high throughput screening
US6994314B2 (en) 2000-12-01 2006-02-07 Biomerieux S. A. Valves activated by electrically active polymers or by shape-memory materials, device containing same and method for using same
WO2002044566A1 (en) 2000-12-01 2002-06-06 Biomerieux S.A. Valves activated by electrically active polymers or by shape-memory materials, device containing same and method for using same
FR2817604A1 (en) 2000-12-01 2002-06-07 Biomerieux Sa VALVES ACTIVATED BY ELECTRO-ACTIVE POLYMERS OR BY SHAPE-MEMORY MATERIALS, DEVICE CONTAINING SUCH VALVES AND METHOD FOR IMPLEMENTING
US9662652B2 (en) 2000-12-29 2017-05-30 Chen & Chen, Llc Sample processing device for pretreatment and thermal cycling
US6964862B2 (en) 2000-12-29 2005-11-15 Chen & Chen, Llc Sample processing device and method
US8148116B2 (en) 2000-12-29 2012-04-03 Chen & Chen, Llc Sample processing device for pretreatment and thermal cycling
US6780617B2 (en) 2000-12-29 2004-08-24 Chen & Chen, Llc Sample processing device and method
US7935504B2 (en) 2000-12-29 2011-05-03 Chen & Chen, Llc Thermal cycling methods
US8486636B2 (en) 2001-04-06 2013-07-16 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US8936764B2 (en) 2001-04-06 2015-01-20 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US7833708B2 (en) 2001-04-06 2010-11-16 California Institute Of Technology Nucleic acid amplification using microfluidic devices
WO2003022435A3 (en) * 2001-09-11 2003-12-04 Iquum Inc Sample vessels
JP2003094395A (en) * 2001-09-26 2003-04-03 Olympus Optical Co Ltd Arrayed micro fluid control device
US8163492B2 (en) 2001-11-30 2012-04-24 Fluidign Corporation Microfluidic device and methods of using same
US7820427B2 (en) 2001-11-30 2010-10-26 Fluidigm Corporation Microfluidic device and methods of using same
JP2003166910A (en) * 2001-11-30 2003-06-13 Asahi Kasei Corp Liquid-feeding mechanism and analyzer provided with the same
JP2003287479A (en) * 2002-03-28 2003-10-10 Asahi Kasei Corp Valve mechanism
US8658418B2 (en) 2002-04-01 2014-02-25 Fluidigm Corporation Microfluidic particle-analysis systems
EP1531936A4 (en) * 2002-07-26 2005-09-07 Applera Corp Actuator for deformable valves in a microfluidic device, and method
US7201881B2 (en) 2002-07-26 2007-04-10 Applera Corporation Actuator for deformable valves in a microfluidic device, and method
EP1531936A2 (en) * 2002-07-26 2005-05-25 Applera Corporation Actuator for deformable valves in a microfluidic device, and method
US9714443B2 (en) 2002-09-25 2017-07-25 California Institute Of Technology Microfabricated structure having parallel and orthogonal flow channels controlled by row and column multiplexors
US8871446B2 (en) 2002-10-02 2014-10-28 California Institute Of Technology Microfluidic nucleic acid analysis
US10328428B2 (en) 2002-10-02 2019-06-25 California Institute Of Technology Apparatus for preparing cDNA libraries from single cells
US10940473B2 (en) 2002-10-02 2021-03-09 California Institute Of Technology Microfluidic nucleic acid analysis
US9579650B2 (en) 2002-10-02 2017-02-28 California Institute Of Technology Microfluidic nucleic acid analysis
JP2004212361A (en) * 2003-01-09 2004-07-29 Yokogawa Electric Corp Cartridge for biochip
JP2004226207A (en) * 2003-01-22 2004-08-12 Asahi Kasei Corp Liquid-feeding mechanism and analyzer provided with the same
US10443050B2 (en) 2003-02-05 2019-10-15 Roche Molecular Systems, Inc. Sample processing methods
US8936933B2 (en) 2003-02-05 2015-01-20 IQumm, Inc. Sample processing methods
US9708599B2 (en) 2003-02-05 2017-07-18 Roche Molecular Systems, Inc. Sample processing methods
US7854897B2 (en) 2003-05-12 2010-12-21 Yokogawa Electric Corporation Chemical reaction cartridge, its fabrication method, and a chemical reaction cartridge drive system
JP2005037368A (en) * 2003-05-12 2005-02-10 Yokogawa Electric Corp Cartridge for chemical reaction, its manufacturing method, and driving system for cartridge for chemical reaction
