WO2002016652A2 - Dispositif de traitement rapide d'echantillons d'adn, par manipulation, thermocyclage et purification integrees de liquides - Google Patents

Dispositif de traitement rapide d'echantillons d'adn, par manipulation, thermocyclage et purification integrees de liquides Download PDF

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Publication number
WO2002016652A2
WO2002016652A2 PCT/US2001/026611 US0126611W WO0216652A2 WO 2002016652 A2 WO2002016652 A2 WO 2002016652A2 US 0126611 W US0126611 W US 0126611W WO 0216652 A2 WO0216652 A2 WO 0216652A2
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Prior art keywords
dialysis
chamber
membrane
sample
nucleotide
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PCT/US2001/026611
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English (en)
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WO2002016652A3 (fr
Inventor
James Benn
Jean-Francois Manchec
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Genome Therapeutics Corporation
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Application filed by Genome Therapeutics Corporation filed Critical Genome Therapeutics Corporation
Priority to AU2001286784A priority Critical patent/AU2001286784A1/en
Publication of WO2002016652A2 publication Critical patent/WO2002016652A2/fr
Publication of WO2002016652A3 publication Critical patent/WO2002016652A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers 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
    • B01L3/50255—Multi-well filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1838—Means for temperature control using fluid heat transfer medium
    • B01L2300/1844—Means for temperature control using fluid heat transfer medium using fans
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • G01N2001/4016—Concentrating samples by transferring a selected component through a membrane being a selective membrane, e.g. dialysis or osmosis

Definitions

  • the invention relates to devices and methods for high speed, low volume automated sample handling of biological samples, which are useful in the field of genomics for a variety of processes, including DNA sequencing, genetic analysis, and gene expression analysis.
  • the invention further relates to devices and methods for preparing and executing assays for high throughput compound screening for pharmaceutical applications.
  • the present invention addresses the foregoing by providing a flat plate dialysis or ultrafiltration cell having a syringe docking port, a sample chamber fluidly coupled to a distal end ofthe syringe docking port, and a vent hole fluidly coupled to the sample chamber.
  • the flat plate cell further includes a membrane for separating or filtering a sample by means of unequal diffusion, e.g., by size exclusion.
  • the present invention provides a flat plate dialysis system, comprising a needle guide having an upper surface and a bottom surface, the upper surface being adapted to receive a needle, an upper channel plate mounted along an upper surface to the bottom surface ofthe needle guide, and at least one dialysis chamber formed along a bottom surface ofthe upper channel plate.
  • the present invention involves a method for performing dialysis on a biological sample comprising the steps of providing a dialysis chamber having a flat dialysis membrane along one side ofthe dialysis chamber, a syringe docking port, and a vent hole each located near an opposite end ofthe dialysis chamber.
  • the method also includes the steps of injecting a sample from a needle through a syringe docking port into a dialysis chamber and into contact with a first side ofthe dialysis membrane, and applying a dialysis solution to a second side ofthe dialysis membrane opposite the first side ofthe dialysis membrane.
  • a flat plate ultrafiltration system is provided.
  • the flat plate ultrafiltration system comprises a syringe docking port, a sample chamber fluidly coupled to a distal end ofthe syringe docking port, an ultrafiltration membrane provided along a portion ofthe sample chamber and a device for applying a pressure differential to a sample in the sample chamber.
  • a method for performing ultrafiltration on a biological sample comprises providing a sample chamber having an ultrafiltration membrane a syringe docking port in fluid communication with said sample chamber, injecting a sample from a needle through the syringe docking port into the sample chamber and into contact with a first side the ultrafiltration membrane, and applying a pressure differential to the sample chamber to perform ultrafiltration of the sample.
  • a method for detecting the presence or absence of a first nucleotide, at a position within a strand of DNA in a sample using the flat plate system is provided.
  • a method of performing a polymerase chain reaction comprises providing a sample chamber having a membrane along one side ofthe sample chamber and having a syringe docking port in fluid communication with the sample chamber, wherein the membrane incorporates appropriate reagents for the performance of a polymerase chain reaction.
  • the method further comprises injecting a biological sample from a needle through the syringe docking port into said sample chamber and into contact with a first side of said membrane and imposing conditions on the sample chamber such that a polymerase chain reaction occurs.
  • a packaged kit for performing a polymerase chain reaction comprising a flat plate separation system comprising a sample chamber having a membrane along one side ofthe sample chamber and having a syringe docking port in fluid communication with the sample chamber, wherein the membrane is impregnated with nucleotide probes and dried, the flat plate separation system packaged with instructions for performing a polymerase chain reaction, is provided.
  • a syringe docking port having a needle stop capable of stopping a needle, a seal mounted against the needle stop and capable of providing a fluid-tight seal after being pierced by the needle and a needle guide mounted against the seal to guide the needle through the seal and toward the needle stop.
  • Figure 1 is a cross-sectional view of one embodiment ofthe invention, illustrated with a syringe needle docking system;
  • Figure 2 is a bottom view of a dialysis chamber ofthe embodiment of Figure 1;
  • Figure 3 is an exploded perspective view of a second embodiment ofthe invention;
  • Figure 4 is a view of a top surface of a needle guide ofthe second embodiment ofthe invention
  • Figure 5 is a cross-sectional view of a portion ofthe needle guide illustrated in Figure 4;
  • Figure 6 illustrates a bottom surface ofthe needle guide illustrated in Figure 4.
  • Figure 7 shows a top surface of an upper channel plate according to the second embodiment of the invention.
