WO2017181100A1 - Éléments chauffants résistifs et transfert thermique anisotropique - Google Patents

Éléments chauffants résistifs et transfert thermique anisotropique Download PDF

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Publication number
WO2017181100A1
WO2017181100A1 PCT/US2017/027753 US2017027753W WO2017181100A1 WO 2017181100 A1 WO2017181100 A1 WO 2017181100A1 US 2017027753 W US2017027753 W US 2017027753W WO 2017181100 A1 WO2017181100 A1 WO 2017181100A1
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WO
WIPO (PCT)
Prior art keywords
heater
heat transfer
heating element
transfer device
heaters
Prior art date
Application number
PCT/US2017/027753
Other languages
English (en)
Inventor
David E. Jones
Michael BILLS
Aaron WERNEREHL
Anson Cole CHAMBERLAIN
Original Assignee
Biofire Defense, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US15/099,721 external-priority patent/US20170297028A1/en
Application filed by Biofire Defense, Llc filed Critical Biofire Defense, Llc
Priority to US16/093,124 priority Critical patent/US11786906B2/en
Publication of WO2017181100A1 publication Critical patent/WO2017181100A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • H—ELECTRICITY
    • H05—ELECTRIC TECHNIQUES NOT OTHERWISE PROVIDED FOR
    • H05B—ELECTRIC HEATING; ELECTRIC LIGHT SOURCES NOT OTHERWISE PROVIDED FOR; CIRCUIT ARRANGEMENTS FOR ELECTRIC LIGHT SOURCES, IN GENERAL
    • H05B1/00—Details of electric heating devices
    • H05B1/02—Automatic switching arrangements specially adapted to apparatus ; Control of heating devices
    • H05B1/0227—Applications
    • H05B1/023—Industrial applications
    • H05B1/0244—Heating of fluids
    • 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
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • 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
    • B01L2200/16—Reagents, handling or storing thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • 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
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • 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
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • 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
    • B01L2300/1883—Means for temperature control using thermal insulation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • B01L2400/0683—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers mechanically breaking a wall or membrane within a channel or chamber
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus

Definitions

  • infectious disease In the United States, Canada, and Western Europe infectious disease accounts for approximately 7% of human mortality, while in developing regions infectious disease accounts for over 40% of human mortality. Infectious diseases lead to a variety of clinical manifestations. Among common overt manifestations are fever, pneumonia, meningitis, diarrhea, and hemorrhagic diarrhea. While the physical manifestations may implicate some pathogens— or eliminate others— as the etiologic agent, it often cannot definitively identify the pathogen, and a clear diagnosis often requires a variety of assays be performed. Traditional microbiology techniques for diagnosing pathogens can take days or weeks, often delaying a proper course of treatment.
  • PCR polymerase chain reaction
  • PCR includes the heating of a sample through one or more heating profiles.
  • the sample is placed in proximity to or in contact with a heater, and the heater is then cycled through the desired temperature profiles to heat, decompose, volatize, or otherwise change the state of the sample.
  • the thermal response of the heater controls the state of the sample during the PCR, and therefore, precise temperature control and rapid changes in the temperature of the heater are desirable.
  • Electric heaters can generate thermal energy by applying a current through a resistive material. For example, the temperature of an electrically conductive wire increases as the current flowing through the wire increases.
  • a heat spreader can be used to control the transmission of the thermal energy from the wire to the sample. The heat spreader can also aid in maintaining uniformity of the thermal energy over the contact surface area with the sample. Efficient thermal transmission between the heating element and the heat spreader is desirable.
  • a system for performing polymerase chain reaction comprises a support member configured to receive a sample vessel having a sample therein, at least one heater associated with the support member and positioned to affect a temperature of the sample vessel, and a heat transfer device disposed between the at least one heater and the sample vessel.
  • the heat transfer device comprises anisotropic fibers axially aligned parallel to one another and positioned to conduct heat from the at least one heater toward the sample vessel in the axial direction of the anisotropic fibers.
  • a heater comprises a body defining one or more channels, a heating element positioned in the one or more channels, and one or more retention members adjacent the one or more channels.
  • the heating element is mechanically interlocked with the channel by the one or more retention members.
  • the one or more retention members are deformable between an open position and a closed position such that at least a portion of the heating element is mechanically interlocked with the channel when the one or more retention members are in the closed position.
  • the one or more channels surround at least 50% of the resistive heating element, as defined by a transverse cross- section of the heater.
  • the one or more channels may surround an area that is less than 50% of the resistive heating element, illustratively 30%, as defined by a transverse cross-section of the heater.
  • a heat transfer device comprises a plurality of anisotropic fibers axially aligned parallel to one another and normal or essentially normal to one or more of a target (e.g., a sample vessel) and/or heater and which is configured to conduct heat from the heater to the target in an axial direction of the anisotropic fibers and a retaining mechanism configured to hold the anisotropic fibers together.
  • the anisotropic fibers comprise carbon or graphite fibers.
  • the heat transfer device has a thermal conductivity in the axial direction between about 200-6600— , between about 300-1200— , between about 300-900— m-K ' m-K ' m-K
  • the anisotropic fibers have a specific heat capacity below 0.9 , preferably between about 0.6-0.8 , and more preferably between about 0.7-0.75 .
  • the anisotropic fibers can be additionally characterized in that each fiber has a thermal conductivity of less than 1 - ⁇ - in the radial direction of the fiber, preferably less than 0.8 - ⁇ — .
  • a method of manufacturing a heater includes providing a body having a channel therein; insulating the body from a heating element by an electrically insulating layer; inserting the heating element into the channel; and deforming at least a portion of the heater to mechanically secure the heating element in the channel.
  • a retention member of the body is plastically deformed to retain the heating element in the channel.
  • Fig. 1 shows a flexible pouch useful for self-contained PCR.
  • FIG. 2 is an exploded perspective view of an instrument for use with the pouch of Fig. 1, including the pouch of Fig. 1, according to an example embodiment of the present invention.
  • FIG. 3 shows a partial cross-sectional view of the instrument of Fig. 2, including the bladder components of Fig. 2, with the pouch of Fig. 1 shown in dashed lines.
  • Fig. 4 shows a motor used in one illustrative embodiment of the instrument of Fig. 2.
  • Figs. 5a-5b show illustrative profiles for an equilibrium paradigm (Fig. 5a) and a kinetic paradigm (Fig. 5b) of PCR.
  • a solid black box represents denaturation
  • a striped box represents annealing
  • a solid white box represents extension of the nucleic acids during thermal cycling.
  • Fig. 6 is an exploded view of an alternative heating embodiment for first-stage PCR for the instrument of Fig. 2.
  • Fig. 7 is a top view of the heating format of Fig. 6.
  • Fig. 8 is a cross-sectional view of the sample vessel positioned on the alternative heating embodiment of Fig. 6.
  • Fig. 9 is a perspective view of an alternative heating embodiment for second- stage PCR for the instrument of Fig. 2
  • Fig. 10 shows results of amplification using a prototype of the instrument of Figs. 6-8 in comparison to amplification using a standard plate-based thermocycler.
  • Fig. 11 shows a graph of the PCR2 Cp that results from running different numbers of cycles for PCR1 in a block thermocycler (circle) and the prototype wiper blade setup (square).
  • Fig. 12 shows a partial side cross-section of an embodiment of a heater, according to the present disclosure.
  • Fig. 13 shows a partial side cross-section of another embodiment of a heater, according to the present disclosure.
  • Fig. 14 shows a partial side cross-section of yet another embodiment of a heater, according to the present disclosure.
  • Fig. 15 shows a partial side cross-section of the embodiment of a heater of Fig. 14 with an electrically insulating layer over a part of the body, according to the present disclosure.
  • Fig. 16 shows a top view of the embodiment of a heater of Fig. 14, according to the present disclosure.
  • Fig. 17 shows a top view of another embodiment of a heater, according to the present disclosure.
  • Fig. 18 shows a top view of yet another embodiment of a heater, according to the present disclosure.
  • Fig. 19 shows a top view of a further embodiment of a heater, according to the present disclosure.
  • Fig. 20 is a flowchart illustrating an embodiment of a method of manufacturing a heater, according to the present disclosure.
  • Fig. 21 is a perspective view of an embodiment of a heat transfer device.
  • Fig. 22 is a perspective view of an embodiment of a heat transfer device made with carbon fibers, with an enlarged view of the top surface of the device shown.
  • Fig. 23 is a perspective view of an embodiment of a heater together with an embodiment of a heat transfer device and sample vessel.
  • Fig. 24 is an exploded view of Fig. 23.
  • Fig. 25 is a perspective view of an embodiment of multiple heaters together with an embodiment of a heat transfer device and sample vessel.
  • Fig. 26 is an elevation view of an embodiment having multiple heaters associated with a heat transfer device.
  • directional and/or arbitrary terms such as “top,” “bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “inner,” “outer,” “internal,” “external,” “interior,” “exterior,” “proximal,” “distal,” “forward,” “reverse,” and the like can be used solely to indicate relative directions and/or orientations and may not be otherwise intended to limit the scope of the disclosure, including the specification, invention, and/or claims.
  • Example embodiments of the present inventive concepts are described herein with reference to cross-sectional illustrations that are schematic illustrations of idealized embodiments (and intermediate structures) of example embodiments. As such, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and/or tolerances, are to be expected. Thus, example embodiments of the present inventive concepts should not be construed as limited to the particular shapes of regions illustrated herein but are to include deviations in shapes that result, for example, from manufacturing. Accordingly, the regions illustrated in the figures are schematic in nature and their shapes are not intended to illustrate the actual shape of a region of a device and are not intended to limit the scope of example embodiments.
  • sample is meant to include an animal; a tissue or organ from an animal; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; a solution containing one or more molecules derived from a cell (prokaryotic and/or eukaryotic), cellular material, or viral material (e.g. a polypeptide or nucleic acid); or a solution containing a non-naturally occurring nucleic acid, which is assayed as described herein.
  • a sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile, or cerebrospinal fluid) that may or may not contain host or pathogen cells, cell components, or nucleic acids.
  • body fluid or excretion for example, but not limited to, blood, urine, stool, saliva, tears, bile, or cerebrospinal fluid
  • nucleic acid refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base pairing.
  • Nucleic acids of the invention can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages).
  • nucleic acids can include, without limitation, DNA, cDNA, gDNA, ssDNA, dsDNA, (+)ssRNA, (- )ssRNA, dsRNA, or any combination thereof.
  • the term "probe,” “primer,” or “oligonucleotide” is meant to include a single-stranded nucleic acid molecule of defined sequence that can base pair to a second nucleic acid molecule that contains a complementary sequence (the "target” sequence).
  • the stability of the resulting hybrid depends upon the length, GC content, and the extent of the base pairing that occurs.
  • the extent of base pairing is affected by parameters such as the degree of complementarity between the probe and target molecules and the degree of stringency of the hybridization conditions.
  • the degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules such as formamide, and is determined by methods known to one skilled in the art.
  • Probes, primers, and oligonucleotides may be detectably- labeled, either radioactively, fluorescently, or non-radioactively, by methods well-known to those skilled in the art.
  • dsDNA binding dyes may be used to detect dsDNA. It is understood that a “primer” is specifically configured to be extended by a polymerase, whereas a “probe” or “oligonucleotide” may or may not be so configured.
  • dsDNA binding dyes includes dyes that fluoresce differentially when bound to double-stranded DNA than when bound to single-stranded DNA or free in solution, usually by fluorescing more strongly. While reference is made to dsDNA binding dyes, it is understood that any suitable dye may be used herein, with some non-limiting illustrative dyes described in U.S. Patent No. 7,387,887, herein incorporated by reference. Other signal producing substances may be used for detecting nucleic acid amplification and melting, illustratively enzymes, antibodies, etc., as are known in the art.
  • the term "specifically hybridizes” is used to describe a probe, primer, or oligonucleotide that recognizes and physically interacts (e.g., base pairs) with a substantially complementary nucleic acid (e.g., a sample nucleic acid) under high stringency conditions and does not substantially base pair with other nucleic acids.
  • high stringency conditions typically occur at about the melting temperature (Tm) minus 5° C (i.e. 5° below the Tm of the probe). Functionally, high stringency conditions are used to identify nucleic acid sequences having at least 80% sequence identity.
  • PCR is the amplification method used in the examples herein, it is understood that any amplification method that uses a primer may be suitable.
  • suitable procedures include strand displacement amplification (SDA); nucleic acid sequence-based amplification (NASBA); cascade rolling circle amplification (CRCA), loop-mediated isothermal amplification of DNA (LAMP); isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN); target based-helicase dependent amplification (HDA); transcription-mediated amplification (TMA), and the like. Therefore, when the term PCR is used, it should be understood to include other alternative amplification methods.
  • reaction time may be used where measurements are made in cycles or Cp, and additional reaction time may be added where additional PCR cycles are added in the embodiments described herein. It is understood that protocols may need to be adjusted accordingly.
  • kits, and devices described herein may be used to detect and sequence a wide variety of nucleic acid sequences from a wide variety of samples, including, human, veterinary, industrial, and environmental samples.
  • Various embodiments disclosed herein use a self-contained nucleic acid analysis pouch to assay a sample for the presence of various biological substances, illustratively antigens and nucleic acid sequences, illustratively in a single closed system.
  • Such systems including pouches and instruments for use with the pouches, are disclosed in more detail in U. S. Patent Nos. 8,394,608; and 8,895,295; and U. S. Patent Publication No. 2014/0283945, which are herein incorporated by reference.
  • pouches are illustrative only, and the nucleic acid preparation and amplification reactions discussed herein may be performed in any of a variety of open or closed system sample vessels, such as multi-well assay plates (e.g., 96-well plates, 384-well plates, etc.), plates of other configurations, arrays, carousels, and the like, using a variety of nucleic acid purification and amplification systems, as are known in the art. While the terms "sample well,” “amplification well,” “amplification container,” or similar are used herein, these terms are meant to encompass wells, tubes, and various other reaction containers, as are used in these amplification systems. In one embodiment, the pouch is used to assay for multiple pathogens.
  • the pouch may include one or more blisters used as sample wells, illustratively in a closed system.
  • various steps may be performed in the optionally disposable pouch, including nucleic acid preparation, primary large volume multiplex PCR, dilution of primary amplification product, and secondary PCR, culminating with optional real-time detection or post-amplification analysis such as melting-curve analysis.
  • optional real-time detection or post-amplification analysis such as melting-curve analysis.
  • Fig. 1 shows an illustrative pouch 510 that can be used in various embodiments disclosed herein, or which can be reconfigured for use in various disclosed embodiments.
  • Pouch 510 is similar to Fig. 15 of U.S. Patent No. 8,895,295 ("the '295 patent"), with like items numbered the same.
  • Fitment 590 is provided with entry channels 515a through 5151, which also serve as reagent reservoirs or waste reservoirs.
  • reagents may be freeze dried in fitment 590 and rehydrated prior to use.
  • Blisters 522, 544, 546, 548, 564, and 566, with their respective channels 514, 538, 543, 552, 553, 562, and 565 are similar to blisters of the same reference numbers disclosed by Fig. 15 of the '295 patent and the associated disclosure.
  • Second-stage reaction zone 580 of Fig. 1 is similar to that of the '295 patent, but the second-stage wells 582 of high density array 581 are arranged in a somewhat different pattern.
  • the more circular pattern of high density array 581 of Fig. 1 eliminates wells in corners and may result in more uniform filling of second-stage wells 582.
  • the high density array 581 is provided with 102 second-stage wells 582.
  • Pouch 510 is suitable for use in the FilmArray® instrument (BioFire Diagnostics, LLC, Salt Lake City, UT). However, it is understood that the pouch embodiment is illustrative only.
  • pouch 510 is formed of two layers of a flexible plastic film or other flexible material, such as polyester, polyethylene terephthalate, polycarbonate, polypropylene, polymethylmethacrylate, and mixtures thereof that can be made by any process known in the art, including extrusion, plasma deposition, and lamination. Metal foils or plastics with aluminum lamination also may be used. Other barrier materials are known in the art that can be sealed together to form the blisters and channels. If plastic film is used, the layers can be bonded together, illustratively, by heat sealing. In some embodiments, the material has low nucleic acid binding capacity.
  • plastic films that exhibit adequately low absorbance and auto-fluorescence at the operative wavelengths are preferred. Such material could be identified by testing different plastics, different plasticizers, and composite ratios, as well as different thicknesses of the film. For plastics with aluminum or other foil lamination, the portion of the pouch that is to be read by a fluorescence detection device can be left without the foil.
  • pouch 510 is made of a clear or translucent material capable of transmitting approximately 80%-90% of incident light.
  • the materials are moved between blisters by the application of pressure, illustratively pneumatic pressure, upon the blisters and channels.
  • the pouch material is flexible enough to allow the pressure to have the desired effect.
  • the term "flexible” is used herein to describe a physical characteristic of the material of pouch.
  • the term “flexible” is defined herein as readily deformable by the operative levels of pressure without cracking, breaking, crazing, or the like.
  • thin plastic sheets, such as SaranTM wrap and Ziploc® bags, as well as thin metal foil, such as aluminum foil are flexible.
  • only certain regions of the blisters and channels need be flexible, even in embodiments employing pneumatic pressure.
  • only one side of the blisters and channels need to be flexible, as long as the blisters and channels are readily deformable.
  • Other regions of the pouch 510 may be made of a rigid material or may be reinforced with a rigid material.
  • a plastic film is used for pouch 510.
  • a sheet of metal such as aluminum, or another suitable material, can be milled or otherwise cut, to create a die having a pattern of raised surfaces.
  • a pneumatic press (illustratively A- 5302-PDS, Janesville Tool Inc., Milton WI), illustratively regulated at an operating temperature of 195° C, the pneumatic press works like a printing press, melting the sealing surfaces of plastic film only where the die contacts the film.
  • Various components such as PCR primers (illustratively spotted onto the film and dried), antigen binding substrates, magnetic beads, and zirconium silicate beads can be sealed inside various blisters as the pouch 510 is formed.
  • reagents for sample processing can be spotted onto the film prior to sealing, either collectively or separately.
  • nucleotide tri-phosphates NTPs
  • NTPs nucleotide tri-phosphates
  • Pouch 510 may be used in a manner similar to that described in the '295 patent.
  • a 300 ⁇ _, mixture comprising the sample to be tested (100 ⁇ ,) and lysis buffer (200 ⁇ ) is injected into an injection port (not shown) in fitment 590 near entry channel 515a, and the sample mixture is drawn into entry channel 515a.
  • Water is also injected into a second injection port (not shown) of the fitment 590 adjacent entry channel 5151, and is distributed via a channel (not shown) provided in fitment 590, thereby hydrating up to eleven different reagents, each of which were previously provided in dry form at entry channels 515b through 5151 via.
  • sample and hydration fluid e.g. water or buffer
  • sample and hydration fluid e.g. water or buffer
  • reagents illustratively may include freeze-dried PCR reagents, DNA extraction reagents, wash solutions, immunoassay reagents, or other chemical entities.
  • the reagents are for nucleic acid extraction, first-stage multiplex PCR, dilution of the multiplex reaction, and preparation of second-stage PCR reagents, as well as control reactions.
  • the sample solution is injected in one injection port and water is injected in the other injection port; all other reagents are contained therein. After injection, the two injection ports may be sealed. Additional information on various configurations of pouch 510 and fitment 590 can be found in the '295 patent, which his already incorporated by reference.
  • Lysis blister 522 is provided with beads or particles 534, such as ceramic beads, and is configured for vortexing via impaction using rotating blades or paddles provided within the FilmArray® instrument. Bead-milling, by shaking or vortexing the sample in the presence of lysing particles such as zirconium silicate beads 534, is an effective method to form a lysate. It is understood that, as used herein, terms such as “lyse,” “lysing,” and “lysate” are not limited to rupturing cells (or contents thereof); those terms should additionally include disruption of non-cellular particles, such as viral capsids (or contents thereof).
  • Fig. 4 shows a bead beating motor 819, comprising blades 821 that may be mounted on a first side 811 of support member 802, of instrument 800 shown in Fig. 2. Blades may extend through slot 804 to contact pouch 510. It is understood, however, that motor 819 may be mounted on other structures of instrument 800.
  • motor 819 is a Mabuchi RC-280SA-2865 DC Motor (Chiba, Japan), mounted on support member 802.
  • the motor is turned at 5,000- 25,000 rpm, more illustratively 10,000-20,000 rpm, and still more illustratively approximately 15,000-18,000 rpm.
  • 7.2V provides sufficient rpm for lysis. It is understood, however, that the actual speed may be somewhat slower when the blades 821 are impacting pouch 510. Other voltages and speeds may be used for lysis depending on the motor and paddles used.
  • controlled small volumes of air may be provided into the bladder 822 adjacent lysis blister 522. It has been found that in some embodiments, partially filling the adjacent bladder with one or more small volumes of air aids in positioning and supporting lysis blister during the lysis process.
  • other structure illustratively a rigid or compliant gasket or other retaining structure around lysis blister 522, can be used to restrain pouch 510 during lysis.
  • motor 819 is illustrative only, and other devices may be used for milling, shaking, or vortexing the sample.
  • the sample is moved to a nucleic acid extraction zone, illustratively through channel 538, blister 544, and channel 543, to blister 546, where the sample is mixed with a nucleic acid-binding substance, such as silica-coated magnetic beads 533.
  • a nucleic acid-binding substance such as silica-coated magnetic beads 533.
  • magnetic beads 533 may be moved through channel 543 to blister 544, and then through channel 538 to blister 522.
  • the mixture is allowed to incubate for an appropriate length of time, illustratively approximately 10 seconds to 10 minutes.
  • a retractable magnet located within the instrument adjacent blister 546 captures the magnetic beads 533 from the solution, forming a pellet against the interior surface of blister 546.
  • incubation takes place in blister 522, multiple portions of the solution may need to be moved to blister 546 for capture.
  • the liquid is then moved out of blister 546 and back through blister 544 and into blister 522, which is now used as a waste receptacle.
  • One or more wash buffers from one or more of injection channels 515c to 515e are provided via blister 544 and channel 543 to blister 546.
  • the magnet is retracted and the magnetic beads 533 are washed by moving the beads back and forth from blisters 544 and 546 via channel 543. Once the magnetic beads 533 are washed, the magnetic beads 533 are recaptured in blister 546 by activation of the magnet, and the wash solution is then moved to blister 522. This process may be repeated as necessary to wash the lysis buffer and sample debris from the nucleic acid-binding magnetic beads 533.
  • elution buffer stored at inj ection channel 515f is moved to blister 548, and the magnet is retracted.
  • the solution is cycled between blisters 546 and 548 via channel 552, breaking up the pellet of magnetic beads 533 in blister 546 and allowing the captured nucleic acids to dissociate from the beads and come into solution.
  • the magnet is once again activated, capturing the magnetic beads 533 in blister 546, and the eluted nucleic acid solution is moved into blister 548.
  • First-stage PCR master mix from injection channel 515g is mixed with the nucleic acid sample in blister 548.
  • the mixture is mixed by forcing the mixture between 548 and 564 via channel 553.
  • the solution is contained in blister 564, where a pellet of first-stage PCR primers is provided— at least one set of primers for each target— and first-stage multiplex PCR is performed. If RNA targets are present, a reverse transcriptase step may be performed prior to or simultaneously with the first-stage multiplex PCR.
  • First-stage multiplex PCR temperature cycling in the FilmArray® instrument is illustratively performed for 15-20 cycles, although other levels of amplification may be desirable, depending on the requirements of the specific application.
  • the first-stage PCR master mix may be any of various master mixes, as are known in the art.
  • the first-stage PCR master mix may be any of the chemistries disclosed in U.S. Patent Publication No. 2015/0118715, herein incorporated by reference, for use with PCR protocols taking 20 seconds or less per cycle.
  • the sample may be diluted, illustratively by forcing most of the sample back into blister 548, leaving only a small amount in blister 564, and adding second-stage PCR master mix from injection channel 515i.
  • a dilution buffer from 515i may be moved to blister 566 then mixed with the amplified sample in blister 564 by moving the fluids back and forth between blisters 564 and 566.
  • dilution may be repeated several times, using dilution buffer from injection channels 515 j and 515k, or injection channel 515k may be reserved for sequencing or for other post-PCR analysis, and then adding second-stage PCR master mix from injection channel 515h to some or all of the diluted amplified sample.
  • level of dilution may be adjusted by altering the number of dilution steps or by altering the percentage of the sample discarded prior to mixing with the dilution buffer or second-stage PCR master mix comprising components for amplification, illustratively a polymerase, dNTPs, and a suitable buffer, although other components may be suitable, particularly for non-PCR amplification methods.
  • this mixture of the sample and second-stage PCR master mix may be pre-heated in blister 564 prior to movement to second-stage wells 582 for second-stage amplification. Such preheating may obviate the need for a hot-start component (antibody, chemical, or otherwise) in the second- stage PCR mixture.
  • the illustrative second-stage PCR master mix is incomplete, lacking primer pairs, and each of the 102 second-stage wells 582 is pre-loaded with a specific PCR primer pair.
  • second-stage PCR master mix may lack other reaction components, and these components may be pre-loaded in the second-stage wells 582 as well.
  • Each primer pair may be similar to or identical to a first-stage PCR primer pair or may be nested within the first-stage primer pair. Movement of the sample from blister 564 to the second-stage wells 582 completes the PCR reaction mixture.
  • Once high density array 581 is filled, the individual second-stage reactions are sealed in their respective second-stage blisters by any number of means, as is known in the art.
  • second-stage PCR master mix contains the dsDNA binding dye LCGreen® Plus (BioFire Diagnostics, LLC) to generate a signal indicative of amplification.
  • LCGreen® Plus BioFire Diagnostics, LLC
  • this dye is illustrative only, and that other signals may be used, including other dsDNA binding dyes and probes that are labeled fluorescently, radioactively, chemiluminescently, enzymatically, or the like, as are known in the art.
  • wells 582 of array 581 may be provided without a signal, with results reported through subsequent processing.
  • a "bladder” When pneumatic pressure is used to move materials within pouch 510, in one embodiment a "bladder" may be employed.
  • the bladder assembly 810 includes a bladder plate 824 housing a plurality of inflatable bladders 822, 844, 846, 848, 864, and 866, each of which may be individually inflatable, illustratively by a compressed gas source. Because the bladder assembly 810 may be subjected to compressed gas and used multiple times, the bladder assembly 810 may be made from tougher or thicker material than the pouch. Alternatively, bladders 822, 844, 846, 848, 864, and 866 may be formed from a series of plates fastened together with gaskets, seals, valves, and pistons. Other arrangements are within the scope of this invention.
  • PCR is dependent upon template generated by the multiplex first-stage reaction.
  • PCR is performed using DNA of high purity.
  • Methods such as phenol extraction or commercial DNA extraction kits provide DNA of high purity.
  • Samples processed through the pouch 510 may require accommodations be made to compensate for a less pure preparation.
  • PCR may be inhibited by components of biological samples, which is a potential obstacle.
  • higher concentration of Taq polymerase enzyme, adjustments in MgCl 2 concentration, adjustments in primer concentration, and addition of adjuvants optionally may be used to compensate for lower nucleic acid purity. While purity issues are likely to be more of a concern with first-stage amplification, it is understood that similar adjustments may be provided in the second-stage amplification as well.
  • the bladder assembly 810 When pouch 510 is placed within the instrument 800, the bladder assembly 810 is pressed against one face of the pouch 510, so that if a particular bladder is inflated, the pressure will force the liquid out of the corresponding blister in the pouch 510.
  • the bladder assembly 810 may have additional pneumatic actuators, such as bladders or pneumatically-driven pistons, corresponding to various channels of pouch 510. Figs.
  • FIGS. 2 and 3 show an illustrative plurality of pistons or hard seals 838, 843, 852, 853, and 865 that correspond to channels 538, 543, 553, and 565 of pouch 510, as well as seals 871, 872, 873, 874 that minimize backflow into fitment 590.
  • hard seals 838, 843, 852, 853, and 865 form pinch valves to pinch off and close the corresponding channels.
  • the hard seals are activated over the channels leading to and from the blister, such that the actuators function as pinch valves to pinch the channels shut.
  • the pinch valve actuator sealing the connecting channel is activated to open the channel, and the pneumatic bladders over the blisters are alternately pressurized, forcing the liquid back and forth through the channel connecting the blisters to mix the liquid therein.
  • the pinch valve actuators may be of various shapes and sizes and may be configured to pinch off more than one channel at a time.
  • pneumatic actuators are discussed herein, it is understood that other ways of providing pressure to the pouch are contemplated, including various electromechanical actuators such as linear stepper motors, motor-driven cams, rigid paddles driven by pneumatic, hydraulic or electromagnetic forces, rollers, rocker-arms, and in some cases, cocked springs.
  • electromechanical actuators such as linear stepper motors, motor-driven cams, rigid paddles driven by pneumatic, hydraulic or electromagnetic forces, rollers, rocker-arms, and in some cases, cocked springs.
  • methods of reversibly or irreversibly closing channels in addition to applying pressure normal to the axis of the channel. These include kinking the plastic across the channel, heat-sealing, rolling an actuator, and a variety of physical valves sealed into the channel such as butterfly valves and ball valves.
  • Fig. 1 small Peltier devices or other temperature regulators may be placed adjacent the channels and set at a temperature sufficient to freeze the fluid, effectively forming a seal.
  • the design of Fig. 1 is adapted for an automated instrument featuring actuator elements positioned over each of the blisters and channels, it is also contemplated that the actuators could remain stationary, and the pouch 510 could be transitioned in one or two dimensions such that a small number of actuators could be used for several of the processing stations including sample disruption, nucleic-acid capture, first and second- stage PCR, and other applications of the pouch 510 such as immuno-assay and immuno- PCR. Rollers acting on channels and blisters could prove particularly useful in a configuration in which the pouch 510 is translated between stations.
  • pneumatic actuators are used in the presently disclosed embodiments, when the term "pneumatic actuator" is used herein, it is understood that other actuators and other ways of providing pressure may be used, depending on the configuration of the pouch and the instrument.
  • FIG. 2 shows an illustrative instrument 800 having heaters 886, 887, 888 that heat a sample, such as that contained within pouch 510.
  • Instrument 800 includes a support member 802 that could form a wall of a casing or be mounted within a casing. Instrument 800 may also include a second support member (not shown) that is optionally movable with respect to support member 802, to allow insertion and withdrawal of pouch 510.
  • a lid may cover pouch 510 once pouch 510 has been inserted into instrument 800.
  • both support members may be fixed, with pouch 510 held into place by other mechanical means or by pneumatic pressure.
  • heaters 886, 887, 888 are mounted on support member 802.
  • Illustrative heaters include Peltiers and other block heaters, resistance heaters, electromagnetic heaters, and thin film heaters, with one or more controller for adjusting electrical current through the heater to thermocycle the contents of blister 864 and second-stage reaction zone 580.
  • Bladder plate 810, with bladders 822, 844, 846, 848, 864, 866, hard seals 838, 843, 852, 853, seals 871, 872, 873, 874 form bladder assembly 808 may illustratively be mounted on a moveable support structure that may be moved toward pouch 510, such that the pneumatic actuators are placed in contact with pouch 510.
  • the various blisters of pouch 510 are in a position adjacent to the various bladders of bladder assembly 810 and the various seals of assembly 808, such that activation of the pneumatic actuators may force liquid from one or more of the blisters of pouch 510 or may form pinch valves with one or more channels of pouch 510.
  • the relationship between the blisters and channels of pouch 510 and the bladders and seals of assembly 808 is illustrated in more detail in Fig. 3.
  • First-stage heater 886 of Fig. 2 may be positioned to heat and cool the contents of blister 564 for first-stage PCR.
  • heater 887 may be provided to control the temperature of the contents of blister 548, where heaters 886 and 887 are controlled together and cycle together.
  • heaters 886 and 887 may be under separate control, illustratively heater 887 may be provided to maintain a suitable annealing temperature, while blister 886 may be provided to maintain a suitable denaturation temperature, although it is understood that this is illustrative only and that the heaters may be reversed. Other configurations are possible. Two temperature PCR using two heating zones is discussed more fully in U.S. Patent No. 9,586,208, already incorporated by reference in its entirety.
  • thermocycling heaters 886, 887, 888 run time for the PCR portions necessarily need to be at least as long as the heater takes to get to a suitable temperature at each transition. It is understood that run time could be reduced if the temperature of the heaters do not need to be changed.
  • Figs. 6-8 show an embodiment for the first-stage PCR amplification having at least two heaters held at constant temperatures.
  • blisters 548 and 564 may be replaced with a single blister 549, and the illustrative instrument is provided with two heaters 986 and 987.
  • Heaters 986, 987 may be Peltiers, resistance heaters, electromagnetic heaters, thin film heaters, printed element heaters, positive temperature coefficient heaters, or other heaters as are known in the art, including any of the heaters described herein.
  • thermocycling and subsequent nucleic acid amplification is performed under an equilibrium paradigm, such as that depicted in Figure 5a.
  • an equilibrium paradigm the sample is brought to a denaturation temperature and maintained at the denaturation temperature for a period of time.
  • the sample is then cooled to an annealing temperature for another period of time followed by heating to a third temperature, the extension temperature.
  • An exemplary equilibrium paradigm protocol could be heating the sample to 94° C for 30 seconds, followed by annealing at 60° C for 1 minute, followed by extending at 72° C for 2 minutes to complete a first amplification cycle.
  • the same three temperatures— denaturing, annealing, and extending— are repeated in the same aforementioned order for each additional amplification cycle.
  • thermocycling and subsequent nucleic amplification is performed under a kinetic paradigm, such as that depicted in Figure 5b.
  • a kinetic paradigm is often associated with rapid cycle PCR protocols and emphasizes temperature transitions rather than discreet, static temperature zones for performing each of denaturation, annealing, or extension.
  • the sample alternates between a denaturation temperature and an annealing temperature with little to no time spent at either extreme temperature.
  • a single heating element is used, and when the heater reaches the denaturation temperature, it cools to the annealing temperature, followed by heating to the denaturation temperature, and so forth.
  • the sample concurrently heats and cools between the denaturation temperature and the annealing temperature, with extension of the primers occurring during the transition between annealing and denaturation temperatures (as exemplified in Figure 5b).
  • multiple heaters can be used.
  • two heaters can be used— a first heater held at the denaturation temperature and the second heater held at the annealing temperature.
  • the sample can transition between heating elements or the heating elements can be cyclically applied to the sample, being switched when the sample reaches the desired temperature.
  • heaters 886 and/or 887 may thermocycle between an annealing and a denaturation temperature
  • heater 986 may be provided at a suitable denaturation temperature, illustratively 94° C
  • heater 987 may be provided at a suitable annealing temperature, illustratively 60° C, although other illustrative denaturation and annealing temperatures may be used, as are known in the art.
  • it may be desirable to set heater 986 higher than 94° C and set heater 987 at a temperature lower than 60° C, as fluid may be circulated through control of each of these heaters quickly as the fluid reaches temperature, thereby increasing ramp rate.
  • an insulating spacer 983 is provided between heater 986 and heater 987.
  • Any suitable insulating material may be used, including foam, plastic, rubber, air, vacuum, glass, or any other suitable material illustratively of low conductivity.
  • run time and energy usage may be substantially reduced.
  • a wiper 989 engages top surface 549b of blister 549.
  • wiper 989 is moved so that body 913 of wiper 989 forces blister 549 into contact with heaters 986, 987, so that a portion of blister 549 is in contact with each of the heaters, to permit thermal transfer from each of the heaters to a portion of blister 549.
  • wiper 989 has an x-shaped blade 949 that divides wiper 989 into four sections 945, 946, 947, 948.
  • blade 949 When engaged, blade 949 contacts blister 549 with enough pressure such that blade 949 divides blister 549 into corresponding four sections, 564a, 564b, 564c, 564d, and rotation of wiper 989 around axis 993 forces fluid within blister 549 into a circular motion around blister 549.
  • blade 949 is a rubber or elastomeric material, or a non-stick material such as Teflon or Delrin having enough stiffness to divide blister 549 into sections and to move fluid within blister 549, but not puncture or tear blister 549, although it is understood that such materials are illustrative only and that other materials may be used, as are known in the art.
  • the blade can also include rollers or other configurations to allow movement of fluid within blister 549.
  • the blade allows portions of the fluid to be heated by each of the heaters simultaneously, and moves portions of fluid from temperature control of one heater while permitting other portions of fluid to be under control of the other heater.
  • Wiper 989 and blade 949 can be moved into position and rotated by any motor, cam, crank, gear mechanism, hydraulics, pneumatics, or other means, as are known in the art. It is understood that wiper body 913 and blade 949 can be a single fixed unit and move as a single fixed unit, or body 913 can be moved into and out of contact with blister 549 independently of movement of blade 949.
  • wiper 989 is provided with an X-shaped blade 849, thereby partitioning wiper into four segments 945, 946, 947, 948, as best seen in Fig. 6, and similarly dividing blister 549 into four segments 549a, 549b, 549c, and 549d, as best seen in Fig. 7.
  • blade 849 may be used, including a single linear blade illustratively substantially corresponding to a diameter of blister 549, a single or multiple non-linear blade including an s-shaped blade or a spiral blade, a single blade corresponding to a radius of blister 549 (e.g., similar to a clock hand), and multiple blades that divide blister 549 into multiple segments.
  • blades that divide blister 549 into multiple similar segments likely provide more controlled heating between different segments where entire segments will be at the annealing and denaturation temperatures at one time, whereas s-shaped, spiral, and radial blades may generate multiple vortexes, eddies, and varied mixing patterns, to move the sample across the thermal surface created by heaters 986, 987. It is also understood that less blade material allows for more of the sample to be in close contact with the heaters, while more blade material better controls fluid movement. Whatever the blade pattern, it is understood that portions of the fluid in blister 549 will be at the annealing temperature, while other portions will be at the denaturation temperature, and yet other portions in transition between the temperatures, all within a single sample container.
  • the choice of shape for blade 949 may depend on size and thickness of the blister and size of the heaters, and the desirability of using wiper 989 for expelling material from blister 549 once first- stage thermal cycling has been completed.
  • heaters 986, 987 provide a flat surface against which blister 549 may be pressed. However, it is understood that this is illustrative only, and heaters 986, 987 may provide a textured surface to aid in mixing for sample uniformity.
  • heaters 986 and 987 are each provided at fixed temperatures, illustratively 94° C and 60° C, respectively. However, it may be desirable to adjust the temperature of heaters 986 and 987 in some embodiments. For example, it may be desirable to increase the temperature of one or both heaters when the sample is first introduced to blister 549, to compensate for a cooler temperature of the fluid as it enters blister 549. Additionally, while two heaters are shown, any number of heaters may be used. One illustrative example uses three heaters, with one set at a denaturation temperature, one set at an annealing temperature, and the third set at an elongation temperature.
  • a first heater is larger than a second heater, so that the sample stays at the first temperature for a longer portion of the cycle.
  • blister 549 and its contents may remain stationary, and heaters 986, 987 may be rotated.
  • fluid may enter blister 549 through channel 552a from a nucleic acid extraction zone, illustratively similar to blister 546 of the pouch of Fig. 1, and channel 552a may then be closed.
  • Body 913 then presses on blister 549, promoting contact of blister 549 with heaters 986 and 987, and blade 949 divides blister 549 into segments 549a, 549b, 549c, and 549d.
  • wiper 989 is rotated, sample in each of the four segments 549a, 549b, 549c, and 549d is moved from contact with heater 986 to contact with heater 987, and back again.
  • the amount of time needed to heat and cool the sample in each of the segments is dependent on a number of factors, including the thickness of film on blister 549, the thickness of the fluid layer within blister 549, mixing of the sample within blister 549, and the amount of contact with the heaters.
  • one full revolution of wiper 989 generally corresponds to one cycle of PCR in this illustrative embodiment.
  • channel 562a may be opened.
  • the direction of wiper 989 may be used to pump fluid from blister 549 into channel 562a.
  • blister 549 may be a stand-alone container for thermocycling a sample, such that blister 549 is sealed after receiving a PCR reaction.
  • Blister 549 may be used for any of a variety of sample types that require thermocycling.
  • each pneumatic actuator is connected to compressed air source 895 via valves 899. While only several hoses 878 are shown in Fig. 2, it is understood that each pneumatic fitting is connected via a hose 878 to the compressed gas source 895.
  • Compressed gas source 895 may be a compressor, or, alternatively, compressed gas source 895 may be a compressed gas cylinder, such as a carbon dioxide cylinder.
  • Compressed gas cylinders are particularly useful if portability is desired.
  • Other sources of compressed gas are within the scope of this invention.
  • Assembly 808 is illustratively mounted on a movable support member, although it is understood that other configurations are possible.
  • a magnet 850 which is mounted on a second side 814 of support member 802, is illustratively deployed and retracted using gas from compressed gas source 895 via hose 878, although other methods of moving magnet 850 are known in the art. Magnet 850 sits in recess 851 in support member 802. It is understood that recess 851 can be a passageway through support member 802, so that magnet 850 can contact blister 546 of pouch 510.
  • recess 851 need not extend all the way through support member 802, as long as when magnet 850 is deployed, magnet 850 is close enough to provide a sufficient magnetic field at blister 546, and when magnet 850 is retracted, magnet 850 does not significantly affect any magnetic beads 533 present in blister 546.
  • retracting magnet 850 it is understood that an electromagnet may be used and the electromagnet may be activated and inactivated by controlling flow of electricity through the electromagnet.
  • withdrawing or retracting the magnet it is understood that these terms are broad enough to incorporate other ways of withdrawing the magnetic field.
  • pneumatic connections may be pneumatic hoses or pneumatic air manifolds, thus reducing the number of hoses or valves required.
  • the various pneumatic pistons 868 of pneumatic piston array 869 are also connected to compressed gas source 895 via hoses 878. Twelve pneumatic pistons 868 are shown. While only two hoses 878 are shown connecting pneumatic pistons 868 to compressed gas source 895, it is understood that each of the pneumatic pistons 868 are directly or indirectly connected to compressed gas source 895.
  • a pair of heating/cooling devices are mounted on a second side 814 of support 802.
  • first-stage heater 886 is positioned to heat and cool the contents of blister 564 for first-stage PCR.
  • second- stage heater 888 is positioned to heat and cool the contents of second-stage blisters 582 of array 581 of pouch 510, for second-stage PCR. It is understood, however, that these heaters could also be used for other heating purposes, and that other heaters may be included, as appropriate for the particular application.
  • Fig. 9 shows an alternative embodiment for second-stage heater 888, which is replaced by heater assembly 988.
  • heater assembly 988 includes three heaters 930, 931, and 932, set in a circular mount 934, driven circularly by motor 933, so that one heater at a time contacts array 581 as each heater is moved sequentially into position adjacent array 581. Types of suitable heaters have been discussed above, with reference to first-stage PCR.
  • heater 930 may be set at an annealing temperature, illustratively 60° C
  • heater 931 may be set at an elongation temperature, illustratively 72° C
  • heater 932 may be set at a denaturation temperature, illustratively 94° C.
  • these temperatures are illustrative only, and that other temperatures and other numbers of heaters may be used.
  • Two heaters are sufficient for many applications. For example, when performing a fast cycle PCR or other nucleic acid amplification protocol under a kinetic paradigm (e.g., Fig. 5b), a two heater assembly may be advantageous.
  • heaters 930, 931, 932 move to contact array 581.
  • Mount 934 may move in one direction only, with each of heaters 930, 931, 932 contacting array 581 in order, or mount may move in both clockwise and counterclockwise directions, illustratively changing direction after each PCR cycle.
  • heaters 930, 931, 932 are provided in mount 934 and are moved relative to array 581, it is understood that this illustrative only, and that two or more stationary heaters may be provided, and array 581 may be rotated relative to the heaters, as with the embodiment shown in Figs. 6-8 for first stage PCR.
  • optical array 890 When fluorescence detection is desired, an optical array 890 may be provided. As shown in Fig. 2, optical array 890 includes a light source 898, illustratively a filtered LED light source, filtered white light, or laser illumination, and a camera 896. Camera 896 illustratively has a plurality of photodetectors, each corresponding to a second-stage well 582 in pouch 510. Alternatively, camera 896 may take images that contain all of the second- stage wells 582, and the image may be divided into separate fields corresponding to each of the second-stage wells 582.
  • a light source 898 illustratively a filtered LED light source, filtered white light, or laser illumination
  • Camera 896 illustratively has a plurality of photodetectors, each corresponding to a second-stage well 582 in pouch 510. Alternatively, camera 896 may take images that contain all of the second- stage wells 582, and the image may be divided into separate fields corresponding to each of the
  • optical array 890 may be stationary, or optical array 890 may be placed on movers attached to one or more motors and moved to obtain signals from each individual second-stage well 582. It is understood that other arrangements are possible.
  • the embodiment for second-stage heaters shown in Fig. 9 provides the heaters on the opposite side of pouch 510 from that shown in Fig. 2. Such orientation is illustrative only and is determined by spatial constraints within the instrument.
  • second-stage reaction zone 580 is provided in an optically transparent material, photodetectors and heaters may be on either side of array 581 or moved into and out of position at the direction of a user or automatically at the direction of a computer.
  • a computer 894 controls valves 899 of compressed air source 895, and thus controls all of the pneumatics of instrument 800.
  • Computer 894 also controls heaters 886 and 888, and optical array 890.
  • Each of these components is connected electrically, illustratively via cables 891, although other physical or wireless connections are within the scope of this invention.
  • computer 894 may be housed within instrument 800 or may be external to instrument 800.
  • computer 894 may include built-in circuit boards that control some or all of the components, and may also include an external computer, such as a desktop or laptop PC, to receive and display data from the optical array.
  • An interface illustratively a keyboard interface, may be provided including keys for inputting information and variables such as temperatures, cycle times, etc.
  • a display 892 is also provided. Display 892 may be an LED, LCD, or other such display, for example.
  • the performance of the illustrative instrument 800 described in relation to Fig. 2 through 11 may be at least partially dependent upon the performance and/or characteristics of the heaters 886, 887, 888, whether the heaters 886, 887, 888 are thermocycled or held at a constant temperature.
  • the PCR and illustrative instrument 800 described herein may include a heater according to the present disclosure.
  • Fig. 12 illustrates a partial side cross-sectional view of an embodiment of a heater 186.
  • the heater 186 may include a thermally conductive body 101 with a channel 103 formed therein.
  • the channel 103 is configured to receive a heating element 105 positioned in the channel 103.
  • the heating element 105 may be mechanically held against the body 101 by retention member 107.
  • the heating element 105 may be electrically insulated from the body 101 and/or the retention member 107 by an electrical insulation layer 109.
  • the body 101 may be made of or include a thermally conductive material, such as a metal, metal alloy, ceramic, polymer, other thermally conductive material, or combinations thereof.
  • the body 101 may include copper, copper alloys, aluminum, aluminum alloys, iron, iron alloys (e.g., steel), titanium, titanium alloys, nickel alloys, tungsten alloys, superalloys, silicon, silicon carbide, ceramics, composites, or combinations thereof.
  • the body 101 may be made of a single material or a combination of materials.
  • the body 101 may include one or more materials laminated together.
  • the heating element 105 illustratively may be a resistive heating element, an inductive heating element, a fluid heating element, or combinations thereof.
  • a resistive heating element 105 may include a nickel -chromium wire that increases in temperature upon an electric current applied therethrough.
  • the resistive heating element may be a copper wire, a steel wire, an aluminum alloy wire, or other metals.
  • An inductive heating element 105 may include a ferromagnetic material that increases in temperature upon exposure to an alternating magnetic field.
  • a fluid heating element 105 may include a thermally controlled fluid that is moved through the heating element 105 to alter the temperature.
  • the channel 103 is shown with a circular cross-section. In other embodiments, the channel 103 may have other cross-sectional shapes, such as elliptical, rectangular, triangular, other polygonal, irregular, or combinations thereof.
  • the heating element 105 is shown with a circular cross-section. In other embodiments, the heating element 105 may have other cross-sectional shapes, such as elliptical, rectangular, triangular, other polygonal, irregular, or combinations thereof. While the channel 103 and the heating element 105 as depicted in Fig. 12 have substantially similar cross-sectional shapes, the cross-sectional shapes of the channel 103 and heating element 105 need not be the same. For example, the channel 103 may be substantially square in cross-section, while the heating element 105 may be circular in cross-section.
  • the heating element 105 may be electrically insulated from the body 101 by an electrical insulation layer 109.
  • the body 101 may be electrically insulating and thermally conductive without the need for an electrical insulation layer 109.
  • the electrical insulation layer 109 may be at least partially located between the heating element 105 and the body 101 to prevent contact and, hence, electrical connection between the live resistive heating element 105 and the body 101.
  • the electrical insulation layer 109 may be made of or include a polyimide film (such as poly (4,4'-oxydiphenylene-pyromellitimide) available as KAPTON from E. I. du Pont de Nemours and Company).
  • the electrical insulation layer 109 may have a thermal conductivity of no less than 0.46 W/m*K.
  • the electrical insulation layer 109 may include other electrically insulating polymers with a thermal conductively of no less than 0.40 W/m*K.
  • the electrical insulation layer 109 may substantially prevent an electrical connection between the heating element 105 and an electrically conductive body 101.
  • a heater 186 may include a heating element 105 connected to the body 101 without an adhesive therebetween.
  • the heater 186 may include one or more retention members 107 connected to the body 101 that may mechanically connect or hold the heating element 105 adjacent to the body 101.
  • the retention members 107 may be integrally formed with the body 101.
  • the retention members 107 may be made of the same material as the body 101.
  • the retention members 107 may be bonded, such as welded, brazed, or otherwise adhered to the body 101.
  • the retention members 107 may be a part of a laminated layer applied to the body 101.
  • the retention members 107 may be movable between an initial open position in which the channel 103 is unobstructed by the retention members 107 and a closed position.
  • the retention members 107 may be made of a malleable or plastically deformable material and deformed from the open position to the closed position shown in Fig. 12. In the closed position, the retention members 107 may enclose at least part of the heating element 105 and, thereby, retain the heating element 105 within the channel 103.
  • the mechanical retention of the heating element 105 in the channel 103 by the retention members 107 may allow the heating element 105 to be secured to the body 101 without the use of an adhesive between the heating element 105 and the body 101.
  • An adhesive may thermally insulate the heating element 105 and reduce the overall efficiency of the heater 186.
  • the body 101 and retention members 107 surround at least 50% of the heating element 105.
  • the body 101 and retention members 107 may surround a percentage of the heating element 105 in a range having upper and lower values including any of 50%, 60%, 70%), 80%), 90%), 100%), or any values therebetween.
  • the body 101 and retention members 107 may surround a percentage of the heating element 105 in a range of 50%o to 100%).
  • the body 101 and retention members 107 may surround a percentage of the heating element 105 in a range of 60%> to 100%>.
  • the body 101 and retention members 107 may surround a percentage of the heating element 105 in a range of 70% to 100%>.
  • the body 101 and retention members 107 may surround a percentage of the heating element 105 in a range of 80%> to 100%).
  • the retention member surrounding percentages are illustrative only, and in an embodiment, the retention member surrounding percentage can be effectively greater than 100%) by, for example, having the retention members overlap the heating element.
  • Fig. 13 illustrates a partial side cross-sectional view of another embodiment of a heater 286 according to the present disclosure.
  • the heater 286 has a body 201 similar to that described in relation to Fig. 12.
  • the body 201 has a channel 203 formed therein, with a heating element 205 positioned in the channel 203.
  • the channel 203 may be rectangular in cross-section (i.e. having a flat bottom with orthogonal sidewalls).
  • the heating element 205 may have a complimentary shape in cross-section, or may be deformed to have a complimentary cross-sectional shape.
  • the channel 203 depicted in Fig. 13 does not have retention members as depicted in Fig. 12.
  • the heater 286 may have a channel 203 into which the heating element 205 may be compressed to mechanically interlock the channel 203 and heating element 205.
  • the mechanical interlock between the heating element 205 and the channel 203 may allow the heating element 205 to be retained in contact and/or adjacent to the body 201 without the use of adhesives.
  • the heating element 205 may be compressed into the channel 203 with a press fit to secure the heating element 205 in the channel 203.
  • the heating element 205 may be deformed into a channel having a wider bottom than opening, such as a dovetail shape in cross- section, producing a mechanical interlock between the heating element 205 and the channel 203.
  • the heating element 205 may be approximately the same shape as the channel 203 prior to deforming the heating element 205 to create the mechanical interlock. In other embodiments, the heating element 205 may have a different cross-sectional shape prior to deformation.
  • the heating element 205 may be a round drawn copper wire initially, and the channel 203 may be substantially rectangular in cross-section until the wire heating element 205 is compressed into the channel 203.
  • the heating element 205 may be a laser-cut nickel chromium foil that is rectangular in cross- section initially, before being deformed into a dovetail-shaped channel 203 in cross-section.
  • the channel 203 may have an electrically insulating layer 209 therein.
  • the electrically insulating layer 209 may be positioned between the heating element 205 and the body 201.
  • the electrically insulating layer 209 may extend around less than the entire heating element 205 in cross-section.
  • Fig. 14 illustrates a partial cross-sectional view of yet another embodiment of a heater 386 with an electrically insulating layer 309 extending over the surface of a body 301 of the heater 386, as well as a channel 303.
  • the body 301 may be an electrically conductive material, as described herein, that is anodized to provide an electrically insulating layer 309 (i.e., an oxide layer) bonded to the surface of the body 301.
  • part of the body 301 may be anodized.
  • the entire surface of the body 301 may be anodized, producing an electrically insulating layer 309 over the entire surface of the body 301.
  • the electrically insulating layer 309 may extend over the body 301, the channel 303, and the retention members 307.
  • Fig. 15 illustrates the embodiment of a heater 386 of Fig. 14 with the electrically insulating layer 309b extending over only a portion of the body 301.
  • the electrically insulating layer 309b may cover the channel 303 only such that part of the retention members 307 are covered by the electrically insulating layer 309b and at least a portion of the retention members 307 are not covered by the electrically insulating layer 309b.
  • the electrically insulating layer 309b may be applied to or deposited on the channel 303 only.
  • the electrically insulating layer 309 may be initially applied to the body 301, the channel 303, and the retention members 307 (such as shown in Fig. 14) and the electrically insulating layer 309 may be removed from substantially all portions of the body 301 and at least a portion of the retention members 307.
  • the electrically insulating layer 309b may be partially removed by laser etching, ion etching, chemical etching, mechanical removal, or combinations thereof.
  • the electrically insulating layer 309b may be partially removed with a C0 2 laser.
  • the electrically insulating layer 309 may be partially applied by sputtering, spraying, or other deposition methods known in the art.
  • Fig. 16 illustrates a top view of the embodiment of a heater 386 of Fig. 15.
  • the heater 386 has a substantially circular body 301.
  • the body 301 may also be rectangular, square, elliptical, triangular, other polygonal, irregular, or combinations thereof.
  • the channel 303 may be configured to provide connection of the heating element 305 distributed across the body 301.
  • the channel 303 shown in Fig. 16 is a spiral that evenly positions the heating element 305 across the circular body 301.
  • the channel 303 may have alternating, parallel passes to evenly distribute the heating element 305 across a rectangular body 301.
  • the heating element 305 has terminals 313-1, 313-2 that may be connected to an energy source to heat the heating element 305.
  • the first terminal 313-1 is located on a first side of the body 301 and the second terminal 313-2 is located on an opposing second side of the body 301.
  • the first terminal 313-1 may be located on the first side of the body 301 and the second terminal 313-2 may also be located on the first side of the body 301, such as terminals 413-1 and 413-2 in the embodiment shown in Fig. 17.
  • Fig. 17 is a top view of another embodiment of a heater 486.
  • the heater 486 includes a channel 403 laid out in concentric spirals on the body 401.
  • the concentric spirals allow the channel 403 and heating element 405 to be distributed evenly about the surface of the body 401 and may aid in averaging out any variations in thermal efficiency of the heating element 405 along a length of the heating element 405.
  • the concentric spirals also may join at or near the center of the body and/or the spirals.
  • One or more thermal sensors 415 such as thermocouples, may be positioned at or near the center.
  • the one or more thermal sensors 415 may be secured or located on a surface of the body 401.
  • the one or more thermal sensors 415 may be positioned through the body 401.
  • the one or more thermal sensors 415 may be located in a bore that extends at least partially through the body.
  • the one or more thermal sensors 415 may be positioned within the bore to monitor the temperature of the body 401 at a selected longitudinal depth of the body 401.
  • the body 401 may have a thickness such that a thermal gradient may be established in the body 401 during heating and/or cooling.
  • the one or more thermal sensors 415 may be located at different depths within the body 401 to monitor the gradient.
  • the one or more thermal sensors 415 may be positioned at or near the interface of interest.
  • the heater 486 may be oriented in the instrument 800 shown in Fig.
  • the one or more thermal sensors 415 may be positioned such that the one or more thermal sensors 415 measure the temperature of the heater 486 at or adjacent to the blisters 522, 544, 546, 548, 564, and 566.
  • FIG. 18 Another embodiment of a heater 986 is shown in FIG. 18.
  • the heater 986 has a body 901 with a square shape.
  • the channel 903 and the heating element 905 are positioned on the body 901 in alternating traces to conduct heat from the heating element 905 to the body 901 substantially evenly across the body 901.
  • FIG. 19 shows another embodiment of a heater 1086 with a body 1001 that has an irregular polygonal shape.
  • the channel 1003 and the heating element 1005 are positioned on the body 1001 in alternating traces to conduct heat from the heating element 1005 to the body 1001 substantially evenly across the body 1001.
  • the illustrative examples provided in Figs. 16- 19 can incorporate any of the embodiments shown in Figs. 12-15, the description associated therewith, or variations thereof.
  • the embodiments of heaters described herein and other heaters according to the present disclosure may be manufactured according a method 717 illustrated in Fig. 20.
  • the method 717 includes providing 719 a body of a heater with a channel.
  • the channel may have one or more retention members, such as described in relation to Fig. 12.
  • the channel may be recessed into the body without retention members, such as described in relation to Fig. 13.
  • the channel may be formed by casting the channel and/or retention members in the body.
  • the channel may be formed by machining the channel and/or retention members in the body.
  • the channel may be formed by stamping or coining the channel and/or retention members in the body.
  • the channel may be formed by etching the channel and/or retention members in the body.
  • the channel and/or retention members may be chemically etched in the body.
  • the channel and/or retention members may be ion etched into the body with a broad beam or focused beam ion source.
  • the channel and/or retention members may be plasma etched into the body.
  • the method 717 further includes electrically insulating 723 the body against a heating element.
  • the electrical insulation may be applied to the heating element, such as a polyimide coating described in relation to Fig. 12, or to the body, such as the anodize coating described in relation to Fig. 14 and Fig. 15. However, it is understood that in some embodiments it may be desirable to omit the insulating layer.
  • the method 717 further includes inserting 725 the heating element into the channel of the body and deforming 727 at least a portion of the heater to mechanically retain the heating element in the channel.
  • deforming 727 at least a portion of the heater may include deforming one or more retention members, illustratively by bending or crimping the retention members. In other embodiments, deforming 727 at least a portion of the heater may include deforming part of the heating element.
  • Mechanically securing a heating element in a heater may allow for elimination of some or all of the adhesives in the heater, thereby reducing the thermal mass of the heater and increasing thermal conductivity between the heating element and a thermally conductive body, heat spreader, or similar (e.g., any of bodies 101, 201, 301, 401, 901, and/or 1001 as shown in Figs. 12-19). Reducing the thermal mass and increasing thermal conductivity increases the heating and cooling rates of the heater, allowing more efficient and faster cycling in applications such as PCR and the PCR instrument described herein.
  • Figs. 21-25 illustrate an embodiment of an anisotropic heat transfer device 601, individually and in connection with the heater 386 and the pouch 510. As illustrated in Figs. 23-25, the anisotropic heat transfer device 601 may be located between the heater 386 and the second stage reaction zone 580 where the sample fluid resides in the second stage wells 582 to facilitate heat transfer between the heater 386 and the sample fluid.
  • the heat transfer device 601 is in the form of a disc or a cylinder with two opposing, cross-sectional circular faces 602a, 602b.
  • the heat transfer device 601 may be formed of anisotropic fibers that are aligned axially normal to the cross- sectional faces and run from one opposing face to the other.
  • the heat transfer device may have any cross-sectional shape. The desired cross-sectional shape may depend on the shape of the heater or the area being heated.
  • the heat transfer device may make contact with, or may be in close proximity to, both the heater/cooler and a target, so as to transfer heat to and from the target.
  • the target includes the second stage wells 582 within the second stage reaction zone 580.
  • Other targets may include various blisters 522, 546, 548, 566 and channels 552, 565, of the pouch, or any surface in contact with or in close proximity to the heat transfer device where temperature regulation is desired.
  • This heat transfer occurs anisotropically, in a direction perpendicular to the top face 602a of the heat transfer device 601. That is, heat is conducted readily and efficiently through faces 602a and 602b of the heat transfer device 601, but heat is conducted very poorly laterally to the edges of the heat transfer device 601.
  • the anisotropic heat transfer is accomplished using carbon fibers.
  • Carbon fibers are one example material for the heat transfer device 601 because of the anisotropic properties of carbon fibers. In other embodiments, other materials such as graphite fibers or pyrolytic graphite fibers may be used as well.
  • Carbon fiber cross sections 603 are illustrated from a top view of the heat transfer device 601, with resin 604 filling the spaces in between the carbon fibers 603. The carbon fibers are aligned perpendicular to the top face 602a of the heat transfer device 601 and normal to the transverse plane of both the heater 386 and the second stage reaction zone 510.
  • Resin 604 such as an epoxy resin
  • the heat transfer device can be formed by lining up carbon fibers so the axes thereof are generally parallel and infusing the fibers with resin. Lining up the fibers and infusing them with resin may be done in a number of ways, including but not limited to pultruding unidirectional tow, stacking and pressing pre-preg lamina, and clamping dry tow and infusing the fibers with resin using a resin transfer molding method.
  • Pre-preg is a term for "pre-impregnated" composite fibers where a matrix material, such as epoxy, is already present.
  • the fibers are often unidirectional, having a straight, constant cross-section and the matrix is used to bond them together and to other components during manufacture. Pre-preg is available in a variety of layups and the fiber composition (e.g., graphene or carbon nanotubes may be included along with typical carbon fiber) may be varied according to manufacturer specifications.
  • Another method of aligning the fibers may be laying fibers that have been cut to the same length in a vat and applying a charge to the bottom surface of the vat. Applying a charge to the bottom surface of the vat can create an opposite charge at the top surface of the vat, which causes the fibers to stand on end. The standing fibers can then be clamped together and infused with resin illustratively using a vacuum bagging method.
  • the resin may be cured and the resulting product can be cut into wafers.
  • Such wafers may have a round, thin shape as shown in Figs. 21-25. The surfaces of the wafer may then be polished smooth.
  • Other thermoset resins and thermoplastic polymers such as polyester resin, vinyl ester resin, phenolic, urethane, and other resins known in the art, can be used to hold the carbon fibers together.
  • retaining mechanisms may include structural mechanisms, such as a ring surrounding the outer circumference of the heat transfer device, may be employed to hold the fibers together.
  • the heat transfer device may be able to transfer heat from one or more heaters (e.g., from more than one temperature zone), simultaneously, without transferring heat through the heat transfer device from one heated cross-sectional area to another.
  • Fig. 25 illustrates an embodiment with multiple heaters 605, 606, 607, 608 transferring heat independently through the heat transfer device 601 to the second stage reaction zone 580.
  • Different second stage sample wells 582, shown in Fig. 24, can be heated to different temperatures independently in this way.
  • This illustrative embodiment of the heat transfer device when made out of commercially available carbon tow, achieves a thermal conductivity in the range of 300-
  • This embodiment of the heat transfer device when made out of commercially available graphite fibers, can achieve a thermal conductivity in the range of about 300-900 A higher thermal conductivity can be achieved using higher strength carbon fibers and increasing the carbon fiber 603 to resin 604 ratio. Carbon fibers
  • Carbon nanotubes which can, for example, be mixed with conventional carbon or graphite fibers to achieve even higher thermal conductivity, have a theoretical maximum thermal conductivity of about
  • aluminum and copper is approximately 200 and 400 , respectively.
  • the fibers have a specific heat capacity between 0.6 and 0.8 or below 0.8 - ⁇ .
  • the density of these fibers is typically about 2.2 g/cm 3 compared to 2.7 g/cm 3 for aluminum and 8.9 g/cm 3 for copper, resulting in a much lower thermal mass. This low thermal mass requires low amounts of energy to change the temperature of the carbon fibers, resulting in efficient thermo-cycling of the sample in the second stage wells 582 and reduced PCR run time.
  • Fig. 23 is a perspective view of an embodiment of the heater 386, the heat transfer device 601, and the second stage reaction zone 580, which resides in the pouch 510.
  • the heat transfer device 601 remains stationary in relation to the second stage reaction zone 580, while the heater 386 may move, making contact with the heat transfer device 601, but not the second stage reaction zone 580. Such movement could occur, for example, in a multi-heater embodiment shown in Fig. 9 and described above.
  • a heat assembly 688 having a plurality of heaters, particularly heaters 386, 630, 631, and 632, and heat transfer device 601, as described above.
  • Heaters 630, 631, and 632 can be any type of heater described above, such as a Peltier device, block heater, resistance heater, electromagnetic heater, or thin film heater.
  • heaters 631 and 632 are Peltier devices, and as illustrated, the Peltier device 631 is in thermal communication with— and in some embodiments coupled to— heater 386. It has been unexpectedly shown that combining a Peltier device with a resistive heating element can create a more efficient heater than either heater alone, and in some embodiments, can allow the temperature to be modulated more accurately and quickly.
  • the energy requirement for both the Peltier device and the resistive heating element may be reduced when combined.
  • the total energy requirement for heating the combined Peltier device and resistive heater to a given temperature e.g., a denaturation temperature
  • the energy requirement for heating a lone Peltier device to the same given temperature is less than the energy requirement for heating a lone Peltier device to the same given temperature and is also less than the energy requirement for heating a lone resistive heater to the same given temperature.
  • the heat transfer device 601 makes contact with, or is in close proximity to, both a target (e.g., a sample or container holding a sample) and one or more heaters 630, 631/386, 632, so as to transfer heat to and from the target.
  • a target e.g., a sample or container holding a sample
  • the target can include the second stage wells 582 within the second stage reaction zone 580.
  • Other targets can include various blisters 522, 546, 548, 566 and channels 552, 565, of the pouch, or any surface in contact with or in close proximity to the heat transfer device where temperature regulation is desired.
  • This heat transfer occurs anisotropically, in a direction perpendicular to a target interaction surface of the heat transfer device 601 and/or parallel to the axially-aligned anisotropic fibers of the heat transfer device 601. That is, heat is conducted readily and efficiently from the heaters 630, 631/386, 632 through the heat transfer device 601, while heat is conducted poorly in a lateral direction (i.e., to the edges of the heat transfer device 601 or in a direction transverse to the axially-aligned anisotropic fibers).
  • Fig. 26 depicts the heaters aligned serially or adjacent to one another
  • the heaters 630, 631/386, and 632 can be part of a multi-heater assembly disposed on a circular mount, similar to that depicted in Figure 9.
  • the heaters can individually be set at a static temperature, and in such an embodiment, the heaters can be rotated and/or selectively positioned such that only one heater is positioned over a target at a given time.
  • the heat transfer device is stationary while the individual heaters are rotated.
  • each heater is associated with an individual heat transfer device that rotates with the heater.
  • a heater assembly can include any number or types of heaters and should not be limited to the specific orientation and type of heater illustrated thereby.
  • a heater assembly can include two Peltier devices that are each in thermal communication with a separate resistive heat element and individual heat transfer devices.
  • a first Peltier device and resistive heat element can be set at a denaturation temperature of, illustratively, 94° C.
  • the second Peltier device and resistive heat element can be set at an annealing temperature of, illustratively, 60° C.
  • the target can be selectively moved between the two temperatures, or alternatively, the heaters can be selectively moved over the target, as described above.
  • the heaters can be used for one or more additional or alternative purposes than that described above.
  • a heater can be positioned and heated to seal a channel or hole.
  • a heater can be positioned proximate to and/or in contact with any of channels 538, 543, 552, 553, 562, 565, or similar of pouch 510 illustrated in Figure 1.
  • the heater can be, illustratively, at a temperature sufficient to thermally couple the opposing sides of the channel, thereby sealing the channel. It should be appreciated that the heater can be pre-heated to a thermal coupling temperature before or after the heater is initially contacted or positioned proximate to the target.
  • a heater according to the present disclosure may be used in other applications outside of PCR and/or laboratory analysis in any application where improved thermal cycling rates are desirable.

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Abstract

L'invention concerne un système, des éléments chauffants et des dispositifs de transfert de chaleur. Par exemple, un système pour effectuer une réaction en chaîne par polymérase comprend un élément de support conçu pour recevoir un récipient d'échantillon et un élément chauffant qui est positionné pour influer sur une température du récipient d'échantillon. Le système comprend en plus un dispositif de transfert de chaleur disposé entre l'élément chauffant et le récipient d'échantillon. Le dispositif de transfert de chaleur comprend de manière illustrative des fibres anisotropes axialement alignées parallèlement les unes aux autres et positionnées pour conduire la chaleur du ou des éléments chauffants vers le récipient d'échantillon dans la direction axiale des fibres anisotropes. Un élément chauffant illustratif comprend un corps définissant un ou plusieurs canaux, un élément chauffant positionné dans l'ou les canaux, et des éléments de retenue adjacents au ou aux canaux. Au moins une partie de l'élément chauffant est mécaniquement verrouillée avec le canal par déformation des éléments de retenue dans une position fermée.
PCT/US2017/027753 2016-04-15 2017-04-14 Éléments chauffants résistifs et transfert thermique anisotropique WO2017181100A1 (fr)

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US15/099,721 2016-04-15
US15/099,721 US20170297028A1 (en) 2016-04-15 2016-04-15 Rapid Response Resistive Heater
US201662357525P 2016-07-01 2016-07-01
US62/357,525 2016-07-01

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* Cited by examiner, † Cited by third party
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WO2021086990A1 (fr) * 2019-10-29 2021-05-06 Quantum-Si Incorporated Pompage péristaltique de fluides pour applications bioanalytiques et procédés, systèmes et dispositifs associés
US12011716B2 (en) 2019-10-29 2024-06-18 Quantum-Si Incorporated Peristaltic pumping of fluids and associated methods, systems, and devices

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US5542471A (en) * 1993-11-16 1996-08-06 Loral Vought System Corporation Heat transfer element having the thermally conductive fibers
US20060288708A1 (en) * 2004-12-08 2006-12-28 George Maltezos Thermal management techniques, apparatus and methods for use in microfluidic devices
US20070173589A1 (en) * 2006-01-20 2007-07-26 Chibante L P Felipe Thermally conductive fibers and fabrics
US20100081191A1 (en) * 2008-09-26 2010-04-01 Marlow Industries, Inc. Anisotropic heat spreader for use with a thermoelectric device
US20140220579A1 (en) * 2011-05-24 2014-08-07 Ingeny PCR B.V. System for and method of changing temperatures of substances

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5542471A (en) * 1993-11-16 1996-08-06 Loral Vought System Corporation Heat transfer element having the thermally conductive fibers
US20060288708A1 (en) * 2004-12-08 2006-12-28 George Maltezos Thermal management techniques, apparatus and methods for use in microfluidic devices
US20070173589A1 (en) * 2006-01-20 2007-07-26 Chibante L P Felipe Thermally conductive fibers and fabrics
US20100081191A1 (en) * 2008-09-26 2010-04-01 Marlow Industries, Inc. Anisotropic heat spreader for use with a thermoelectric device
US20140220579A1 (en) * 2011-05-24 2014-08-07 Ingeny PCR B.V. System for and method of changing temperatures of substances

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021086990A1 (fr) * 2019-10-29 2021-05-06 Quantum-Si Incorporated Pompage péristaltique de fluides pour applications bioanalytiques et procédés, systèmes et dispositifs associés
US12011716B2 (en) 2019-10-29 2024-06-18 Quantum-Si Incorporated Peristaltic pumping of fluids and associated methods, systems, and devices

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