US9061280B2 (en) 2003-05-12 2015-06-23 Yokogawa Electric Corporation Chemical reaction cartridge, its fabrication method, and a chemical reaction cartridge drive system
US7170594B2 (en) 2003-05-28 2007-01-30 Smiths Detection, Inc. Device for polymerase chain reactions
WO2005009617A1 (en) * 2003-05-28 2005-02-03 Smiths Detection Inc. Device for polymerase chain reactions
US7648835B2 (en) 2003-06-06 2010-01-19 Micronics, Inc. System and method for heating, cooling and heat cycling on microfluidic device
US10865437B2 (en) 2003-07-31 2020-12-15 Handylab, Inc. Processing particle-containing samples
US11078523B2 (en) 2003-07-31 2021-08-03 Handylab, Inc. Processing particle-containing samples
US11441171B2 (en) 2004-05-03 2022-09-13 Handylab, Inc. Method for processing polynucleotide-containing samples
US7473551B2 (en) 2004-05-21 2009-01-06 Atonomics A/S Nano-mechanic microsensors and methods for detecting target analytes
EP1625888A3 (en) * 2004-08-13 2006-06-07 Alps Electric Co., Ltd. Test plate and test method using the same
EP1625888A2 (en) * 2004-08-13 2006-02-15 Alps Electric Co., Ltd. Test plate and test method using the same
WO2006045619A1 (en) * 2004-10-28 2006-05-04 Directif Gmbh Process and device for processing biopolymers in parallel
CN102929309A (en) * 2005-01-25 2013-02-13 欧西里其有限责任公司 Temperature controller for small fluid samples having different heat capacities
US9568424B2 (en) 2005-06-23 2017-02-14 Biocartis Nv Cartridge, system and method for automated medical diagnostics
EP2409767A1 (en) 2005-06-23 2012-01-25 Biocartis SA Modular cartridge, system and method for automated medical diagnosis
EP2520367A1 (en) * 2005-10-03 2012-11-07 Rheonix, Inc. Microfluidic membrane pump and valve
EP2520368A1 (en) * 2005-10-03 2012-11-07 Rheonix, Inc. Microfluidic membrane pump and valve
US7815868B1 (en) 2006-02-28 2010-10-19 Fluidigm Corporation Microfluidic reaction apparatus for high throughput screening
US8420017B2 (en) 2006-02-28 2013-04-16 Fluidigm Corporation Microfluidic reaction apparatus for high throughput screening
US10821436B2 (en) 2006-03-24 2020-11-03 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using the same
US11141734B2 (en) 2006-03-24 2021-10-12 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
US11806718B2 (en) 2006-03-24 2023-11-07 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
US11666903B2 (en) 2006-03-24 2023-06-06 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using same
US11142785B2 (en) 2006-03-24 2021-10-12 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US11085069B2 (en) 2006-03-24 2021-08-10 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US10913061B2 (en) 2006-03-24 2021-02-09 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using the same
US10900066B2 (en) 2006-03-24 2021-01-26 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US10857535B2 (en) 2006-03-24 2020-12-08 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using same
US10843188B2 (en) 2006-03-24 2020-11-24 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using the same
US10821446B1 (en) 2006-03-24 2020-11-03 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
US10799862B2 (en) 2006-03-24 2020-10-13 Handylab, Inc. Integrated system for processing microfluidic samples, and method of using same
US10744469B2 (en) 2007-06-21 2020-08-18 Gen-Probe Incorporated Multi-chambered receptacles
US8735055B2 (en) 2007-06-21 2014-05-27 Gen-Probe Incorporated Methods of concentrating an analyte
WO2009002447A1 (en) * 2007-06-21 2008-12-31 Gen-Probe Incorporated Instrument and receptacles for use in performing processes
US8491178B2 (en) 2007-06-21 2013-07-23 Gen-Probe Incorporated Instruments and methods for mixing the contents of a detection chamber
US8480976B2 (en) 2007-06-21 2013-07-09 Gen-Probe Incorporated Instruments and methods for mixing the contents of a detection chamber