  • Figure 8 shows a cross-sectional view of a portion ofthe upper channel plate illustrated in Figure 7;
  • Figure 9 provides a bottom surface view ofthe upper channel plate illustrated in Figure 7;
  • Figure 10 is an upper surface view of a lower channel plate according to the second embodiment ofthe invention;
  • Figure 11 is a detailed view of a portion ofthe upper surface ofthe lower channel plate illustrated in Figure 10;
  • Figure 12 provides a bottom surface view ofthe lower channel plate illustrated in Figure 10 according to the second embodiment ofthe invention.
  • Figure 13 provides an upper surface view of a manifold according to the second embodiment ofthe invention.
  • Figure 14 provides a bottom surface view ofthe manifold illustrated in Figure 13; and Figure 15 is a cross-sectional view of an alternate embodiment ofthe invention, wherein the flat plate system is used for ultrafiltration of a sample.
  • Figure 16 provides dialysis results of an example of an embodiment of the present invention.
  • biological sample refers to a sample comprising one or more cellular or extracellular components of a biological organism.
  • Such components include, but are not limited to, nucleotides (e.g., DNA, RNA, fragments thereof and plasmids), peptides (e.g., structural proteins and fragments thereof, enzymes, etc.), and carbohydrates, etc.
  • the biological samples described herein may also include transport media, biological buffers and other reagents well known in the art for carrying out the processes described above.
  • biological samples in accordance with the invention typically have microliter ( ⁇ L) volumes and therefore can be referred to as microsamples, e.g., biological microsamples.
  • the methods ofthe invention are advantageously practiced with biological samples having volumes ranging between about 10 ⁇ l and about 0.05 ⁇ L, and preferably between about 0.1 ⁇ L and about 3 ⁇ L.
  • dialysis is art-recognized and is understood to refer to the separation or filtering of substances in solution by means of their unequal diffusion through a membrane, including the following forms of dialysis.
  • equilibrium dialysis refers to dialysis which occurs without exchange or flow of dialysate, e.g. dialysis solution.
  • Flow dialysis refers to dialysis which occurs with a flow (or counterflow) of dialysate.
  • Exchange dialysis refers to dialysis which includes at least one change ofthe dialysate surrounding the membrane.
  • membrane refers to both dialysis membranes and ultrafiltration membranes, as appropriate, to accomplish dialysis or ultrafiltration.
  • the membrane is a material of any suitable composition and size which may used to separate or filter substances in solution by means of unequal diffusion, e.g., by size exclusion.
  • dialysis membranes and ultrafiltration membranes typically are semipermeable, the term “membrane” as used herein is not so limited.
  • Dialysis membranes and ultrafiltration membranes are closely related and are interchangeable as used herein. In most applications, ultrafiltration membranes are generally designed to withstand elevated pressures.
  • purification is intended to encompass, in its various grammatical forms and synonyms (e.g., purification, purifying, clean up, etc.) any operation whereby an undesired component(s) is/are separated or filtered from a desired component(s). Such operations include, but are not limited to, filtration, ultrafiltration, dialysis/equilibrium dialysis, chromatography, and the like.
  • purification is achieved by molecular size discrimination among the components ofthe biological sample. Purification by molecular size discrimination can be achieved using any number of materials of varying porosity well known in the art including, but not limited to, filters, membranes, and semipermeable ultrafiltration filter materials.
  • sample chamber refers to a chamber suitable for holding a sample to be processed.
  • dialysis chamber refers to a chamber used to hold a sample for performing dialysis
  • ultrafiltration chamber refers to a chamber used to hold a sample for performing ultrafiltration.
  • temperature processing refers to the application of a variety of temperature conditions to the sample, depending on the particular process underway and include, but are not limited to, continuous and discontinuous heating regimens, e.g., denaturation, annealing, incubation, precipitation, and the like.
  • continuous and discontinuous heating regimens e.g., denaturation, annealing, incubation, precipitation, and the like.
  • thermocycling associated with PCR and similar processes.
  • ultrafiltration refers to any method of purification, separation or filtration wherein the sample is under positive or negative pressure.
  • a flat plate dialysis cell 10 is provided as shown in Figure 1.
  • the flat plate dialysis cell 10 includes a syringe docking port 20 fluidly coupled with a sample chamber 30 and a vent hole 40.
  • a membrane 50 is provided along a portion ofthe sample chamber 30.
  • the syringe docking port 20 preferably includes a needle guide 60, a seal 70 and a needle stop 80.
  • the syringe docking port 20 includes an entry portion 22 opposite a distal end 24.
  • the optional needle guide 60 defines an insertion axis 90, which is preferably perpendicular to the membrane 50.
  • the needle guide 60, formed near entry portion 22, is preferably funnel shaped so as to guide a syringe along a path intersecting the insertion axis 90.
  • the needle guide 60 is preferably formed of polyethylene. However, other non-reactive materials may be used to form the needle guide 60.
  • the optional seal 70 is preferably formed to provide a fluid-tight seal within the syringe docking port 20.
  • the seal 70 is designed to be repeatedly pierced by a syringe while maintaining the ability to provide a fluid-tight seal.
  • the seal 70 is preferably pierced upon manufacture. Alternatively, the seal 70 may be manufactured without piercing and later pierced by a sharp needle during use.
  • the seal 70 may be formed of silicone, rubber, silicone rubber, or other elastic material, although silicone rubber is preferred.
  • the optional needle stop 80 can be formed near the distal end 24 ofthe syringe docking port 20 to prevent a needle from piercing the membrane 50, preferably by preventing the needle from entering the sample chamber 30.
  • the needle stop 80 is formed of a non-reactive material, such as polyethylene.
  • the flat plate dialysis cell 10 includes the sample chamber 30.
  • the sample chamber 30 is preferably formed with the distal end 24 ofthe syringe docking port 20 fluidly coupled to one end ofthe sample chamber 30.
  • a vent hole 40 is also fluidly coupled to the sample chamber 30, preferably near an opposite end ofthe sample chamber 30.
  • An optional seal 45 or valve may be provided in or in fluid communication with vent hole 40 to provide for the control of pressure within the sample chamber 30.
  • the membrane 50 is preferably provided along a portion ofthe sample chamber 30.
  • a plastic barrier may be mounted to the bottom surface ofthe upper channel plate to allow thermocycling ofthe sample chamber.
  • An optional heat exchanger may also be mounted to the bottom ofthe plastic barrier.
  • the membrane 50 ofthe flat plate dialysis cell 10 may incorporate enzymes and other reagents necessary for performing PCR (polymerase chain reaction), to allow use ofthe flat plate dialysis cell 10 in performing in situ PCR.
  • PCR polymerase chain reaction
  • a wide variety of other membranes may be provided to conduct additional processes.
  • a membrane is formed to provide a molecular weight cutoff about 100 Kdal.
  • the flat plate dialysis cell 10 ofthe invention may be used to purify biologic samples less than one microliter by the use ofthe membrane 50.
  • the membrane 50 may be used to retain molecules of interest and allow unwanted molecules to pass through the membrane, out ofthe sample, by means of dialysis.
  • the sample chamber 30, shown in Figure 2 is preferably formed with a diameter of less than 1 mm and greater than 0.1 mm, preferably having a volume of less than 1 microliter. A diameter of approximately 0.5 is preferred.
  • the sample chamber is preferably formed in the shape of an elongated tube cut along its longitudinal axis, thereby forming a flat portion along substantially all its length.
  • the sample chamber may be formed in a serpentine shape, such as an S shape as is shown in Figure 2, or may be straight.
  • the sample chamber 30 shown in Figure 2 shows a lower portion 67 ofthe guide channel 65 in fluid communication with the sample chamber 30, near an end of the sample chamber 30. A vent hole 40 is also illustrated in fluid communication near an opposite end ofthe sample chamber 30.
  • a needle containing a sample is introduced into the syringe docking port 20.
  • the needle guide 60 guides the needle onto insertion axis 90 and into seal 70.
  • the needle stop 80 prevents the needle from being inserted too far.
  • the needle introduces the sample into the sample chamber 30 preferably through needle stop 80.
  • the vent hole 40 allows for the escape of air from the sample chamber 30 as the sample is introduced.
  • the portion ofthe sample chamber 30 having the membrane 50 is exposed to a dialysis solution.
  • the dialysis solution allows smaller molecules to pass through the membrane 50 from the sample out ofthe sample chamber 30.
  • the needle previously used to insert the sample, or a different needle removes the sample from the sample chamber through needle stop 80.
  • the needle may optionally be removed from syringe docking port 20 during dialysis and reinserted upon completion of dialysis to effect removal ofthe sample.
  • washing ofthe flat plate dialysis cell 10, or any part thereof may then be performed.
  • an alcohol-based solution is used. Washing may be performed with or without disassembly ofthe flat plate dialysis cell 10.
  • the flat plate dialysis cell 10 is easily optionally multiplied into an array of multiple flat plate dialysis cells, allowing each flat plate dialysis cell 10 to use a portion of a single, continuous dialysis membrane 50.
  • the invention is capable of processing many samples in parallel, if desired, using standard micro-titer plates as reagent sources.
  • the system can be used to retrieve, mix and dispense fluids by integration with air or liquid-filled volumetric devices, such as piezoelectric elements, movable pistons or syringe-type plungers.
  • a syringe needle docking system 95 may be used to automate the insertion and removal of samples.
  • the syringe needle docking system 95 may optionally include automated syringe needle movement and automated syringe plunger actuation.
  • the dimensions ofthe sample chamber 30 provide for the use of small sample volumes while providing a large surface area for the sample to be in contact with the membrane 50. It is desirable to maximize the surface area ofthe sample chamber 30 along the membrane 50 for a given sample chamber 30 volume. However, the surface tension ofthe sample is an important consideration to allow for the maximum recovery of a sample from the sample chamber by a needle through the needle stop 80.
  • the sample chamber 30 diameter is between 1.0 mm and 0.1 mm. Specifically, approximately 0.5mm is preferred.
  • a large surface area along the membrane 50 allows for more rapid dialysis of a sample. This large surface area is provided without need for additional components, such as those disclosed in U.S. Patent 5, 679,310 to Manns.
  • thermocycling can be performed involving a hot and/or cold air or liquid to change the sample temperature.
  • Simple air blowers or blowing ambient air and air heated by resistance heaters over the sample chamber 30 may be used to change the temperature.
  • a plastic membrane is used along a portion of sample chamber 30.
  • a temperature controlled gas, such as air, or fluid is then passed along the plastic membrane to control the temperature ofthe sample chamber 30 and a sample therein.
  • the temperature may be measured and controlled by standard controllers.
  • the heating rate may be increased as desired by using, for example, superheated air for the first part ofthe heating cycle, then cooler air to avoid excessive overshoot ofthe temperature ofthe sample chamber 30.
  • the present invention is well suited to thermocycling of submicroliter samples, as the temperature of each ofthe sample chambers 30 can quickly and easily be controlled as described above.
  • the present invention provides for effective removal of contaminants from a thermocycling reaction. Once the reaction mixture is thermocycled, purification may be achieved by placing the mixture into the sample chamber 30 with the membrane 50, which is in contact with a dialysis solution having a lower concentration of ionic components. The difference in osmotic pressure across the membrane 50 forces contaminants in the product to migrate across the membrane 50 into the dialysis solution, effectively removing them from the product.
  • in situ PCR can be performed using the flat plate dialysis cell ofthe illustrative embodiment.
  • the membrane 50 ofthe flat plate dialysis cell 10 may further be impregnated (on the sample chamber side) with nucleotide probes.
  • nucleotide probes refers to suitable reagents for performing a polymerase chain reaction, including the labeled primers used in the SNP (single nucleotide polymorphism) assay described in U.S.
  • oligonucleotide primers labeled or unlabeled nucleotides, and labeled or unlabeled dideoxynucleotides.
  • These probes can generally be dried down on the membrane 50 and stored for months or more on the membrane surface.
  • the membrane 50 is then covered with a thin water-impermeable membrane, such as a mylar membrane, to allow eventual thermocycling ofthe sample chamber 30 without loss of water due to evaporation through the membrane.
  • the target DNA sample is loaded into the sample chamber 30 and conditions for performing a polymerase chain reaction are imposed such that a polymerase chain reaction occurs.
  • condition refers to the addition of enzymes, e.g. a proofreading polymerase, magnesium ions, heat and other materials, for the performance of a polymerase chain reaction, as described in, for example, U.S. Patent No. 5,391,480 and U.S. Patent No. 4,683,195, the contents of which are incorporated herein by reference.
  • enzymes e.g. a proofreading polymerase, magnesium ions, heat and other materials
  • the thin mylar membrane is peeled off or removed from the surface ofthe membrane 50, and a dialysis or ultrafiltration protocol as described herein is performed, such that the primers or unincorporated tagged nucleotides are removed from the PCR reaction, providing for further processing ofthe DNA sample or for detection ofthe presence or absence of a particular nucleotide in the DNA sample.
  • the flat plate dialysis system 100 shown in Figure 3 preferably includes a needle guide 200, a seal 300, an upper channel plate 400, a membrane 500, a lower channel plate 600 and a manifold 700.
  • a plurality, such as 96 or 384 or more, flat plate dialysis cells 10 are provided in the flat plate dialysis system 100, as described below. Those of ordinary skill will recognize that any suitable number of cells can be employed.
  • a needle guide 200 is provided with a plurality of holes. For each flat plate dialysis cell 10, an entry portion 22 and a vent hole 40 are preferably provided within needle guide 200. Alignment holes 210 are also preferably provided to aid in mounting ofthe various components ofthe flat plate dialysis system 100 to each other.
  • a cross-section ofthe needle guide 200 shown in Figure 4 is provided in Figure 5. Entry portions 22 are fluidly coupled via an upper portion 66 ofthe guide channel 65, preferably to an annular seal receiving portion 220.
  • the bottom surface of needle guide 200 preferably provides an annular seal receiving portion 220 for each flat plate dialysis cell 10.
  • Figure 6 also illustrates a vent hole 40 corresponding to each annular seal receiving portion 220.
  • the optional seal 300 is preferably provided between the needle guide 200 and the upper channel plate 400.
  • the seal 300 is preferably configured so as to mate with the annular seal receiving portion 220 to provide a fluid-tight seal along the guide channel 65.
  • the upper channel plate 400 is described with reference to Figure 7.
  • Figure 7 illustrates a pattern of holes similar to those provided in the needle guide 200 in that a pair of two holes is provided for each flat plate dialysis cell 10.
  • the upper channel plate 400 differs from the needle guide 200 in that the upper channel plate 400 preferably provides a needle stop, analogous to needle stop 80 ofthe first embodiment ofthe invention.
  • An upper portion 66 ofthe guide channel 65 corresponding to a flat plate dialysis cell 10 is shown in Figure 7.
  • a corresponding vent hole 40 is also provided, as shown in Figure 7.
  • a cross-section of a portion ofthe upper channel plate 400 is provided in
  • a guide channel 65 is shown having an upper portion 66 and a lower portion 67.
  • the lower portion 67 ofthe guide channel 65 preferably has a needle stop formed by a reduced diameter so as to prevent a needle from traveling within the lower portion 67 ofthe guide channel 65.
  • a vent hole 40 is also provided within the upper channel plate 400. The vent hole 40 may be provided with a varying diameter.
  • Figure 8 also illustrates a cross-section ofthe sample chamber 30 in fluid communication with the lower portion 67 ofthe guide channel 65 and the vent hole 40.
  • FIG. 9 illustrates a bottom surface ofthe upper channel plate 400.
  • a sample chamber 30 is provided for each flat plate dialysis cell 10.
  • a vent hole 40 and a lower portion 67 ofthe guide channel 65 are illustrated in Figure 9 and correspond to those shown in Figure 7.
  • Alignment holes 410 are preferably provided within the upper channel plate 400 to correspond to the alignment holes 210 ofthe needle guide 200. It is within the scope ofthe invention to integrally form the needle guide
  • the membrane 500 is provided between the upper channel plate 400 and the lower channel plate 600.
  • the membrane 500 may optionally be bonded to upper channel plate 400 or may be mounted by a compressive force applied to keep the upper channel plate 400 and the lower channel plate 600 together. Bonding may be performed by ultrasonic welding, heat bonding or a variety of adhesives. A wide variety of membranes 500 may be used depending on the desired operation for flat plate dialysis system 100.
  • an upper surface ofthe lower transfer plate 600 provides fluid transfer chambers 620 to correspond to the sample chambers 30 ofthe upper channel plate 400 shown in Figure 9.
  • the surface areas and volumes of each corresponding sample chamber 30 and fluid transfer chamber 620 are equal, respectively. Equalization ofthe surface area on each side ofthe membrane 500 provides an improved structure for equilibrium dialysis by minimizing any pressure differential across the membrane 500.
  • Another variation involves using an inverted upper channel plate 400. Such a variation could also involve a second needle guide 200, resulting in a virtually identical structure on each side ofthe membrane 500.
  • Each fluid transfer chamber 620 is provided with a first port 630 and a second port 640.
  • a detailed view ofthe fluid transfer chamber 620 is provided in Figure 11.
  • Figure 12 shows a bottom surface view ofthe lower channel plate 600.
  • Alignment holes 610 are optionally provided within the lower channel plate 600 to correspond to the alignment holes in other components ofthe flat plate dialysis system 100.
  • a manifold 700 is provided under the lower channel plate 600.
  • Manifold 700 as shown in Figure 13, provides on an upper surface, a first and a second trough 730, 740.
  • First and second troughs 730, 740 are fluidly coupled to first and second ports 630, 640, respectively, ofthe lower channel plate 600.
  • First trough 730 fluidly communicates with a first external port 735.
  • Second trough 740 preferably does not communicate with an external port.
  • Both first and second troughs 730, 740 allow fluid communication among first and second ports 630, 640 along a row of flat plate dialysis cells 10 within the flat plate dialysis system 100.
  • alignment holes 710 are provided within the manifold 700 to correspond to alignment holes ofthe other components of the flat plate dialysis system 100.
  • Figure 14 provides a view of a bottom surface ofthe manifold 700.
  • the lower channel plate 600 and the manifold 700 are both optional components ofthe flat plate dialysis system 100. Processing of a sample, such as conducting dialysis or thermocycling, can be performed in the sample chamber 30 by passing a dialysis solution along the membrane 500 with or without the fluid transfer chamber 620 ofthe lower channel plate 600.
  • the flat plate dialysis system 100 is adapted to be used with a syringe needle docking system 95 or a multi-channel pipettor system, such as a 96 or 384 or more channel pipettor.
  • Pipettor syringes are provided to align with the entry portions 22 shown in Figures 3, 4 and 5.
  • the needles ofthe pipettor syringes are inserted into the needle guide 200 each along an insertion axis 90, shown in Figure 5.
  • the needles travel along the guide channel 65.
  • the guide channel 65 is provided with a larger diameter along an upper portion 66 and a narrower diameter along lower portion 67.
  • the lower portion 67 ofthe guide channel 65 preferably does not allow the needle to pass within it.
  • a fluid-tight seal is provided by the seal 300 preferably seated within the annular seal receiving portion 220, illustrated in Figures 3, 5 and 6.
  • the sample chamber 30 is preferably formed with a diameter of less than 1 mm and greater than 0.1 mm. A diameter of approximately 0.5 is preferred.
  • the sample chamber is preferably formed in the shape of an elongated tube cut along its longitudinal axis, thereby forming a flat portion along substantially all its length.
  • the sample chamber may be formed in a serpentine shape, such as an S shape, or may be straight.
  • the sample flows freely into the sample chamber 30 due to vent hole 40 allowing the release of air contained within the sample chamber 30.
  • an optional seal 45 or valve may be provided within or in fluid communication with vent hole 40 to regulate the flow through vent hole 40.
  • Dialysis is conducted by the introduction of dialysis solution into the first trough 730 ofthe manifold 700.
  • the dialysis solution passes through the first trough 730 into the first port 630, thereby entering the fluid transfer chamber 620.
  • Dialysis solution is introduced sufficiently to allow the dialysis solution to enter the second trough 740 after passing through the fluid chamber 620 and second port 640.
  • first and second troughs 730, 740 may be provided such that a continual flow path is provided.
  • second trough 740 may be fluidly coupled to an external second port to provide for release of dialysis solution introduced into the first external port 735.
  • An increase in flow ofthe dialysis solution across the membrane 500 typically increases the rate of dialysis. It is within the scope ofthe invention to integrally form the lower channel plate 600 and manifold 700 in a unitary piece.
  • a needle is used to remove the sample from sample chamber 30.
  • the dialysis solution present in the fluid transfer chamber 630 may be removed in advance of, concurrently with or after removal ofthe sample from sample chamber 30.
  • the invention is ideally suited for use with equilibrium dialysis, requiring no pressure differential across the membrane 500.
  • alternative dialysis and purification methods can be used with the invention.
  • the invention may be used to perform ultrafiltration of a sample, as illustrated in Figure 15.
  • Ultrafiltration involves applying a pressure differential to a sample chamber across the membrane to drive the filtration process.
  • the applied pressure differential may comprise a positive pressure or a negative pressure.
  • the amount of pressure applied in the ultrafiltration process depends upon particular parameters ofthe flat plate system, the rate of ultrafiltration desired, and the sample being used. For example, the type of membrane being used, the active filtration area ofthe membrane, the molecular cutoff of the membrane, the strength and thickness ofthe membrane, the amount of sample to be filtered and the level of polarization ofthe sample are all factors that affect the amount of pressure used in the ultrafiltration process.
  • a positive pressure differential between about 0.5 and about 80 pounds per square inch (PSI) may be used, and a negative pressure differential between about 0.5 and about 15 psi may be used to effect filtration of a sample through the membrane.
  • PSI pounds per square inch
  • a pressure plenum 800 for applying a pressure differential to the sample may be utilized to provide ultrafiltration ofthe sample in the flat plate system ofthe invention.
  • pressure may be increased within the fluid transfer chamber 630 or the sample chamber 30.
  • the vent hole 40 ofthe flat plate system must be sealed using the seal 45 or a valve.
  • the vent is first sealed and additional liquid or gas, such as water or air, can be injected into the sample chamber 30 containing a sample, providing ultrafiltration.
  • a pressure plenum 800 providing a positive or negative pressure, may be positioned in fluid communication with the sample chamber to increase or decrease the pressure in the sample chamber, thereby achieving ultrafiltration ofthe sample.
  • the membrane 50, 500 is preferably configured to prevent DNA from passing through the membrane 50, 500, while allowing impurities to escape.
  • a vacuum may also be applied to either the fluid transfer chamber 620 or sample chamber 30 to provide ultrafiltration, using the device 800 for applying pressure, such as a vacuum manifold or other suitable device.
  • a syringe used to inject a sample into the flat plate system may be used to apply a pressure differential to the sample, by expelling a gas or a liquid into the sample chamber 30 containing the sample.
  • a variety of processes are within the scope ofthe invention.
  • the components ofthe flat plate dialysis cell 10 and flat plate dialysis system are preferably formed of non-reactive plastic.
  • components such as the needle guide 200, upper channel plate 400, lower channel plate 600 and manifold 700 may preferably be formed of hydrophobic materials, such as polystyrene, polycarbonate, TEFLONTM, or DELPJNTM.
  • Optional coatings of TEFLONTM or silane may also be used to enhance hydrophobic properties of these materials.
  • the membrane 50, 500, for use in dialysis may preferably be formed of cellulose, cellulose ester, TEFLONTM, polysulfone and polyethersulfone.
  • alternative membranes may be used in place ofthe membrane 50, 500, such as MYLARTM for thermocycling.
  • a further variation ofthe invention involves the use of transparent components ofthe flat plate dialysis system 100 to allow fluoroscopy.
  • the sample and/or the dialysate may be fluorometrically analyzed.
  • a further variation ofthe invention allows the use of alignment holes 210, 410, 610 and 710 for the passage of a temperature-controlled solution so as to vary the temperature ofthe sample chamber 30 and/or fluid transfer chamber 620. Thermocycling may be achieved, for example, by blowing air of different temperatures, although a liquid medium could also be used for heat transfer. Alternatively, additional holes or passages may be provided to allow for the distribution of a temperature controlled fluid to effect the temperature of sample chamber 30 or fluid transfer chamber 620 and the membrane 500.
  • An additional application ofthe invention involves using the flat plate dialysis system to detect the presence or absence of a particular nucleotide sequence in a strand of DNA.
  • the flat plate dialysis system can be used with a SNP (single nucleotide polymorphism) assay to detect the presence of a SNP in a strand of DNA.
  • the flat plate dialysis cell 10 may be utilized in conjunction with a SNP assay as described in U.S. Patent Number 5,391,480, U.S. Patent Number 5,888,819, U.S. Patent Number 6,004,744 and U.S.
  • the SNP assay described in U.S. application number 60/266,035 provides a method for detecting the presence or absence of a first nucleotide, at a position within a DNA molecule in a sample by forming an admixture of a primer and a strand of DNA ofthe sample and imposing conditions such that a hybridization product is formed between the primer and the DNA strand.
  • the primer comprises a sequence of DNA which hybridizes with the strand of DNA adjacent to the first nucleotide position and has a second nucleotide opposite the first nucleotide position.
  • the second nucleotide has an associated label (e.g., a fluorescent label, a radioactive label or a mass-tag) and hybridizes to the first nucleotide in the event that the second nucleotide is complementary to the first nucleotide.
  • the second nucleotide does not hybridize to the first nucleotide in the event that the second nucleotide is not complementary.
  • a proofreading polymerase is applied to the hybridization product under conditions in which the second nucleotide is preferentially excised to form a labeled nucleotide product in the event that the second nucleotide is not hybridized to the first nucleotide, and in which the second nucleotide is preferentially incorporated into a primer extension product in the event that the second nucleotide is hybridized to the first nucleotide.
  • the presence or absence of a label in excised nucleotides and extension products may then be detected using the dialysis system ofthe illustrative embodiment.
  • the admixture is injected, e.g., from a needle, through the syringe docking port 30, into the sample chamber 30 of a flat plate dialysis cell 10 and into contact with a first side of the membrane 50.
  • the membrane 50 ofthe flat plate dialysis cell 10 is selected to have a molecular weight cut-off such that the labeled nucleotide excision product may pass through (or passes through quickly), the primer may not pass through (or passes through slowly), and the extension product may not pass through.
  • a dialysis solution is applied to a second side ofthe membrane 50 opposite the first side ofthe membrane to effect separation ofthe components.
  • the dialysate from the flat plate dialysis cell 10 may then be collected and the presence ofthe label in the dialysate may be determined using any suitable detection means, e.g., direct fluorescence measurement, or mass spectrometry.
  • the sample on the first side may also be monitored for the presence of a label after providing sufficient time for dialysis ofthe various components ofthe sample to occur.
  • the presence of a label in the dialysate in concentrations greater than a background amount after a first predetermined time period is indicative ofthe absence ofthe first nucleotide, and the presence of a label remaining in the sample chamber in concentrations greater than a background amount after a second predetermined time period that is greater than the first predetermined time period
  • extension product is indicative ofthe presence ofthe first nucleotide.
  • ultrafiltration may be used to separate the labeled nucleotide excision product, the primer, and the extension product by applying a pressure differential to the sample chamber to effect separation and subsequently detecting the sample or the filtrate for the presence of a label.
  • the membrane has a molecular cutoff of 100 kDaltons.
  • the filtrate is monitored at a time period of between about two minutes and about thirty minutes after the process begins. Preferably, the filtrate is detected about fifteen minutes after the separation ofthe sample begins. At about fifteen minutes, the ratio of fluorescence between a positive background and a negative result is about 1.5, when a fluorescent label is used.
  • the sample may be detected for the presence of a label after between about thirty minutes and about an hour. Preferably, the sample is detected after about forty-five minutes. At about forty-five minutes, the ratio of fluorescence between a positive result and a negative result is about 1.25 for the sample.
  • compression bolts may be provided within the alignment holes 210, 410, 610, 710 of the invention to compress the flat plate dialysis system. Screws may also be used in place of or in combination with compression bolts.
  • Other devices such as C-clamps or large hose clamps may be used to hold the needle guide 200, upper channel plate 400, membrane 50, lower channel plate 600 and manifold 700 together. Any ofthe above-described items may also be used with a subset of components ofthe flat plate dialysis system.
  • Another variation ofthe invention involves the use of a beveled corner on each ofthe needle guide 200, the upper channel plate 400, lower channel plate 600 and manifold 700, or any subset thereof, to aid in alignment of these components ofthe flat plate dialysis system, as shown in Figure 3.
  • the present invention can be used with a conventional fluid dispensing unit, such as a Hydra dispenser, manufactured by Robbins Scientific.
  • a conventional fluid dispensing unit such as a Hydra dispenser, manufactured by Robbins Scientific.
  • Those of ordinary skill will also recognize that other fluid dispensing and sample handling units, whether in modular or discrete forms, can be employed to work with the invention.
  • the present invention provides benefits over a capillary-type dialysis system.
  • the present invention is less expensive, more durable and more easily constructed and cleaned than a dialysis system using capillaries.
  • the sample chambers 30 preferably have internal volumes that accommodate fluid sizes of less than about 1 microliter. This sample conservation advantage significantly reduces the sample volumes necessary to achieve selected processing ofthe sample.
  • Another advantage of employing submicroliter sample chambers 30 is use of minimal amounts of expensive sequencing reagents and relatively small volumes of biological samples in an automated sample handling format.
  • the invention can be used to perform purification procedures on polymerase chain reaction (PCR) products, preparing sequencing ladders, and injecting the sequencing ladders into appropriate microtiter plates, or aspirating the biological products. Further applications include removal of primers, single nucleotides, fluorescent-labels and salts from PCR reactions, SNP assays and sequencing reactions. Studies on the purification of DNA sequencing reactions by capillary dialysis have demonstrated longer read lengths and higher quality scores than are obtained by conventional alcohol precipitation. Several processing regimens which can be accommodated by the invention are described in detail below.
  • Two different flat-sheet dialysis membranes were used for sequencing reaction clean-up.
  • One membrane was a 500,000 MWCO PBVK polysulphone-type membrane, the other a PLCMK cellulosic- type membrane of 300,000 MWCO, both manufactured by MiUipore Corp. of Bedford, MA. Standard 1/4X BigDye Terminator Sequencing Reactions were transferred to the dialysis cassette sample chambers using a Hamilton syringe (Hamilton Company, Reno, Nevada). Distilled water was recycled through the dialysate chambers. After a specified period of time the samples were removed from the dialysis cassette and then run on an ABI 377.
  • EtOH PPT ethanol precipitation
  • purification of a sample may be achieved by a variety of methods, including dialysis, filtration and ultrafiltration.
  • the invention further provides various configurations to achieve purification, depending on the method of purification selected.
  • the apparatus ofthe invention provides at least one sample chamber 30 with a membrane 50 in operative contact with a dialysate, such as, for example, water.
  • a dialysate such as, for example, water.
  • the dialysate is contained in fluid transfer chambers 620.
  • the contents ofthe fluid transfer chambers 620 may be changed, or the lower channel plate 600 may be removed and optionally replaced by another with fresh dialysate, or by an open bath of dialysate
  • DNA sequencing products are purified to remove excess salt, nucleotides and primers from the biological sample.
  • the membrane 50 ofthe present invention can be employed to purify DNA, excluding the desired products, while concomitantly allowing undesired components to pass therethrough.
  • the DNA sample is cycled through the membrane 50 by pressure optionally formed within the sample chamber 30, thereby resulting in relatively small components being filtered out ofthe sample.
  • the present invention includes dialysis techniques, which may be used effectively to "clean up" polymerase chain reaction (PCR) and cycle sequencing reactions.
  • PCR polymerase chain reaction
  • the typical PCR or sequencing reaction generally utilizes sample volumes of approximately 10 ⁇ L or less, significantly smaller than the sample volumes in conventional dialysis techniques and well suited to the invention.
  • the invention is usable for processes such as aspirating, mixing, incubating, assaying, cleaning, dialysis, purification, filtering, ultrafiltration, toxicology, thermocycling, such as heating or cooling, performing PCR, detecting the presence or absence of particular nucleotides in a strand of DNA and delivery ofthe biological sample alone or in a biologically compatible carrier fluid in a selected manner.
  • the invention can be used in place of or in combination with centrifugal separation and/or charge separation.
  • the present invention is also usable within automated sample handling systems, such as hotel systems, allowing for large-scale automated processing.
  • automated sample handling systems such as hotel systems
  • the present invention has been described by way of example, and modifications and variations ofthe exemplary embodiments will suggest themselves to skilled artisans in this field without departing from the spirit ofthe invention.
  • Features and characteristics ofthe above-described embodiments may be used in combination.
  • the preferred embodiments are merely illustrative and should not be considered restrictive in any way.
  • the scope ofthe invention is to be measured by the appended claims, rather than the preceding description, and all variations and equivalents that fall within the range ofthe claims are intended to be embraced therein.

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Abstract

L'invention concerne un système et une unité d'ultrafiltration et de dialyse à plaques horizontales, comportant une chambre d'échantillons, un orifice de positionnement de seringue, pourvu d'un joint capable de constituer un scellement étanche aux fluides après son percement à l'aide d'une aiguille, un arrêt d'aiguille empêchant une aiguille de trop pénétrer dans la chambre d'échantillons, ainsi qu'un guide d'aiguille, du type entonnoir, formé dans l'orifice de positionnement de seringue, de manière à guider une aiguille en direction de la chambre d'échantillons. Cette chambre est également dotée, le long d'une de ses portions, d'une membrane de dialyse ou d'ultrafiltration et elle est couplée de manière fluidique à une extrémité distale de l'orifice de positionnement de seringue et d'un trou d'aération.
PCT/US2001/026611 2000-08-25 2001-08-24 Dispositif de traitement rapide d'echantillons d'adn, par manipulation, thermocyclage et purification integrees de liquides WO2002016652A2 (fr)

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WO2007120515A1 (fr) * 2006-04-07 2007-10-25 Corning Incorporated Microplaque fermée à écoulement continu et modes d'utilisation et procédés de fabrication de celle-ci
WO2008106960A1 (fr) * 2007-03-08 2008-09-12 Scienova Gmbh Dispositif de réception, de traitement et de conservation d'échantillons de faible volume
US7445893B2 (en) 2002-04-12 2008-11-04 Primera Biosystems, Inc. Sampling method for amplification reaction analysis
US7550266B2 (en) 2001-10-24 2009-06-23 Primera Biosystems, Inc. Methods and systems for dynamic gene expression profiling
US7674582B2 (en) 2002-04-12 2010-03-09 Primeradx, Inc. Methods for amplification monitoring and gene expression profiling, involving real time amplification reaction sampling
WO2011009073A1 (fr) * 2009-07-16 2011-01-20 Abbott Laboratories Procédé d'amplification d'acides nucléiques
US9062342B2 (en) 2012-03-16 2015-06-23 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module
EP3875574A4 (fr) * 2018-10-29 2022-01-05 NOK Corporation Puce microfluidique et dispositif microfluidique
WO2022167617A1 (fr) * 2021-02-05 2022-08-11 Synhelix Dispositif de réactions en plusieurs étapes thermocyclée amélioré

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DE102005010096B3 (de) * 2005-03-04 2006-11-09 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Assay mit osmotisch induzierter Abtrennung und Anreicherung hochmolekularer nachzuweisender Substanzen und fluidisches Mikrosystem zu seiner Durchführung

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US7550266B2 (en) 2001-10-24 2009-06-23 Primera Biosystems, Inc. Methods and systems for dynamic gene expression profiling
US8182995B2 (en) 2002-04-12 2012-05-22 Primeradx, Inc. Real time gene expression profiling
US7445893B2 (en) 2002-04-12 2008-11-04 Primera Biosystems, Inc. Sampling method for amplification reaction analysis
US7674582B2 (en) 2002-04-12 2010-03-09 Primeradx, Inc. Methods for amplification monitoring and gene expression profiling, involving real time amplification reaction sampling
EP1563097A2 (fr) * 2002-11-21 2005-08-17 Primera Biosystems Procede et dispositif d'echantillonnage permettant d'analyser des reactions d'amplification
EP1563097A4 (fr) * 2002-11-21 2006-03-15 Primera Biosystems Procede et dispositif d'echantillonnage permettant d'analyser des reactions d'amplification
WO2007120515A1 (fr) * 2006-04-07 2007-10-25 Corning Incorporated Microplaque fermée à écoulement continu et modes d'utilisation et procédés de fabrication de celle-ci
US8758706B2 (en) 2007-03-08 2014-06-24 Scienova Gmbh Device for receiving, treating, and storing small volume samples
WO2008106960A1 (fr) * 2007-03-08 2008-09-12 Scienova Gmbh Dispositif de réception, de traitement et de conservation d'échantillons de faible volume
WO2011009073A1 (fr) * 2009-07-16 2011-01-20 Abbott Laboratories Procédé d'amplification d'acides nucléiques
US9062342B2 (en) 2012-03-16 2015-06-23 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module
US9334528B2 (en) 2012-03-16 2016-05-10 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module
US9757725B2 (en) 2012-03-16 2017-09-12 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module
US9914119B2 (en) 2012-03-16 2018-03-13 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module
EP3875574A4 (fr) * 2018-10-29 2022-01-05 NOK Corporation Puce microfluidique et dispositif microfluidique
WO2022167617A1 (fr) * 2021-02-05 2022-08-11 Synhelix Dispositif de réactions en plusieurs étapes thermocyclée amélioré

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AU2001286756A1 (en) 2002-03-04
WO2002015949A2 (fr) 2002-02-28
WO2002015949A9 (fr) 2003-03-27

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