US9744506B2 (en) 2007-06-21 2017-08-29 Gen-Probe Incorporated Instruments for mixing the contents of a detection chamber
US8765367B2 (en) 2007-06-21 2014-07-01 Gen-Probe Incorporated Methods and instruments for processing a sample in a multi-chambered receptacle
US11235295B2 (en) 2007-06-21 2022-02-01 Gen-Probe Incorporated System and method of using multi-chambered receptacles
US11235294B2 (en) 2007-06-21 2022-02-01 Gen-Probe Incorporated System and method of using multi-chambered receptacles
US8828654B2 (en) 2007-06-21 2014-09-09 Gen-Probe Incorporated Methods for manipulating liquid substances in multi-chambered receptacles
US7767447B2 (en) 2007-06-21 2010-08-03 Gen-Probe Incorporated Instruments and methods for exposing a receptacle to multiple thermal zones
US10086342B2 (en) 2007-06-21 2018-10-02 Gen-Probe Incorporated Multi-channel optical measurement instrument
US7780336B2 (en) 2007-06-21 2010-08-24 Gen-Probe Incorporated Instruments and methods for mixing the contents of a detection chamber
US8221705B2 (en) 2007-06-21 2012-07-17 Gen-Probe, Incorporated Receptacles for storing substances in different physical states
US8048375B2 (en) 2007-06-21 2011-11-01 Gen-Probe Incorporated Gravity-assisted mixing methods
US8052929B2 (en) 2007-06-21 2011-11-08 Gen-Probe Incorporated Gravity-assisted mixing methods
US9458451B2 (en) 2007-06-21 2016-10-04 Gen-Probe Incorporated Multi-channel optical measurement instrument
US8784745B2 (en) 2007-06-21 2014-07-22 Gen-Probe Incorporated Methods for manipulating liquid substances in multi-chambered receptacles
US10688458B2 (en) 2007-06-21 2020-06-23 Gen-Probe Incorporated System and method of using multi-chambered receptacles
US11254927B2 (en) 2007-07-13 2022-02-22 Handylab, Inc. Polynucleotide capture materials, and systems using same
US10875022B2 (en) 2007-07-13 2020-12-29 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US11845081B2 (en) 2007-07-13 2023-12-19 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US11266987B2 (en) 2007-07-13 2022-03-08 Handylab, Inc. Microfluidic cartridge
US11060082B2 (en) 2007-07-13 2021-07-13 Handy Lab, Inc. Polynucleotide capture materials, and systems using same
US10844368B2 (en) 2007-07-13 2020-11-24 Handylab, Inc. Diagnostic apparatus to extract nucleic acids including a magnetic assembly and a heater assembly
US11466263B2 (en) 2007-07-13 2022-10-11 Handylab, Inc. Diagnostic apparatus to extract nucleic acids including a magnetic assembly and a heater assembly
US11549959B2 (en) 2007-07-13 2023-01-10 Handylab, Inc. Automated pipetting apparatus having a combined liquid pump and pipette head system
US8216832B2 (en) 2007-07-31 2012-07-10 Micronics, Inc. Sanitary swab collection system, microfluidic assay device, and methods for diagnostic assays
DE102010003782B4 (en) 2010-04-08 2023-09-28 Ist Innuscreen Gmbh Device for detecting nucleic acids
US9399793B2 (en) 2010-04-08 2016-07-26 Aj Innuscreen Gmbh Device for detecting nucleic acids
WO2011124688A1 (en) * 2010-04-08 2011-10-13 Aj Innuscreen Gmbh Device for detecting nucleic acids
US10641707B2 (en) 2011-02-24 2020-05-05 Gen-Probe Incorporated Systems and methods for distinguishing optical signals of different modulation frequencies in an optical signal detector
US9915613B2 (en) 2011-02-24 2018-03-13 Gen-Probe Incorporated Systems and methods for distinguishing optical signals of different modulation frequencies in an optical signal detector
US11788127B2 (en) 2011-04-15 2023-10-17 Becton, Dickinson And Company Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection
US10865440B2 (en) * 2011-10-21 2020-12-15 IntegenX, Inc. Sample preparation, processing and analysis systems
US11684918B2 (en) 2011-10-21 2023-06-27 IntegenX, Inc. Sample preparation, processing and analysis systems
US20150024436A1 (en) * 2011-10-21 2015-01-22 Integenx Inc, Sample preparation, processing and analysis systems
US11453906B2 (en) 2011-11-04 2022-09-27 Handylab, Inc. Multiplexed diagnostic detection apparatus and methods
DE102011056273A1 (en) * 2011-12-12 2013-06-13 sense2care GmbH Fluid reservoir for a device for analyzing patient samples
DE102011056273B4 (en) * 2011-12-12 2013-11-21 sense2care GmbH Fluid reservoir for a device for analyzing patient samples
WO2013087567A1 (en) 2011-12-12 2013-06-20 sense2care GmbH Fluid reservoir for a device for analyzing patient samples
WO2014191519A1 (en) * 2013-05-30 2014-12-04 Commissariat à l'énergie atomique et aux énergies alternatives Fluidic card comprising a fluidic channel provided with an opening resealable by means of a flexible film
US10086371B2 (en) 2013-05-30 2018-10-02 Commisariat à l'énergie atomique et aux énergies alternatives Fluidic card comprising a fluidic channel provided with an opening resealable by means of a flexible film
FR3006207A1 (en) * 2013-05-30 2014-12-05 Commissariat Energie Atomique FLUID CARD COMPRISING A FLUIDIC CHANNEL HAVING A REFERMABLE OPENING BY A FLEXIBLE FILM
US10821435B2 (en) 2014-09-02 2020-11-03 Canon Medical Systems Corporation Nucleic acid detection cassette
JP2016052253A (en) * 2014-09-02 2016-04-14 株式会社東芝 Nucleic acid detection cassette
JP2016052254A (en) * 2014-09-02 2016-04-14 株式会社東芝 Nucleic acid detection cassette
US11027281B2 (en) 2015-02-02 2021-06-08 Binx Health Limited Instrument for performing a diagnostic test on a fluidic cartridge
US11666919B2 (en) 2015-02-02 2023-06-06 Binx Health Limited Instrument for performing a diagnostic test on a fluidic cartridge
WO2016124908A1 (en) * 2015-02-02 2016-08-11 Atlas Genetics Limited Instrument for performing a diagnostic test on a fluidic cartridge
US11813613B2 (en) 2015-02-02 2023-11-14 Binx Health Limited Instrument for performing a diagnostic test on a fluidic cartridge
WO2016128570A1 (en) * 2015-02-13 2016-08-18 Espci Paper device for genetic diagnosis
WO2018052768A1 (en) * 2016-09-16 2018-03-22 General Electric Company Compact valve array with actuator system
US10525466B2 (en) 2016-09-16 2020-01-07 General Electric Company Compact valve array with actuation system
WO2018153950A1 (en) * 2017-02-22 2018-08-30 Selfdiagnostics Deutschland Gmbh Microfluidic test device
DE102022203778A1 (en) 2022-04-14 2023-10-19 Robert Bosch Gesellschaft mit beschränkter Haftung Microfluidic cartridge with a trench-shaped depression to prevent heat conduction in the outer wall

Also Published As

Publication number Publication date
AU1825197A (en) 1997-08-20
US5863502A (en) 1999-01-26

Similar Documents

Publication Publication Date Title
US5863502A (en) Parallel reaction cassette and associated devices
US5882903A (en) Assay system and method for conducting assays
KR19990067304A (en) Assay system and method for performing the assay
US6875619B2 (en) Microfluidic devices comprising biochannels
US9498776B2 (en) Microfluidic devices with removable cover and methods of fabrication and application
US9316331B2 (en) Multilevel microfluidic systems and methods
US8323887B2 (en) Miniaturized fluid delivery and analysis system
US5863801A (en) Automated nucleic acid isolation
US8986927B2 (en) Reaction system for performing in the amplification of nucleic acids
JP5250669B2 (en) Microfluidic structure, pathogen detection system and method for pathogen analysis
US8067176B2 (en) Microchemistry reaction method
US6319472B1 (en) System including functionally separated regions in electrophoretic system
KR20120051709A (en) Microfluidic devices and uses thereof
CA2393690A1 (en) Multilayered microfluidic devices for analyte reactions
WO2010077618A1 (en) Programmable microfluidic digital array
KR20110030415A (en) Universal sample preparation system and use in an integrated analysis system
ZA200504838B (en) Method and apparatus for pathogen detection and analysis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UG UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97526336

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase