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WO2025064904A1 - Systèmes de pcr numériques intégrés - Google Patents

Systèmes de pcr numériques intégrés Download PDF

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Publication number
WO2025064904A1
WO2025064904A1 PCT/US2024/047803 US2024047803W WO2025064904A1 WO 2025064904 A1 WO2025064904 A1 WO 2025064904A1 US 2024047803 W US2024047803 W US 2024047803W WO 2025064904 A1 WO2025064904 A1 WO 2025064904A1
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WO
WIPO (PCT)
Prior art keywords
sample
chip
pcr
instrument
zone
Prior art date
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Pending
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PCT/US2024/047803
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English (en)
Inventor
Yansu Christie JU
Kin Wong
Yanzheng Xu
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Astrabios Inc
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Astrabios Inc
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Publication of WO2025064904A1 publication Critical patent/WO2025064904A1/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
    • B01L7/525—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 with physical movement of samples between temperature zones
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • 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
    • B01L2300/0883—Serpentine channels
    • 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
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present disclosure relates to polymerase chain reaction (PCR) for nucleic acid amplification, particularly to a digital PCR chip and its detection system.
  • PCR polymerase chain reaction
  • PCR Polymerase Chain Reaction
  • a chip processing instrument comprises: (a) a plurality of heat blocks, each configured to maintain a constant temperature during thermal cycling; (b) a flow inductio unit configured to induce a flowing fluid through a microfluidic channel in a PCR chip engager with the chip processing instrument; (c) an optical detector; (d) a receptacle sized and shaped t ⁇ engage the PCR chip in a fixed position with respect to: (i) the flow induction unit, (ii) the optical detector, (iii) at least one of the plurality of heat blocks; or (iv) any combination thereol and (e) a signal processor configured to receive signals from the optical detector, wherein the signals comprise information about amplification of nucleic acids in sample droplets of the o.,;u
  • the plurality of heat block comprise (i) a high temperature heat block configured to maintain a first region of the PCR chip at a first temperature or
  • the first temperature or first temperature range is greater than the second temperature or second temperature range.
  • at least one of the plurality of heat blocks is configured to maintain a temperature that facilitates hot start polymerase activation, nucleic acid denaturing, nucleic acid annealing, nucleic acid extension, or any combination thereof.
  • the plurality of heat blocks comprises a high temperature heat block configured to facilitate hot start polymerase activation and/or nucleic acid denaturing.
  • such high temperature heat block comprises a first region proximate a sample inlet configured to facilitate polymerase activation and a second region configured to facilitate nucleic acid denaturing.
  • the first and second regions are held at same temperature during thermal cycling.
  • the first and second regions together have a “T” shape.
  • the plurality of heat blocks comprises a low temperature heat block configured to facilitate sample inlet heating, nucleic acid annealing, nucleic acid extension, or any combination thereof.
  • such low temperature heat block comprises a section configured to heat a sample reservoir to a temperature that activates a UNG enzyme and/or a reverse transcription reaction.
  • the plurality of heat blocks comprises a polyimide heater or a rod heater.
  • the flow induction unit comprises a negative pressure source.
  • the negative pressure source comprises a vacuum pump.
  • the optical detector comprises a plurality of optical submodules, each configured to detect a different wavelength.
  • each of the plurality of optical submodules comprises a light source and a light detector.
  • the plurality of optical submodules comprises 2 to 10 optical submodules.
  • the signal processor is further configured to computationally process the signals in a manner that conducts a digital PCR analysis.
  • the digital PCR analysis provides an absolute concentration of nucleic acid in the sample.
  • the instrument additionally includes a control processor configured to control the flow induction unit, the plurality of heat blocks, the optical detector, or any combination thereof.
  • the instrument has a longest dimension of not greater than about 500 mm or less.
  • PCR chips may be characterized by the following features: (a) a sample inlet configured to receive a sample comprising a nucleic acid; (b) a nozzle configured to generate sample droplets comprising the sample, wherein the sample droplets are separated from one another by an immiscible phase of a flowing fluid; (c) a fluid flow channel configured to contain the flowing fluid; and (d) an optical detection zone configured for optical interrogation the sample droplets flowing in an optical detection segment of the fluid flow channel.
  • the chip further comprises a sample reservoir in fluid communication with the sample inlet.
  • the sample reservoir comprises a cylindrical container.
  • the chip further comprises an immiscible phase inlet for receiving a liquid comprising the immiscible phase. In certain embodiments, the chip further comprises an immiscible phase reservoir in fluid communication with the immiscible phase inlet.
  • the sample droplets comprise reagents for performing digital PCR.
  • the sample droplets have a diameter of about 160 to 230 micrometers.
  • the nozzle is in contact with the sample and the immiscible phase.
  • the nozzle comprises a first conduit for delivering the sample toward the fluid flow channel, a second conduit for delivering the immiscible phase toward the fluid flow channel, and a junction of the first conduit and the second conduit where, during operation, the sample droplets in the flowing fluid are produced.
  • the nozzle comprises a plurality of second conduits, each for delivering the immiscible phase to the junction.
  • the nozzle is configured to, during operation, produce the flowing fluid under the influence of a vacuum source.
  • the fluid flow channel has a cross-sectional width of about 100 to 300 micrometers. In certain embodiments, the fluid flow channel is about 1500 to 3000 mm in total length.
  • the fluid flow channel has a serpentine path.
  • the serpentine path when the PCR chip engages with a chip processing instrument, the serpentine path repeatedly passes between at least one high temperature heat block and at least one low temperature heat block of the chip processing instrument.
  • the serpentine path comprises a plurality of parallel serpentine segments of the fluid flow channel, and wherein each serpentine segment has an upstream portion over a first low temperature heat block, a downstream portion over a second low temperature heat block, and an intermediate portion over a high temperature heat block, wherein the intermediate portion is between the upstream and downstream portions.
  • the plurality of parallel serpentine segments comprises about 30 to 50 parallel serpentine segments.
  • the flowing fluid in the serpentine path experiences at least about 50 thermal cycles within the PCR chip.
  • the optical detection zone is located adjacent to an optical detector configured to obtain optical signals from the sample droplets in the flowing fluid.
  • the optical detection zone is downstream from a denaturing zone and an annealing and extension zone.
  • the PCR chip additionally includes a polymerase activation zone configured to perform hot-start polymerase activation in the sample droplets when the sample droplets are flowing in at least one polymerase activation segment of the fluid flow channel within the polymerase activation zone.
  • the polymerase activation zone when the PCR chip engages with a chip processing instrument, is located over a high temperature heat block configured to operate at a temperature that facilitates hot start polymerase activation in the sample in the at least one polymerase activation segment of the fluid flow channel.
  • the PCR chip further comprises a first annealing and extension zone for annealing and extending nucleic acids in the sample droplets when flowing in a plurality of first segment portions of the fluid flow channel, wherein the plurality of first segment portions is located in the first annealing and extension zone.
  • the first annealing and extension zone when the PCR chip engages with a chip processing instrument, is located over a low temperature heat block configured to operate at a temperature that facilitates annealing and extending nucleic acids in the sample while the sample is flowing in the first segment portions over the first annealing and extension zone.
  • the first annealing and extension zone is located downstream from a polymerase activation zone in the fluid flow channel.
  • the PCR chip further comprises a denaturing zone for denaturing doublestranded nucleic acids in the sample droplets when flowing in a plurality of second segment portions of the fluid flow channel, wherein the plurality of second segment portions is located in the denaturing zone.
  • the denaturing zone when the PCR chip engages with the chip processing instrument, the denaturing zone is located over a high temperature heat block configured to operate at a temperature that facilitates denaturing the nucleic acids in the sample in the second segment portions of the fluid flow channel.
  • at least some of the plurality of second segment portions are located downstream from at least some of the first segment portions in the first annealing and extension zone.
  • a polymerase activation zone and the denaturing zone are both located over a high temperature heat block of the chip processing instrument.
  • the PCR chip further comprises a temperature buffer zone disposed between first annealing and extension zone and the denaturing zone.
  • the PCR chip further comprises a second annealing and extension zone for annealing and extending nucleic acids in the sample droplets when flowing in a plurality of third segment portions of the fluid flow channel, wherein the plurality of third segment portions is located in the second annealing and extension zone.
  • the second annealing and extension zone when the PCR chip engages with the chip processing instrument, is located over a low temperature heat block configured to operate at a temperature that facilitates annealing and/or extending double-stranded nucleic acids in the sample in the third segment portions.
  • at least some of the plurality of third segment portions are located downstream from at least some of the plurality of second segment portions in the denaturing zone.
  • the first and second annealing and extension zones are both located over a low temperature heat block of the chip processing instrument.
  • the PCR chip further comprises a temperature buffer zone disposed between the denaturing zone and the second annealing and extension zone.
  • the PCR chip further comprising a waste chamber coupled to an outlet of the fluid flow channel.
  • the PCR chip has a longest dimension of not greater than about 200 mm or less.
  • Some aspects of the disclosure pertain to methods that may be characterized by the following operations: (a) introducing a fluid in a channel, wherein the fluid comprises sample droplets separated from one another in a carrier fluid, wherein the sample droplets comprise a sample including a nucleic acid, and wherein the sample is substantially immiscible in the carrier fluid; (b) while in the channel, flowing the fluid alternately between at least one denaturing zone having a first temperature and at least one annealing and extension zone having a second temperature that is lower than the first temperature, wherein the nucleic acid in the sample undergoes amplification while flowing alternately between the at least one denaturing zone and the at least one annealing and extension zone; and (c) after flowing the fluid alternately between the at least one denaturing zone and the at least one annealing and extension zone, detecting an optical signal from the sample droplets while continuing to flow in the channel, wherein the optical signal is associated with a quantity of amplified nucleic acid in the sample droplets.
  • the sample droplets comprise reagents for performing digital PCR. In certain embodiments, the sample droplets have a diameter of about 160 to 230 micrometers.
  • the methods further comprise producing the fluid by contacting the sample with the carrier fluid at a nozzle upstream of the channel.
  • the nozzle comprises a first conduit for delivering the sample toward the channel, a second conduit for delivering the carrier fluid toward the channel, and a junction of the first conduit and the second conduit where the sample droplets in the fluid are produced.
  • the nozzle comprises a plurality of second conduits, each for delivering the carrier fluid to the junction.
  • introducing the fluid in a channel comprises applying a vacuum to draw the fluid into the channel.
  • the fluid flow channel has a cross-sectional width of about 100 to 300 micrometers. In certain embodiments, the fluid flow channel is about 1500 to 3000 mm in total length. In certain embodiments, the fluid flow channel has a serpentine path. In some implementations, flowing the fluid alternately between the at least one denaturing zone and the at least one annealing and extension zone comprises flowing the fluid through the serpentine path, which repeatedly passes between the at least one denaturing zone and the at least one annealing and extension zone.
  • the serpentine path comprises a plurality of parallel serpentine segments of the fluid flow channel, and wherein each serpentine segment has an upstream portion in a first annealing and extension zone, a downstream portion in a second annealing and extension zone, and an intermediate portion in a first denaturing zone, wherein the intermediate portion is between the upstream and downstream portions.
  • the plurality of parallel serpentine segments comprises about 30 to 50 parallel serpentine segments.
  • flowing the fluid through the serpentine path causes the sample droplets to experience at least about 50 thermal cycles.
  • detecting an optical signal from the sample droplets comprises detecting a fluorescence associated with the nucleic acid.
  • the fluorescence is produced by a fluorophore coupled to an amplification primer.
  • the fluorescence is produced by a plurality of fluorophores, each emitting light at a different wavelength, and each associated with a different nucleic acid.
  • the method further comprises analyzing the optical signal to provide a digital PCR analysis.
  • the digital PCR analysis provides an absolute concentration of the nucleic acid in the sample.
  • the method further comprises, before flowing the fluid alternately between the at least one denaturing zone and the at least one annealing and extension zone comprises, flowing the fluid though a polymerase activation zone that performs hot-start polymerase activation on the sample droplets.
  • the method the polymerase activation zone is held at the first temperature.
  • the method further comprises, after detecting the optical signal, recovering the sample droplets and conducting further analysis of the sample.
  • the method further comprises diluting a biological specimen and adding PCR reagents to produce the sample.
  • FIG. 1 depicts a general architecture of the integrated digital PCR chip reader provided in this present application.
  • FIG. 2 is a flowchart showing the first part of the steps for obtaining quantitative PRC results according to the same example embodiment of Fig. 1.
  • FIG. 3 is a flowchart showing the continued second part of the steps after steps of FIG. 2 for obtaining quantitative PRC results according to the same example embodiment of Fig. 1.
  • FIGs. 4A-4D illustrate components of a digital PCR instrument in accordance with some embodiments.
  • FIG. 5A presents a cross-sectional view of a digital PCR instrument having spring loaded heating blocks configured to facilitate good thermal contact between the heat blocks and a microfluidic chip.
  • FIG. 5B presents a cross-sectional view of a digital PCR instrument having thermal insulation between high temperature and low temperature heating blocks.
  • FIGs. 6A-6E depict a digital PCR instrument in accordance with certain embodiments.
  • Figures 6F and 6G depict an embodiment of a digital PCR instrument having thermal control blocks disposed on a top cap of the instrument.
  • Figures 6H and 61 depict an embodiment of a digital PCR instrument having a sealing connector for connecting an outlet of a microfluidic chip to a vacuum line of the instrument.
  • FIGs. 7 A and 7B present views of the digital PCR microfluidic chip, showing different functional area of the chip, as provided in this application.
  • FIGs. 8A and 8B depict a nozzle structure suitable for generating sample droplets in a digital PCR chip.
  • Figure 9A depicts a PCR microfluidic chip having a waste chamber with internal structured defining a flow path within the waste chamber.
  • Figure 9B depicts a waste chamber having sample droplets arranged in a self-forming array.
  • FIG. 10 depicts an embodiment having oil and sample ports in a single well.
  • Figure 11 A depicts a microfluidic PCR chip having a waste chamber including a plurality of walls or barriers.
  • Figure 1 IB presents and image of a collection chamber of a microfluid chip, where a plurality of individual droplets have collected before exiting the chip.
  • FIG. 12 is a depiction of digital PCR multi-color optical assembly with respect to an installed microfluidic chip.
  • FIG. 13 depicts the orientation of the temperature regulation module with respect to an installed microfluidic chip.
  • FIG. 14 depicts the sample temperature profile of each microdroplet as it moves along the microchannels in the reaction chamber.
  • FIG. 15 depicts a multiple flow channel microfluidic chip.
  • FIG. 16 depicts a sequence of sample droplets flowing in a channel of a microfluidic chip.
  • FIGs. 17A and 17B depict forms of modular high throughput digital PCR systems.
  • FIGs. 18A-B show the linearity of ASF Results obtained from digital PCR illustrating the relationship between the number of positive droplets and the targeted DNA copies.
  • FIGs. 19A-B show the linearity of ASF Results obtained from qPCR illustrating the relationship between the qPCR CT value and the log of template dilution.
  • amplifying and “amplification” are used interchangeably and refer to generating one or more copies or “amplified product” of a nucleic acid.
  • DNA amplification generally refers to generating one or more copies of a DNA molecule or “amplified DNA product.”
  • reverse transcription generally refers to the generation of deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template via the action of a reverse transcriptase.
  • denaturing and “denaturation” are used interchangeably and generally refer to the full or partial unwinding of the helical structure of a double- stranded nucleic acid, and in some cases the unwinding of the secondary structure of a single stranded nucleic acid.
  • Conditions at which denaturation may occur include a “denaturation temperature” that generally refers to a temperature at which denaturation is permitted to occur and a “denaturation duration” that generally refers to an amount of time allotted for denaturation to occur.
  • extension generally refers to the incorporation of nucleotides to a nucleic acid in a template directed fashion. Extension may occur via the aid of an enzyme, such as, for example, a polymerase or reverse transcriptase. Conditions at which extension may occur include an “extension temperature” that generally refers to a temperature at which extension is permitted to occur and an “extension duration” that generally refers to an amount of time allotted for extension to occur.
  • nucleic acid generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleo tides or ribonucleotides, or analogs thereof.
  • Nucleotides may be nucleoside triphosphate, such as deoxyribonucleotide triphosphate (dNTP).
  • Nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown. Non- limiting examples of nucleic acids include DNA, and RNA.
  • Nucleic acids can include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a nucleic acid may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure may be made before or after assembly of the nucleic acid.
  • the sequence of nucleotides of a nucleic acid may be interrupted by non-nucleotide components.
  • a nucleic acid may be further modified after polymerization, such as by conjugation or binding with a reporter agent.
  • reaction mixture generally refers to a composition comprising reagents necessary to complete nucleic acid amplification (e.g., DNA amplification, RNA amplification), with non-limiting examples of such reagents that include primer sets having specificity for target RNA or target DNA, DNA produced from reverse transcription of RNA, a DNA polymerase, a reverse transcriptase (e.g., for reverse transcription of RNA), suitable buffers (including zwitterionic buffers), co-factors (e.g., divalent and monovalent cations), dNTPs, and other enzymes (e.g., uracil-DNA glycosylase (UNG)), etc).
  • reaction mixtures can also comprise one or more reporter agents.
  • a “reporter” refers to a composition that yields a detectable signal, such as an optical signal, the presence or absence of which can be used to detect the presence of amplified product.
  • the reporter is a fluorophore.
  • Optical analysis techniques include any technique that obtains optical signals from a sample. Such signals may be generated through interaction of the sample with a stimulus such as incident light. Samples may interact with stimulus to produce or modify light through, for example, absorption, emission, scattering, refraction, diffraction, etc. In some cases, an optical analysis technique contains information about fluorescence chemical species introduced into a PCR device.
  • target nucleic acid generally refers to a nucleic acid molecule in a starting population of nucleic acid molecules having a nucleotide sequence whose presence, amount, and/or sequence, or changes in one or more of these, are desired to be determined.
  • a target nucleic acid may be any type of nucleic acid, including DNA, RNA, and analogues thereof.
  • the term “fluid” generally refers to a liquid or a gas. A fluid cannot maintain a defined shape and will flow during an observable time frame to pass through and/or fill a container in which it is put. The fluid may have any suitable viscosity that permits flow. If two or more fluids are present, each fluid may be independently selected among essentially any fluids (liquids, gases, and the like) by those of ordinary skill in the art.
  • aqueous fluid generally refers to a fluid that is made with, of, or from water, or a fluid that contains water.
  • an aqueous fluid may be an aqueous solution with water as the solvent.
  • An aqueous fluid of the present disclosure may comprise reagents necessary for conducting a desired chemical reaction, e.g., PCR.
  • non-aqueous fluid generally refers to a fluid that is made from, with, or using a liquid other than water.
  • non-aqueous fluid include, but are not limited to, oils such as hydrocarbons, silicon oils, fluorocarbon oils, organic solvents, etc.
  • junction generally refers to a point or area, where one channel crosses or meets another channel.
  • the term “droplet” generally refers to an isolated portion of a first fluid (e.g., an aqueous fluid) that is surrounded by a second fluid (e.g., a non-aqueous fluid).
  • An emulsion may include a dispersion of droplets of a first fluid (e.g., liquid) in a second fluid.
  • the first fluid may be immiscible in the second fluid.
  • the first fluid and the second fluid are substantially immiscible.
  • a droplet of the present disclosure may be spherical or assume other shapes, such as, for example, shapes with elliptical cross-sections.
  • the diameter of a droplet, in a non-spherical droplet is the diameter of a perfect mathematical sphere having the same volume as the non-spherical droplet.
  • a droplet of the present disclosure may be a single emulsion, a double emulsion, or a triple emulsion, etc.
  • microfluidic generally refers to a chip, area, device, article, or system including at least one fluid channel having a cross-sectional dimension of less than about 1 mm, 0.5 mm, or 0.1 mm.
  • a “cross-sectional dimension” of a channel may be measured perpendicularly with respect to the general direction of fluid flow within the channel.
  • channel generally refers to a feature on or in a device or substrate (e.g., a chip) that at least partially directs flow of a fluid.
  • a channel may have any cross- sectional shape (circular, oval, triangular, irregular, square or rectangular, etc.) and may be covered or uncovered. When a channel is completely covered, at least one portion of the channel may have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlets and/or outlets or openings.
  • a channel of the present disclosure may be of any suitable length.
  • the channel may be straight, substantially straight, or it may contain one or more curves, bends, etc.
  • the channel may have a serpentine or a spiral configuration.
  • the channel includes one or more branches, with some or all of which connected with one or more other channel(s).
  • the corner or turning point may be rounded so that a fluid or a partition would not be trapped in the corner or at the turning point.
  • An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics that can exert a force (e.g., a containing force) on a fluid.
  • force actuators that can produce suitable forces include piezo actuators, pressure valves, electrodes to apply AC electric fields etc.
  • the fluid within the channel may partially or completely fill the channel. When an open channel is used, the fluid may be held within the channel, for example, using surface tension (i.e., a concave or convex meniscus).
  • sample generally refers to any composition containing or suspected of containing a nucleic acid molecule.
  • a sample may be provided to a PCR device such as PCR chip for purposes of amplifying the nucleic acid molecule.
  • a sample may be or may be derived from any of many types of biological specimens.
  • a sample can be derived from biological specimen containing one or more nucleic acid molecules.
  • the biological specimen can be obtained (e.g., extracted or isolated) from a bodily sample of a subject. Examples of biological specimens include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears.
  • a bodily specimen may be a fluid or tissue sample (e.g., skin sample) of the subject.
  • the sample is obtained from a cell-free bodily fluid of the subject, such as whole blood.
  • the sample can include cell-free DNA and/or cell-free RNA.
  • the sample is or is derived from an environmental sample (e.g., soil, waste, ambient air and etc.), an industrial sample (e.g., samples from any industrial processes), a food sample (e.g., dairy products, vegetable products, and meat products), and the like.
  • Controllers, processors, and any of various associated computational elements may be integrated circuits, microcontrollers, etc. They may contain memory, instructions, routines, models, or other components. Controllers, processors, and other computational elements may be implemented as hardware that is manipulated by software and/or firmware (e.g., an FPGA or a general-purpose processor executing software) to operate in manner that is capable of performing the recited task(s).
  • software and/or firmware e.g., an FPGA or a general-purpose processor executing software
  • Elements, units, or components herein are sometimes described or claimed as “configured to” perform an operation or operations.
  • the phrase “configured to” is used to connote structure by indicating that the element, unit, or component includes structure or structural features and optionally control elements (e.g., processors, stored instructions, circuitry, etc.) that perform in a particular way during operation.
  • an element, unit, or component can be said to be configured to perform in that way even when the specified component is not necessarily currently operational (e.g., is not in use).
  • qPCR real-time quantitative PCR
  • this technology typically provides only relative quantification rather than absolute quantification. Due to its insufficient sensitivity, qPCR technology has difficulty detecting rare single nucleotide site mutations, which are significant for cancer screening. Moreover, the amplification efficiency of nucleic acids in qPCR is easily interfered by inhibitors (such as hemoglobin, heparin, EDTA, bilirubin, etc.). Deficiencies in the reaction system affect the sensitivity, stability, and inter-laboratory reproducibility of the results, thereby slowing down the progress of this class of technology in clinical applications.
  • inhibitors such as hemoglobin, heparin, EDTA, bilirubin, etc.
  • the principle of digital PCR is to disperse the sample containing the PCR reaction solution into many aliquots (e.g., droplets) of consistent volume. Each aliquot is treated as an independent PCR reaction cavity, in which one or more target nucleic acid sequences may exist, while no target sequences exist in the remaining cavities.
  • the endpoint reaction that occurs in the cavities housing the aliquots can achieve reliable amplification of the target sequences.
  • the total amount of target sequences in the sample can be accurately calculated, achieving absolute quantitative analysis.
  • qPCR provides relative quantitative results through comparison with standard curves.
  • Digital PCR due to its absolute quantitative advantages, allows highly specific detection of rare gene mutations in a complex wild-type allele background, which is critical for evaluating tumor samples, liquid biopsy samples, and viruses. It also provides high precision to distinguish allelic copy number variations distributed throughout the human genome, which is crucial to understanding their roles in genome structure and diseases.
  • microfluidic chip technology platform for digital PCR
  • existing product implementation methods generally involve the use of two or more devices for droplet generation, target sequence amplification, and fluorescence signal reading. Additionally, these devices involve dedicated operations and maintenance, thereby requiring skilled personnel. The coordination between multiple devices increases the likelihood of potential failures. These complex experimental steps ultimately hinder the speed of target sequence detection. Therefore, there is an urgent need to develop a compact, cost-effective, highly integrated, and rapid digital PCR detection platform.
  • a system that addresses one or more of the deficiencies of the existing digital PCR system. For example, some implementations consolidate all or most procedures of digital PCR into a microfluidic chip and a detection instrument, both of which have a small footprint. This system may be characterized by a simplified design, compact size, and cost- effectiveness, thereby reducing the overall cost of digital PCR experiments.
  • a sample is introduced into a sample reservoir filled with a pre-existing oil phase solution of lower density than the sample. This causes the sample to settle at the base of the reservoir, which is thermally regulated, enabling the activation/deactivation of the UNG enzyme and the reverse transcription reaction.
  • the sample reservoir may be linked to the oil phase reservoir via a microchannel at the microdroplet generating nozzle.
  • a vacuum pump applies pressure, inducing fluid movement towards the end of the microchannel.
  • the sample and oil phases flow in a controlled manner, with the sample phase breaking at the nozzle to create uniformly sized water-in-oil microdroplets.
  • the oil phase acts as a spacer, preventing microdroplet contact during the reaction period.
  • one or more thermal control blocks are tightly connected to the top of the microfluidic chip via, e.g., a spring-loaded mechanism, resulting in at least two, three, or four thermally regulated zones on the chip.
  • a uniform flow rate which is determined by, e.g., the vacuum pressure, the respective viscosities of the sample and oil phases, and the length and cross-sectional area of the microchannels.
  • microdroplets remain physically separated throughout the thermal cycling process, effectively preventing the merging of microdroplets.
  • thermal cycling steps in existing digital PCR technologies are often conducted in a test tube, a microplate, or an untreated flat chip, where the generated microdroplets are in physical contact with each other.
  • This requires a complex and costly oil phase composition to minimize large- scale merging of microdroplets during thermal cycles.
  • Certain embodiments herein employ a common oil phase composition for the generation of water-in-oil microdroplets.
  • microdroplets once the microdroplets are generated, they flow through a network of one or more microchannels, with the oil phase composition acting as spacers. This greatly reduces the risk of microdroplet merging during thermal cycling.
  • Some existing digital PCR technologies involve loading samples onto a flat microfluidic chip containing pre-etched microwells, followed by oil phase composition, with each microdroplet trapped in a microwell. However, this process is typically cumbersome, and the manufacture of such a microfluidic chip is complex and costly.
  • high and low-temperature zones on a thermal control block are closely packed and separated by thin thermal insulation layers.
  • the temperature setting of each thermal control block can be adjusted based on user or assay requirements.
  • thermal cycling in some existing digital PCR technologies is conducted on a Peltier device capable of temperature ramping and cooling.
  • this design requires high-powered electronics and a complex control algorithm, which are better suited to larger scale instrumentation.
  • the time required for temperature ramping and stabilization is significant relative to the overall digital PCR reaction time, thereby increasing the time needed to complete each thermal cycle and the overall reaction time.
  • inefficient temperature cooling during DNA annealing increases the likelihood of unspecific binding of the primer to the DNA template, thus increasing the probability of false positive results.
  • each microdroplet experiences the same temperature profile as they follow the same path until they reach an optical detection zone on the microfluidic chip.
  • some existing digital PCR technologies spread microdroplets across the bottom of a flat chip surface, followed by thermal cycling with a Peltier device.
  • an optical detection zone is integrated into the microfluidic chip. Microdroplets that have completed thermal cycling enter this zone one at a time, and their fluorescence is recorded by a high-speed camera.
  • some existing digital PCR technologies propose obtaining the fluorescent intensity of each microdroplet through planar fluorescent imaging using highly sensitive and expensive enhanced CCDs. This requires sophisticated calibration, both at the software and hardware levels, to ensure each microdroplet receives the same activation light intensity.
  • Microdroplets that have completed the reaction and fluorescent signal reading may be collected in a waste collection chamber at the end of the microfluidic chip, preventing contamination through aerosol or direct contact with the amplified DNA solution.
  • the microfluidic chip can be made from any of a variety of materials such as PMMA (polymethyl methacrylate), PC (polycarbonate), PDMS (polydimethylsiloxane), COC (cyclic olefin copolymer), COP (cyclo olefin polymer), or any other optically transparent materials capable of withstanding the temperatures required for digital PCR reactions.
  • PMMA polymethyl methacrylate
  • PC polycarbonate
  • PDMS polydimethylsiloxane
  • COC cyclic olefin copolymer
  • COP cyclo olefin polymer
  • FIG. 1 presents a block diagram of an example integrated digital PCR chip reader or instrument.
  • Components of the instrument include a central processing unit (CPU) 2, a microcontroller unit (MCU) 3, a pressure control module 4, a temperature control module 5, one or more light sources 6, an optical detection module 7, a signal processing system 8, and a user interface 9.
  • CPU 2 and MCU 3 are located on the same PCB board and communicate with each other via on-board connectors. Together, they facilitate all necessary internal operations within the chip reader instrument.
  • the pressure control module 4 connected to the MCU 3 through on-board connectors and power cables, applies a uniform negative pressure to the digital PCR microfluidic chip. This negative pressure triggers the unidirectional movement of fluid inside the chip, facilitating the generation of sample droplets.
  • these light sources excite the fluorophores within the droplets, enabling the optical detection module 7 to detect the fluorescent signals emitted by each droplet.
  • the optical detection module 7 is connected to the CPU 2 through a data cable, and the data generated by the detection module 7 is recorded and analyzed by the signal processing system 8. The results obtained are then displayed on the user interface 9.
  • the CPU 2 of the integrated digital PCR chip reader interacts with the operator through a user interface 9, receiving experimental parameters and assay type inputs. Based on these inputs, the CPU 2 automatically adjusts the chip reader settings by transmitting commands to the microcontroller board. This leads to the appropriate modification of pressure and temperature settings within the pressure control module and temperature control module, respectively. In some embodiments, modification to pressure and/or temperature is made automatically via feedback and/or feed forward control logic.
  • a method includes an initial operation of introducing a fluid into a channel such as a microfluidic channel on a PCR chip.
  • the fluid comprises sample droplets separated from one another in a carrier fluid in which the sample is substantially immiscible (e.g., an oil).
  • the sample droplets comprise a sample including a nucleic acid, which may be amplified during the method.
  • the sample droplets are aqueous.
  • the fluid flows alternately between at least one denaturing zone having a first temperature and at least one annealing and extension zone having a second temperature, which is lower than the first temperature.
  • the method detects an optical signal from the sample droplets while they continue to flow in the channel.
  • the optical signal is associated with a quantity of amplified nucleic acid in the sample droplets.
  • the method may computationally process the optical signal to provide a digital PCR analysis. Also, the digital PCR analysis may provide an absolute concentration of the nucleic acid in the sample.
  • the sample droplets may comprise reagents such as a polymerase and primers for performing digital PCR.
  • the sample droplets may have a diameter of about 150 to 250 micrometers.
  • the fluid may be produced by contacting the sample with the carrier fluid at a nozzle upstream of the channel.
  • the nozzle may include a first conduit for delivering the sample toward the channel, a second conduit for delivering the carrier fluid toward the channel, and a junction of the first conduit and the second conduit where the sample droplets in the fluid are produced.
  • the nozzle comprises a plurality of second conduits, each for delivering the carrier fluid to the junction.
  • the droplet generation and fluid flow in a channel may be induced by applying a vacuum to draw the fluid into the channel.
  • the fluid may be induced to flow into the channel to by applying a positive pressure to sources of the sample and/or the carrier fluid upstream from the channel.
  • the fluid flow channel has a serpentine path, so that during PCR, the fluid flows in the serpentine path. While flowing through the serpentine path, the fluid may repeatedly encounter at least one denaturing zone and at least one annealing and extension zone.
  • the serpentine path of a channel may comprise parallel serpentine segments, with each serpentine segment having an upstream portion in a first annealing and extension zone, a downstream portion in a second annealing and extension zone, and an intermediate portion in a first denaturing zone.
  • the sample droplets flow in the serpentine path, they may experience at least about 50 thermal cycles.
  • a method for conducting PCR may detect an optical signal from the sample droplets.
  • Such signal may comprise fluorescence associated with the nucleic acid.
  • the fluorescence may be produced by a fluorophore coupled to an amplification primer.
  • the method detects fluorescence or other optical signal produced by a plurality of fluorophores (or other sources), each emitting light at a different wavelength, and each associated with a different nucleic acid.
  • the sample is produced via a simple preprocessing stage.
  • preprocessing may involve diluting a biological specimen and adding PCR reagents to produce the sample.
  • the sample in the flowing fluid undergoes an initial polymerase activation.
  • the fluid before flowing alternately between the at least one denaturing zone and the at least one annealing and extension zone comprises, the fluid may flow through a polymerase activation zone that performs hot-start polymerase activation on the sample droplets.
  • the polymerase activation is performed at the same temperature as the denaturing described above. In fact, the same heating element may be employed to perform polymerase activation and denaturing.
  • the sample may be recovered after it has been subject to PCR and optical detection.
  • the recovered sample may be used for conducting further analysis such as sequencing.
  • Figure 2 illustrates an example method for conducting digital PCR. As illustrated, the method begins in operation 201 where the sample to be analyzed is prepared from a biological specimen and a PCR reaction mixture. As known to those of skill in the art the reaction mixture may include primers for generating and amplified from a target sequence, polymerase, and the like.
  • the samples mixed with a carrier fluid, which is typically a fluid in which an aqueous sample is substantially immiscible. See operation 202.
  • a carrier fluid typically a fluid in which an aqueous sample is substantially immiscible.
  • the density of the sample may be greater than that of the carrier fluid, which causes the sample to settle to the base of a sample reservoir.
  • the carrier fluid may be an oil.
  • the temperature of the sample reservoir is elevated and sustained at a level that (a) facilitates activation of UNG enzyme that may be present and (b) facilitates reverse transcription.
  • UNG uracil-DNA glycosylase
  • Reverse transcription is the process of generating DNA from an RNA template. Thus, if a sample contains RNA having a putative target sequence, reverse transcription is performed prior to PCR in order to convert the RNA in the sample to DNA, which can be amplified by PCR.
  • the sample is converted into microdroplets, which potentially contain target DNA.
  • individual sample microdroplets are separated from one another while flowing with a carrier fluid in a microfluidic channel.
  • the flowing fluid which contains the sample microdroplets and the carrier fluid, are generated via a nozzle.
  • the fluid formed at the nozzle initially flows into a hot-start zone where any reverse transcriptase is deactivated and DNA polymerase for PCR is concurrently activated.
  • the sample microdroplets in the flowing fluid are subjected to thermal cycling between high and low-temperature zones. This thermal cycling amplifies any target DNA present within the microdroplets.
  • end-point analysis is conducted by measuring fluorescent intensity from each microdroplet. From these intensity values, an initial quantity of nucleic acid in the sample may be calculated using a Poisson distribution.
  • Figure 3 presents a data collection and processing sequence in a digital PCR embodiment.
  • the example method begins in an operation 307 where sample micro droplets, having completed the thermal cycling process described above, individually enter an optical scanning zone via the microchannel in which the sample droplets are flowing.
  • each micro droplets undergoes fluorescence scanning via one, two, or more high-speed cameras.
  • Each camera detects a distinct fluorescence signal.
  • a sample comprising multiple potential target nucleic acid sequences may analyze sample for each of those target sequences.
  • Each such target sequence is associated with a different fluorescence channel.
  • a region of interest may be a rectangular area designated for the measurement of optical signal (e.g., fluorescence).
  • optical signal e.g., fluorescence
  • a mean fluorescence intensity within this area may be determined.
  • a time-series chart may then be generated for each channel, depicting the average fluorescence over time, which is used for analyzing and identifying peak patterns subsequently.
  • a baseline fluorescence and a fluorescence threshold of the microdroplets are continuously analyzed while the fluid flows in the optical scanning zone.
  • an initial count of nucleic acid copies in the sample is updated in real-time, based on the principles of the Poisson distribution.
  • a preliminary positive qualitative result is optionally delivered once a specified quantity of positive microdroplets has been detected.
  • a positive microdroplet is one in which the baseline fluorescence detected exceeds the threshold fluorescence.
  • quantitative results are output using a comprehensive analysis of the entire microdroplet population. An initial count of nucleic acid copies in the sample is calculated based on the proportion of positive results among the results of all analyzed microdroplets.
  • PCR PCR-specific PCR
  • an instrument may work with a PCR chip to conduct digital PCR.
  • PCR chips are disposable while PCR instruments are permanent, being able to conduct PCR in multiple PCR chips, sequentially.
  • a PCR instrument is a “module,” which is one of two or more such instruments that operate together, sharing resources. See Figures 8 A and 8B for examples of the modular approach.
  • a PCR chip processing instrument comprises at least the following components: (a) a plurality of heat blocks, each configured to maintain a constant temperature during thermal cycling; (b) a flow induction unit configured to induce a flowing fluid through a microfluidic channel in a PCR chip engaged with the chip processing instrument; (c) an optical detector; and (d) a receptacle sized and shaped to engage the PCR chip in a fixed position with respect to: (i) the flow induction unit, (ii) the optical detector, (iii) at least one of the plurality of heat blocks; or (iv) any combination thereof.
  • the instrument also comprises a signal processor configured to receive signals from the optical detector, where the signals comprise information about amplification of nucleic acids in sample droplets of the flowing fluid.
  • processing optical signals is conducted remotely, outside the instrument.
  • the heat blocks include (i) a high temperature heat block configured to maintain a first region of the PCR chip at a first temperature or a first temperature range, and (ii) a low temperature heat block configured to maintain a second region of the PCR chip at a second temperature or a second temperature range, where the first temperature or first temperature range is greater than the second temperature or second temperature range.
  • the high-temperature heat block(s) may be configured to maintain a temperature that facilitates hot start polymerase activation and/or nucleic acid denaturing.
  • the first temperature or temperature range is within a range from about 90 to 95° C.
  • the low- temperature heat block(s) may be configured to maintain a temperature that facilitates sample inlet heating, nucleic acid annealing, nucleic acid extension, or any combination thereof.
  • the second temperature or temperature range is within a range from about 50 to 72° C.
  • a high temperature heat block comprises a first region proximate a sample inlet configured to facilitate polymerase activation and a second region configured to facilitate nucleic acid denaturing.
  • the first and second regions are held at same temperature during PCR. As an example, when the first and second regions are viewed together from above (where the PCR chip engages the instrument), they have a “T” shape.
  • a low temperature heat block comprises (i) a first region configured to heat a sample reservoir to a temperature that activates a UNG enzyme and/or a reverse transcription reaction, and (ii) a second region configured to facilitate nucleic acid annealing and extension.
  • the first and second regions of the low temperature block are held at same temperature during PCR.
  • one or more of the heat blocks comprises a polyimide heater or a rod heater.
  • the flow induction unit may operate by applying a negative pressure or a positive pressure to the sample.
  • a flow induction unit examples include vacuum pumps, plungers, syringe drives, and the like.
  • the flow induction unit may induce fluid flow using a driving force other than pressure. Examples, of such driving forces include electrostatic forces such as electrocapillary forces.
  • the optical detector may include two or more optical submodules, each configured to detect electromagnetic radiation at a different wavelength.
  • an optical detector may comprise two to ten optical submodules (e.g., two or six optical submodules).
  • each submodule includes a light source and a light detector.
  • the light source emits radiation at an excitation frequency for a fluorescent moiety under consideration
  • the light detector detects the intensity of light at the emission frequency of the fluorescent moiety under consideration.
  • an optical submodule employes a light emitting diode (LED) or other broadband light source.
  • the submodule may employ a filter such as a notch filter that reduces the bandwidth of the excitation radiation that is directed onto the sample droplets.
  • the filter excludes frequencies away from the excitation frequency of the fluorescent moiety under consideration.
  • an optical submodule employs laser or other narrow band excitation source. In such cases, the submodule may not include a filter to narrow the wavelength range of the excitation radiation directed onto the sample droplets.
  • the instrument may support up to six or up to ten different optical submodules.
  • an instrument may six channels, each for distinct fluorophore:
  • the cameras or detectors are synchronized.
  • the instrument may measure and characterize multiple fluorescence channels (such as those for FAM, HEX, and CY5) present in each droplet.
  • fluorescence channels such as those for FAM, HEX, and CY5
  • every droplet may be imaged by distinct color channels at separate instances in time, thus requiring an alignment of the channel outputs to avoid misrepresentation in fluorescence signals.
  • This may be achieved by synchronizing the cameras or sensors to maintain an identical frame rate, thereby enabling precise alignment of image frames for accurate result computation.
  • a relatively simpler digital PCR instrument supports detection of only a single nucleic acid sequence, i.e., a single amplicon.
  • the signal processor may be configured to computationally process optical signals in a manner that conducts a digital PCR analysis, which may provide an absolute concentration of nucleic acid in the sample.
  • some embodiments involve processing optical signals outside the instrument such as via a cloud computing device.
  • the instrument may include a control processor configured to control the flow induction unit, the plurality of heat blocks, the optical detector, or any combination thereof.
  • the instrument has design features that permit home or lay user usage.
  • the overall footprint or dimensions of the instrument are sufficiently small to make the instrument portable or suitable for countertop or desktop operation.
  • FIG. 4A shows an example exterior view of a digital PCR instrument 401.
  • instrument 401 has a casing 403 that including a removable top portion 405 and a bottom portion 407.
  • instrument 401 is shown with a PCR chip 409 inserted in a slot 411 that serves as a guide for the microfluidic chip, enabling the user to easily slide it in.
  • PCR chip 409 includes an oil reservoir 413 and sample reservoir 415. Note that most of PCR chip 409 is inside module 401 to the right of slot 411.
  • Figure 4B presents an exploded view of digital PCR instrument 401.
  • the instrument generally includes components for optical data collection, heating, and driving fluid flow.
  • instrument 401 includes an optical module 421, including six optical submodules.
  • instrument 401 includes a high temperature heater block 423, which has a generally T shape and a low temperature heater block 425 which has a generally U shape.
  • the high and low temperature blocks 423 and 425 may align with regions of the fluid flow channel in the PCR chip as illustrated in Figure 13.
  • Instrument 401 additionally includes various components for applying a vacuum to the PCR chip 409 during operation.
  • instrument 401 includes a vacuum pump 427, a connection429 for engaging the vacuum pump and PCR chip 409 during operation, and a vacuum valve 431.
  • the connection 429 is attached to an actuator such as a solenoid configured to move the connection vertically to allow the PCR chip 409 to be inserted into position in module 401 and subsequently connect chip 409 to vacuum pump 427.
  • the up position is depicted in Figure 4C, and the down position is depicted in Figure 4D.
  • a solenoid is initially positioned downward, and valve 431 is closed.
  • the PCR instrument 401 also includes a printed circuit board 433 on which is mounted electronics for operating the module. Such electronics may include a device controller and/or analysis logic. As explained, these elements may be implemented as integrated circuits.
  • the lower portion 405 of digital PCR instrument 401 has a receptacle 435 sized and shaped to engage with PCR chip 409 during amplification and analysis.
  • one or more thermal control blocks are tightly connected to the top of the microfluidic chip.
  • the bottom of the heat blocks may be loaded with springs, which exert a substantially constant upward pressure to the blocks. Installation of the microfluidic chip results in its bottom make contact with these spring-loaded heat blocks, causing the springs to compress. This compression generates a force that maintains the chip firmly against the heat blocks, facilitating efficient thermal transfer between the chip and the heat blocks.
  • FIG. 5A see the cross-sectional view of a digital PCR instrument 501 as illustrated in Figure 5A, which provides a crop view of a spring-loaded high and low temperature heat blocks 502 and 504 for provided good thermal conductance between a microfluidic chip 506 and the heat blocks.
  • instrument 501 includes a spring 508 for supporting low temperature heat block 504 and springs 510 for supporting high temperature heat block 502.
  • high and low-temperature zones on a thermal control block are closely packed and separated by thin thermal insulation layers.
  • the thin thermal insulation layers function as barriers between the heat blocks having differing temperatures. These layers serve to impede thermal transfer between the high and low-temperature zones.
  • the thermal insulation layers are constituted from materials such as EVA (ethylene-vinyl acetate), EPE (expanded polyethylene), XPE (cross-linked polyethylene), or other insulative substances. Some thermal insulation layers may be porous. As an example, see the cross-sectional view of a digital PCR instrument as illustrated in Figure 5B including thermal insulation layer 512.
  • microdroplets As microdroplets move from high to low- temperature zones, their temperature rapidly decreases and stabilizes to a desired level. Conversely, as microdroplets move from low to high- temperature zones, their temperature rapidly increases and stabilizes to a desired level.
  • the magnitude of temperature variation depends on the speed of droplet movement and the gap separating the zones of high and low temperature. For example, with a 35 °C temperature differential between the two zones, a 5mm separation, and droplet speeds varying from 1.0 to 5.0mm/s, the rate at which the temperature ramps can vary from 7°C/s to 35°C/s.
  • Figures 6A-E illustrate aspects of a digital PCR instrument 601.
  • Figure 6A presents a perspective view
  • Figure 6B presents a front view
  • Figure 6C presents a back view
  • Figure 6D presents a side view
  • Figure 6E presents a top view (with an optical cover removed) of the instrument.
  • instrument 601 includes a chassis 603, an optics cover 605, a user interface including a display screen 607, support feet 609, a microfluidic chip holder 611, a collection vessel 613, a tube 615 for connecting an outlet of a fluidic channel in a microchip to the collection vessel, and a tube 617 for connecting a negative pressure source (not shown) to the collection vessel
  • the display screen may be touchscreen with interactive user interface.
  • Other components of the user interface may include audio, tactile, and/or visual interface elements.
  • vessel 613 in bottle or similar device configured to collect processed sample and/or waste from the chip.
  • vessel 613 serves as a pressure chamber that may buffer pressure fluctuations from operation of a vacuum pump that would otherwise be felt by the chip during operation.
  • Figure 6 A and 6E show chip holder 611 with a microfluidic chip inserted therein, as would be the case during dPCR operation.
  • Figure 6E shows instrument 601 with optics cover 605 removed and optics submodules 619A and 619B exposed.
  • the embodiment of Figures 6A-E illustrates a two-channel instrument.
  • the instrument can be configured to additional optics submodules to allow processing additional channels, as is the case in highly multiplexed dPCR processing.
  • a digital PCR instrument has a top cap with hinged connection to a main chassis of the instrument. When closed, the top cap allows the PCR reaction and analysis to proceed while protecting the microfluidic chip from outside interferences from, e.g., air currents, optical interference, and the like.
  • one or more of the heat blocks is disposed on the top cap of the dPCR instrument.
  • the heat blocks may be spring-loaded with the top cap.
  • the heat blocks are attached to the top cap with springs, so that when the top cap closes on the main body of the instrument, the heat blocks gently engage with the microfluidic chip. Nevertheless, the area of contact between the microfluidic chip and the heat block is maintained, ensuring consistent thermal performance.
  • the microfluidic chip is placed on a stable, solid surface (in the main body of the instrument), ensuring its vertical position remains constant. In some cases, this approach may facilitate optical imaging of flowing sample droplets by maintaining the microfluidic chip in the optical focal plane of the imaging elements.
  • FIGs 6F and 6G illustrate a dPCR instrument 651 with a chassis 653 and a top cap 655 attached to one another by a hinge 657.
  • Chassis 653 includes, among other elements, a user interface 659, optical modules 661, and a chip holder 663.
  • a microfluidic chip 665 is disposed in chip holder 663 while top cap 655 is in an open position. See Figure 6F.
  • Top cap 655 includes a low temperature heat block 667 and a high-temperature heat block 669.
  • Figure 6G illustrates instrument 651 with the top cap 655 closed and the microfluidic chip 665 engaged for operation. As illustrated, oil and sample ports 671 and 673 of microfluidic chip 665 are accessible for delivery of oil and sample during operation.
  • the heat blocks on the top cap have the same or substantially the same two-dimensional sizes, shapes, and orientations as those in embodiments discussed elsewhere herein such as with respect to Figure 13.
  • a preheater portion of a heat block is provided in the main body of the instrument, as opposed to being part of the preheaters in the top cap.
  • preheater 1313 in the Figure 13 design may be provided in the body of the heat block, under the chip, while all other heater portions are provided in the top cap.
  • a separate preheater block may be employed, which is no longer part of the first low temperature block.
  • the top cap includes a preheater, which may be part of the first low temperature block.
  • a microfluidic chip holder includes a negative pressure vacuum line connection, optionally located within the top cap itself.
  • the vacuum line connection may provide a mechanical/fluidic connection between the outlet of the chip and a tube from the vacuum source.
  • the vacuum line connection comprises an elastic suction cup, which can be made of various flexible materials such as rubber, silicone rubber, PVC, or PE. When the cap is closed, the vacuum line connection compresses against the outlet of the microfluidic chip. This motion deforms the vacuum line connection, resulting in a firm seal that effectively connects the microfluidic chip to the negative pressure source.
  • the vacuum line connection is disposed in the body of the instrument rather than in a top cap. Such embodiments may be appropriate when the distal end of a vacuum line is disposed within the body of the instrument rather than in the top cap.
  • Figure 6H and 61 illustrate a dPCR instrument 681, having features in common with dPCR instrument 651 illustrated in Figures 6F and 6G.
  • Figures 6H and 61 focus on a top cap 683 connected to an instrument chassis 685 by a hinge 695.
  • Figure 6H depicts instrument 681 with top cap 683 in an open position with a microfluidic chip 687 installed
  • Figure 61 depicts instrument 681 with top cap 683 closed on chip 687.
  • the bottom of top cap 683 includes a vacuum line connection 689 (e.g., a suction cup) positioned over a fluid outlet 691 of chip 687.
  • a vacuum line connection 689 e.g., a suction cup
  • vacuum line connection 689 engages with fluid outlet 691 to provide a continuous path from a vacuum source (not shown) to microfluidic chip 687.
  • a vacuum line 693 connecting vacuum line connection 689 to the vacuum source is shown in Figure 61.
  • a PCR instrument may work with a PCR chip to implement digital PCR.
  • the PCR chip is disposable.
  • a PCR chip may include: (a) a sample inlet configured to receive a sample comprising a nucleic acid; (b) a nozzle configured to generate sample droplets comprising the sample; (c) a fluid flow channel configured to contain a flowing fluid including the sample droplets; and (d) an optical detection zone configured for optical interrogation the sample droplets flowing in an optical detection segment of the fluid flow channel.
  • the PCR chip additionally includes a waste chamber coupled to an outlet of the fluid flow channel.
  • the sample droplets are separated from one another by an immiscible phase of the flowing fluid.
  • the immiscible phase may serve as a carrier fluid for the sample droplets, which may be aqueous.
  • the sample droplets may comprise reagents for performing digital PCR.
  • the sample inlet may include a port, conduit, reservoir, or similar structure configured to receive sample into the PCR chip.
  • the PCR chip includes a separate sample reservoir in fluid communication with the sample inlet.
  • the reservoir which may comprises a cylindrical container, holds the sample and makes it available for producing the sample droplets in the flowing fluid.
  • the PCR chip may include an immiscible phase inlet for receiving a liquid comprising the immiscible phase. It may also include an immiscible phase reservoir in fluid communication with the immiscible phase inlet.
  • the nozzle of the PCR chip may be in contact with the sample and the immiscible phase.
  • the nozzle comprises a first conduit for delivering the sample toward the fluid flow channel, a second conduit for delivering the immiscible phase toward the fluid flow channel, and a junction of the first conduit and the second conduit. During operation, the sample droplets in the flowing fluid are produced at the junction.
  • the nozzle comprises a plurality of second conduits, each for delivering the immiscible phase to the junction. Figures 8A and 8B illustrate an example of such a nozzle.
  • the nozzle is configured to produce the flowing fluid, including the sample droplets, under the influence of a vacuum source.
  • a vacuum may draw both the sample and the immiscible phase from their respective reservoirs to the nozzle.
  • the sample droplets may have a diameter or other dimension that is larger than the size of the nozzle.
  • the nozzle may have a diameter that is about 100 to 150 micrometers.
  • the fluid flow channel has a serpentine path on the PCR chip. Each time the channel traverses the chip, it passes over two or more heating zones. In this manner, thermal cycling is accomplished.
  • the serpentine path may repeatedly pass between at least one high temperature heat block and at least one low temperature heat block of the chip processing instrument.
  • the serpentine path may be characterized by a plurality of parallel serpentine segments of the fluid flow channel, and each such serpentine segment may have an upstream portion over a first low temperature heat block, a downstream portion over a second low temperature heat block, and an intermediate portion over a high temperature heat block, where the intermediate portion is between the upstream and downstream portions.
  • the plurality of parallel serpentine segments may comprise about 30 to 50 parallel serpentine segments.
  • the fluid flowing in the serpentine path may experience at least about 50 thermal cycles within the PCR chip.
  • the chip When the PCR chip engages with a chip processing instrument, the chip’s optical detection zone is located adjacent to an optical detector configured to obtain optical signals from the sample droplets in the flowing fluid.
  • the optical detection segment of the optical detection zone is located downstream from fluid flow channel segments of a denaturing zone and an annealing and extension zone.
  • the fluid flow channel passes through multiple zones or regions of different temperatures.
  • the serpentine path facilitates repeated cycles of temperature increase and decrease needed for denaturing, annealing, and extension.
  • the PCR chip may include a polymerase activation zone configured to perform hot-start polymerase activation in the sample droplets when the sample droplets are flowing in at least one polymerase activation segment of the fluid flow channel within the polymerase activation zone.
  • the polymerase activation zone when the PCR chip engages with a chip processing instrument, is located over a high temperature heat block configured to operate at a temperature that facilitates hot start polymerase activation in the sample in the at least one polymerase activation segment of the fluid flow channel.
  • Some PCR chips have fluid flow channels that pass through three primary temperature regions during each pass of the flow channel in a serpentine path. For example, each pass (or segment) may start in a first low temperature region, then pass over a high temperature region, and finally pass though a second low temperature region.
  • Figure 13 provides an example of this channel configuration.
  • the PCR chip may include a first annealing and extension zone for first annealing and extending nucleic acids in the sample droplets when they flow in a plurality of first segment portions of the fluid flow channel, wherein the first segment portions are located in the first annealing and extension zone.
  • the first annealing and extension zone may be located over a low temperature heat block configured to operate at a temperature that facilitates annealing and extending nucleic acids in the sample while the sample is flowing in the first segment portions over the first annealing and extension zone.
  • first “segment portions” of the fluid flow channel are portions of the full-length channel that, during operation, are located over the annealing zone, and fluid flowing within those segment portions has a temperature dictated by the low temperature heat block.
  • the first annealing and extension zone is located downstream from a polymerase activation zone in the fluid flow channel.
  • the PCR chip also contains a denaturing zone for denaturing double-stranded nucleic acids in the sample droplets when flowing in a plurality of second segment portions of the fluid flow channel, where the second segment portions are located in the denaturing zone.
  • the denaturing zone may be located over a high temperature heat block configured to operate at a temperature that facilitates denaturing the nucleic acids in the sample in the second segment portions of the fluid flow channel.
  • at least some of the plurality of second segment portions are located downstream from at least some of the first segment portions in the first annealing and extension zone.
  • the polymerase activation zone and the denaturing zone are both located over a high temperature heat block of the chip processing instrument.
  • the PCR chip additionally includes a temperature buffer zone disposed between first annealing and extension zone and the denaturing zone.
  • the PCR chip also contains a second annealing and extension zone for annealing and extending nucleic acids in the sample droplets when flowing in a plurality of third segment portions of the fluid flow channel, where the third segment portions are located in the second annealing and extension zone.
  • the second annealing and extension zone is located over a low temperature heat block configured to operate at a temperature that facilitates annealing and/or extending double-stranded nucleic acids in the sample in the third segment portions.
  • at least some of the plurality of third segment portions are located downstream from at least some of the plurality of second segment portions in the denaturing zone.
  • the first and second annealing and extension zones are both located over a low temperature heat block of the chip processing instrument.
  • the PCR chip additionally includes a temperature buffer zone disposed between the denaturing zone and the second annealing and extension zone.
  • an example of a digital PCR microfluidic chip 711 is comprised of two layers, a top and a bottom layer 715 and 716.
  • the top layer 715 of the digital PCR microfluidic chip contains a network of microfluidic channels that may be fabricated by, e.g., etching with a high precision laser module or molding through injection molding.
  • Oil inlet 712 and sample inlet 713 are located upstream of the microfluidic channels and are accessed through holes.
  • Cylindrical containers are situated on top of the sampleinlet and the oil inlet, serving as reservoirs for sample and oil.
  • Sample and oil microfluidic channels merge at the microdroplet generating nozzle 714, where uniform-sized microdroplets are formed through the application of either positive or negative pressure on the microchannel.
  • An embodiment employing a positive driving force may optionally apply pressure to the oil and sample reservoirs on the PCR chip.
  • FIG 7B an upper panel depicts a cropped top view of chip 711, while a lower panel depicts a cross-sectional side view of chip 711 cut through the cylindrical reservoirs.
  • a hot-start polymerase activation zone 715 Downstream of the microdroplet generating nozzle is a hot-start polymerase activation zone 715, where microdroplets undergo a sustained high temperature treatment while flowing along corresponding microchannels.
  • the hot-start zone is connected to a microdroplet reaction chamber 716, where a series of microchannels alternate between high and low temperature zones, resulting in, e.g., about 30-45 temperature cycles within the digital PCR microfluidic chip.
  • an optical detection zone 717 Following the reaction chamber is an optical detection zone 717, where the fluorescent intensity of each of the microdroplets is detected.
  • the optical detection zone is elongated such that multiple fluorescence color channels can be placed to achieve multiplexing detection.
  • the detection signal can be analyzed using on- board electronics or transmitted to external devices for further data processing and analysis.
  • the end of the microchannels is connected to a waste chamber 718 where all the reacted microdroplets are collected.
  • the waste outlet is a connection port 719 that attach
  • a PCR microfluidic chip processes a stream of sample droplets produced upstream of PCR zones on the chip.
  • Many types of device structures may be suitable for generating the sample droplets.
  • a nozzle or similar structure is one example. More generally, the structure can be referred to as a sample droplet generation feature.
  • a sample droplet generator is configured to bring into contact with the sample, which is typically aqueous, and a carrier fluid, which is typically immiscible with the sample.
  • the generator may comprise separate lines for transporting these two liquids from their respective sources or reservoirs to a junction of contact point, where the two liquids mix in a manner that produces the stream of sample droplets in the immiscible carrier fluid.
  • the sample liquid is transported to the contact point in a first line and the carrier fluid is transported to the contact point in two or more second lines that straddle or surround the first line.
  • the combination of the sample and carrier liquids may be such that carrier fluid surrounds the droplets as a sheath or that the carrier fluid merely separates the droplets from one another, in which case the droplets may contact the channel wall without being surrounded by the carrier fluid.
  • the droplet size and shape may be dictated by any number of parameters such as the nozzle size and shape; the reception channel diameter; the fluid flow rate; and sample and carrier fluid hydrodynamic properties such as viscosity, density, and wetting; and any combination thereof.
  • oil reservoir 803 is disposed upstream of sample reservoir 805. Acting under the influence of a pressure differential, oil from oil reservoir 803 flows out of a channel 807 connected to reservoir 803 and enters a branching flow path 809 that straddles at least a portion of sample reservoir 805.
  • Figure 8B depicts in more detail a portion of sample reservoir 805 and the various flow channels where the oil phase and sample combine to form the sample droplets. Collectively, some or all the channels that connect to produce the sample droplets 817 may be referred to as a nozzle.
  • sample droplets 817 forming at junction 813 initially enter a wide-diameter portion 819 of channel 815. It has been found wide-diameter portion 819 may facilitate sample droplet generation in a manner that prevents the sample droplets from sticking to the channel walls. In certain embodiments, wide-diameter portion 819 has diameter of about 300 to 500 micrometers and a length of about 3 to 8 millimeters.
  • Figures 9A and 9B show a microfluidic PCR chip 950 having a parallel flow design to facilitate sample droplet generation.
  • one or more microchannels 951 for flowing an immiscible phase 961 run in parallel with and straddle an inner channel 953 for flowing an aqueous sample phase 963.
  • the one or more immiscible phase microchannels 951 are provided as single channel surrounds the inner channel 953 to form a sheath around channel 953. All such designs, whether the one or more microchannels 951 form a sheath or merely form two or more channels parallel to inner channel 953, can be considered co-axial.
  • a nozzle exit 955 shear forces caused by the immiscible phase 961 flowing past the aqueous sample phase lead to the destabilization and breakup of the sample stream into discrete droplets 959.
  • the size of these droplets is dictated by parameters such as the magnitude of vacuum pressure applied to the microfluidic channel, flow rates of oil and aqueous phases, the viscosity and interfacial tension of the fluids, and the geometry of the chip, such as the diameter of the nozzle.
  • fluidic and design parameters applied to conduct PCR on a chip may be as follows.
  • Vacuum pressure applied to the microfluidic channel about IkPa to lOOkPa, or about WkPa to 30kPa.
  • Flow rates of immiscible and aqueous phases about 0.5mm/s to lOmm/s or about about 1.5mm/s to 4.5mm/s.
  • Viscosity and interfacial tension of the immiscible phase fluid @ 25°C about 5 to 150 centistokes (cSt) or about about 25 to 75 cSt.
  • Width about 30 - 50mm
  • Diameter of the nozzle about 40nm to 200nm or about 70nm to 140nm.
  • Figure 10 depicts an alternative embodiment having a combined oil and sample port 1001.
  • Figure 10 includes a left panel showing a top view and a right panel showing a side cross- sectional view of sample port 1001.
  • sample port 1001 includes an outer well wall 1003, and inner sample well wall 1005, and two oil ports 1005 straddling a sample port 1007.
  • Sample port 1007 is bounded by sample well wall 1005.
  • oil is present in an annular space between outer wall 1003 and inner sample well wall 1005.
  • the sample port 1007 and two oil ports 1005 feed three microchannels 1009, each configured to delivering sample and oil from their respective ports.
  • microchannels 1009 flow sample and oil horizontally until they reach a junction point (not shown) where the sample forms droplets in the oil, which serves as a carrier fluid.
  • the junction point and downstream channel has structure similar to that depicted in Figure 8B.
  • the immiscible phase or carrier fluid is manually introduced (e.g., pipetted) by the operator at the start of a run.
  • the immiscible phase or carrier fluid is pre-filled into the microfluidic chip, optionally as part of the chip’s fabrication process.
  • the operator need not introduce such fluid, cutting down on the time it takes to prepare a dPCR run. Moreover, it lessens the amount of manual operation needed for the run.
  • the chip design may be similar to that in which carrier fluid is not pre-filled.
  • the structure is essentially the same as that shown in Figure 8A.
  • the pre-filled fluid may be stored on the microfluidic chip in reservoir 1203 and may move to the nozzle via channels 807 and 809 during operation.
  • a similar pre-filled chip may use the concentric reservoir design of Figure 10.
  • Figures 7A and 11 A show the position of a carrier fluid reservoir in the context of the entire microfluidic chip.
  • a reservoir holding pre-filled carrier fluid may be sealed or partially sealed from the atmosphere.
  • a microfluidic PCR chip may include a waste chamber.
  • the waste chamber is an open or hollow area without internal features.
  • the waste chamber in the microfluidic chip includes one or more features such as channels, barriers, sub-chambers, walls, etc. Such features may cause the oil and reacted sample to utilize much or all the volume of the chamber before exiting the microfluidic chip.
  • the chamber is configured with walls that create a maze-like pattern (e.g., a serpentine pattern) that may increase utilization of surface area within the waste chamber.
  • a maze-like pattern e.g., a serpentine pattern
  • This design forces fluid (both oil and reacted sample) to navigate through the entire chamber prior to reaching the outlet. As a result, it ensures that the complete volume of the waste chamber is utilized, optimizing the efficiency of waste handling within the microfluidic system.
  • Figure 1 A illustrates a microfluidic PCR chip 1101 having features in common with microfluidic PCR chip 711 of Figure 7A (and uses some common reference numbers).
  • microfluidic PCR chip 1101 has a waste chamber 1103 including a plurality of walls or barriers 1105, interior to chamber 1103, that cause fluid within chip 401 to traverse a tortuous path (e.g., a serpentine path) before exiting chip 1101 at an outlet 407.
  • a tortuous path e.g., a serpentine path
  • the microfluidic chip is designed so that a reacted sample, which typically contains some droplets with amplified nucleic acid segments encapsulated within droplets, gradually collects in a collection chamber, which may also serve as a waste chamber, where the droplets spontaneously and orderly settle into a layer at the chamber's base.
  • a collection chamber which may also serve as a waste chamber, where the droplets spontaneously and orderly settle into a layer at the chamber's base.
  • Embodiments making use of a collected layer of processed droplets may employ a different or additional optical system from those described elsewhere herein, such as with respect to Figure 12, which detects fluorescence of the droplets, one-by-one, sequentially in time, as they flow within a channel of the chip.
  • embodiments utilizing a layer of collected droplets may employ a single image or a small group of images to capture the entire array of droplets. Such image(s) may be captured at one time, after the sample is done flowing through the microfluidic chip near the heater blocks. Note that the presence or absence of amplified nucleic acids in all the droplets may be accomplished with appropriate image analysis routines such as segmentation routines.
  • Figure 1 IB presents and image of a collection chamber 1111 of a microfluid chip, where a plurality of individual droplets 1113 have collected before exiting the chip. As shown, droplets 1113 spread and pack into a single layer of droplets 1115 in the chamber.
  • an example optical detection module 1220 of the instrument is composed of, e.g., one to six distinct optical submodules 1221 and is located beneath the microfluidic chip 1211.
  • Each submodule may include a single fluorescent emission/detection channel.
  • a submodule comprises a light source 1222, which can be a laser or light-emitting diode (LED); a fluorescent detector, such as a charge-coupled device (CCD), complementary metal-oxide- semiconductor (CMOS), or photodiode; and optionally other optical elements such as an objective lens, emission filters, and excitation filters, and/or a dichroic lens.
  • any two submodules have two different light sources, which may emit radiation of frequencies that excite two different fluorophores.
  • a horizontally emitted light beam is bent at a 90-degree angle by the dichroic lens and subsequently focused, through an objective lens, on the optical detection zone 1217 of the microfluidic chip 1211, forming a light band.
  • the fluorescence within each droplet is sequentially excited by the light beams emitted by each of the optical submodules.
  • the fluorescent intensity is captured by a fluorescent detector board 1223 situated at the base of each optical submodule 1221.
  • a temperature regulation module 1324 comprises separated temperature blocks, each equipped with a dedicated temperature sensor and a heater such as a rod heater or a polyimide film heater.
  • the power supplied to the heater may be regulated by a microcontroller unit (MCU) using a Proportional-Integral-Derivative (PID) algorithm. It ensures accurate and stable temperature conditions within the temperature blocks, allowing for efficient thermal cycling and DNA amplification.
  • MCU microcontroller unit
  • PID Proportional-Integral-Derivative
  • Two temperature buffer zones 1327 are positioned between the high 1326 and low-temperature heat blocks 1325 and can prevent heat neutralization between them. These buffer zones, thermally insulated from their surroundings, maintain a temperature intermediary between that of the high 1326 and low- temperature heat blocks 1325. Heat neutralization is an undesired heat transfer from the zone of high temperature to the low temperature zone via convection, which results in the excessive heating of the zone that is supposed to remain cooler.
  • each temperature block may be constructed using a specific metal or alloy, such as silver, copper, or aluminum. These metals may be chosen for their excellent heat transfer properties and thermal conductivity, which allow for efficient and precise temperature control. The selection of the metal or alloy depends on factors such as the desired temperature range, thermal stability, and the specific requirements of the application.
  • each microdroplet journeys through the entire length of the microchannel at a consistent flow rate. Consequently, all microdroplets are subjected to identical thermal cycling parameters, ensuring uniform processing.
  • the temperature within each thermal control zone is preset and stabilized prior to the experiment. As microdroplets navigate across the two thermal control zones, they experience nearly instantaneous temperature ramping, either upwards or downwards.
  • a first thermal cycle starts when the sample droplet moves along the flow channel in the hot- start polymerase activation zone 1315.
  • the sample experiences a high temperature at which double stranded nucleic acids in the sample denature. This may represent the first phase of the first PCR thermal cycle.
  • the sample droplet After the sample droplet leaves activation zone 1315 via the flow channel, it continues to travel in the flow channel where it enters the lower right corner of the left portion of low temperature region 1325. While in the left portion of region 1325, the sample undergoes anneal and extension. In other words, anneal and extension occur all the while the sample flows from the entry point, first in leftward direction within region 1325, then changes direction in a hairpin turn rightward at the far left side of region 1325, and finally in a rightward direction until the fluid leaves region 1325 while continuing to flow in a rightward direction. At this point, the sample may be considered to have completed its first thermal cycle.
  • the sample droplet then flows beyond the right side of region 1327 and enters the left side of the right portion of low temperature region 1325.
  • the droplet enters the right portion of region 1325, where the sample droplet undergoes another anneal and extension operation.
  • anneal and extension in the second thermal cycle occurs all the while the sample droplet flows from the entry point in region 1325, first in rightward direction, then changes direction in a hairpin turn leftward at the far right side of region 1325, and finally in a leftward direction until the droplet leaves region 1325.
  • the sample may be considered to have completed its second thermal cycle.
  • sample droplet enters the right side of high temperature region 1327, where it begins the third thermal cycle, and the nucleic acids in the sample droplet is again denatured.
  • a fluid flow channel of a PCR chip typically has defined cross-sectional dimensions.
  • such channel has depth (measured in the z-direction from a surface plane of the chip to a bottom of the channel) of about 20 to 200 micrometers or about 50 to 100 micrometers.
  • such channel has a width (measured in the x-direction along the surface plane of the chip) of about 100 to 300 micrometers or about 160 to 200 micrometers.
  • these cross-sectional dimensions are constant or nearly constant over the entire length of the channel in PCR chip, or at least within certain regions of the PCR chip such as the denaturing and annealing/e tension zones.
  • the channel cross-section is often rectangular, this is not necessarily the case.
  • the walls of the channel need not be formed at right angles.
  • the cross-section may have a non-rectangular polygonal shape such as triangular, or the cross-section may have curved walls such as circularly or elliptically shaped walls.
  • the total length of a channel in a PCR chip is about 1500 to 3000 mm or about 2100 to 2400 millimeters. This total length includes the entire channel passage from sample droplet entrance (e.g., from a nozzle) mm a sample recovery or waste exit.
  • the total length may include a hot-start zone, a low temperature annealing/extension zone, a high temperature denaturing zone, and an optical detection zone.
  • the channel path may be serpentine, although other path designs are possible.
  • some chip designs have one or more of the following regional channel lengths:
  • Buffer section between droplet generation and hot start section about 10 to 20 mm (e.g., about 15 mm)
  • Hot start section about 50mm to 200mm
  • Length of channel in the two low temperature zone in each serpentine pass about 10 to 20 mm (e.g., about 15mm) or about 20 to 40 mm total in each pass
  • Length of high temperature zone in each serpentine pass about 5 to 20 mm (e.g., about 10mm)
  • Length of the temperature buffer zone (between the high and low temperature zones) in each serpentine pass about 3 to 8 mm (e.g., about 5mm) in each segment of a pass, or about 6 to 16 mm total in each pass
  • Optical detection zone about 50 to 150 mm (e.g., about 85mm)
  • the number of serpentine passes in a hot-start section may be include 1-5 passes. Also, in some serpentine embodiments, the number of serpentine passes in the thermocycling section (including at least one denaturing zone, at least one annealing and extension zone, and optionally one or more temperature buffer zones) is about 30 to 50 passes, e.g., about 40 passes.
  • a microfluidic chip includes two or more parallel channels in which one, two, or more samples can flow in parallel.
  • Figure 15 illustrates one such embodiment 1501 with a first channel 1503 and a second flow channel 1505, with the flow direction in such channels indicated by arrows.
  • the first and second channels may alternate between being the inner and outer channels with each turn of the serpentine.
  • multiple channel embodiments allow a greater volume of sample to flow through the chip, and be analyzed, at a given instance in time.
  • the optics components may be configured to capture optical signal at separate locations for respective parallel channels, and/or the components and/or associated processor may be configured to synchronize signal capture to discriminate between sample flowing in the parallel channels.
  • different samples flow in different ones of the parallel channels.
  • Sample droplets produced in certain embodiments have characteristic sizes and shapes.
  • the droplet size and shape may be dictated by the nozzle size, the reception channel size, the flow rate, the sample and oil hydrodynamic properties such as viscosity, density, and wetting, etc.
  • the diameter of each sample droplet is about 100 to 300 micrometers, e.g., about 150 to 300 micrometers or about 160 to 230 micrometers. In some cases, the droplet diameters depends on the size of the droplet generation nozzle.
  • the nozzle of a PCR chip may have a diameter that is slightly smaller than the diameter of the droplet. In certain embodiments, the nozzle diameter is about 100-150 micrometers.
  • the shape of the sample droplets may be cylindrical or ellipsoidal, as they are constricted by microfluidic channel that has a rectangular cross-section.
  • the depth of each sample droplet is about 50 to 100 micrometers, depending on the depth of the microfluidic channel.
  • the depth of a droplet is its vertical or y-direction dimension. See e.g., Figure 16.
  • the droplets are separated from one another in the flowing fluid by a distance of about 1 to 3 droplet diameters.
  • droplet separation distance is about 150 to 900 micrometers, or about 300 to 600 micrometers.
  • the minimum droplet separation distance equals to the diameter of each droplet, which, as an example, may be about 160 to 230 micrometers.
  • the flow rate of the fluid (including the sample droplets and carrier liquid) in a microfluid PCR chip is about 1 to 10 mm/s, or about 2 to 4 mm/s. In some implementations, the flow rate is adjustable by the user.
  • Sample droplets produced in certain embodiments have defined temperatures in different zones as they flow through a PCR chip.
  • a pre-heated sample droplet produced from the nozzle may have a temperature of about 50°C to 72°C; e.g., about 50°C.
  • a sample droplet flowing in a hot-start polymerase activation region may have a temperature of about 75°C to 98°C; e.g., about 95°C.
  • a sample droplet flowing over a high temperature heat block may have a temperature of about 75 °C to 98°C; e.g., about 95 °C.
  • a sample droplet flowing over a low temperature heat block may have a temperature of about 45°C to 75°C. In some cases, the temperature of the droplet in the low temperature region is adjustable, ranging from about 50°C to 72°C. In certain embodiments, sample droplets flowing in buffer regions have a temperature gradient that is bounded by the temperatures of the high and low temperature blocks. The gradient is not necessarily linear. In certain embodiments, the sample droplets in the buffer regions have a temperature that varies within a range from the temperatures of the low temperature and high temperature heat blocks, e.g., from about 50°C to 95°C.
  • compositions of the sample and carrier fluid phases may be chosen to facilitate PCR and analysis in particular PCR chip designs.
  • the sample phase may be aqueous and substantially immiscible in the carrier fluid phase.
  • the sample phase may be denser that the carrier fluid phase.
  • the sample phase is prepared by mixing a raw, diluted, or otherwise preprocessed biological specimen with a PCR reaction mix.
  • the PCR reaction mix is a standard PCR reaction mixture.
  • the PCR reaction mix is customized with, e.g., custom salt concentrations.
  • a final concentration of about Img/mL of BSA Bovine serum albumin
  • the PCR reaction mix comprises about 0.1 to 0.5 mg/mL BSA to serve as a protective layer at the water-oil boundary.
  • the PCR reaction mix is enhanced with glycerin to augment the viscosity of the reaction mixture.
  • the carrier phase fluid has a density of about 0.8 to 0.97 g/cm 3 . In some embodiments, the carrier phase fluid has a viscosity of about 1 to 1000 cP at 25°C.
  • a sample comprising DNA or RNA molecules is thoroughly mixed with a magnetic bead solution, causing any DNA present in the sample to attach to the magnetic beads.
  • the solution is then added to the sample port of the microfluidic chip.
  • a solenoid encircles the sample port on the microfluidic chip. When activated, the solenoid creates a magnetic field that draws the magnetic beads toward it, causing them to adhere to the sample port’s side walls. Vacuum pressure is then applied within the microfluidic channel, pulling the undesired suspension solution into the chip's waste chamber.
  • PCR systems and methods described herein may have many applications in research, clinical settings, point of care settings, and even home use.
  • Home use allows lay patients to conveniently test for or monitor various genetic and health conditions indicated by the presence and/or quantity of nucleic acid markers such as those associated with infectious pathogens, cancers, gene therapies, ancestry, and the like.
  • Some PCR systems may have features designed or configured for home use. Such features include a relatively small footprint, a user interface tailored for lay users, etc.
  • a PCR instrument such as depicted in Figures 4 A and 4B, which controls operation of a PCR chip, has design features that permit home or lay user usage.
  • the overall footprint or dimensions of the instrument are sufficiently small to make the instrument portable or suitable for countertop or desktop operation.
  • the instrument has a longest dimension of not greater than about 500 mm, or not greater than about 300 mm, or not greater than about 150 mm.
  • An example instrument has the following dimensions: (W)70mm x (D)130mm x (H)80mm.
  • a PCR chip may have a correspondingly small footprint.
  • a PCR chip may have a longest side dimension of about 200 mm or less or about 100 mm or less.
  • a PCR chip such as illustrated in Figures 7 A, 12, and 13 may have a width of about 20 to 50 mm and/or a length of about 50 to 200 mm.
  • a PCR chip has a width of 35 mm and a length of 100 mm.
  • a modular digital PCR instrument may have a relatively small size or footprint.
  • a PCR system as shown in Figure 17A may have a width of about 500 mm or less, a depth of about 300 mm or less, and/or a height of about 350 mm or less.
  • a PCR System such as shown in Figure 17A has the following dimensions: (W)445mm x (D)200mm x (H)240mm.
  • a rack configuration as shown in Figure 17B has the following dimensions: (W)600 mm x (D)1500 mm x (H)500 mm.
  • a digital PCR system as described herein employs all or almost all the sample made available to it. In other words, there is little or no “dead volume” in the sample. This should be contrasted with other existing commercial systems, where often 50% or more of the sample cannot be used. Such high quantities of dead volume present a significant problem when working with samples that are hard to obtain, valuable, or in short supply. Also, systems that require significant dead volumes often suffer from poor sensitivity. In contrast, digital PCR systems such as those described herein that have relatively little or no dead volume provide quantitative results with much better sensitivity. In some digital PCR systems and methods described herein, at most about 10% of the original sample is unused (dead volume). In some embodiments, at most about 5% is unused, or at most about 1% of the sample is unused.
  • digital PCR systems and methods described herein can be performed in a manner that recovers all or nearly all the sample that has undergone processing on a PCR chip. This allows for further analysis or research to be conducted on the sample.
  • an amplified sample is collected as PCR chip output that would otherwise be waste.
  • the recovered sample may comprise extended and labeled nucleic acid segments that are now available for additional sequencing or other biochemical analysis.
  • at least about 90% of the sample is recovered, or at least about 95% of the sample is recovered, or at least about 99% of the sample is recovered.
  • systems and methods herein allow multiplexed digital PCR.
  • the sample used in a PCR chip employs nucleic acids from two or more different sources. These nucleic acids are mixed with each other in the sample phase and may co-occur within individual sample droplets in a single PCR run. The results of amplification(s) of these nucleic acids from different sources may be isolated by different optical detection signals such as different fluorescent wavelengths.
  • a digital PCR instrument as described herein may support multiple optical channels. Each such channel may detect amplified nucleic acid from distinct source such as a distinct biological specimen.
  • a relatively simpler digital PCR instrument supports detection of only a single nucleic acid sequence, i.e., a single amplicon.
  • PCR amplification is typically a power-hungry process, particularly due to the power required to repeatedly and rapidly change the temperature of a heater in the thermal cycler.
  • digital PCR instruments described herein consume substantially less power.
  • In situ optical detection in the instruments described herein may further reduce the power consumed.
  • the instrument is configured to perform digital PCR powered with a battery or battery pack.
  • a battery powered digital PCR instrument as described herein is portable.
  • PCR reaction mix probe, primers, dNTPs, buffer
  • human epidermal growth factor receptor 2 gene HER2
  • a pipette was used to transfer a specific volume of dilution buffer into each of the sterile test tubes. Then, a specific volume of standard solution is transferred to the first test tube dilution buffer and be mixed thoroughly. The resulting solution has a dilution factor of D 1. Next, the serial dilution process was repeated by transferring 50% of the volume from DI into the second test tube, creating a new solution of dilution factor of D2. The dilution factor of D2 is therefore 2X relative to that of DI. The stepwise dilution process continues for several dilution steps until the desires number of dilutions are achieved.
  • the luL of diluted samples are mixed with 24uL of HER2 PCR reaction mix, creating a 25uL reaction sample volume for each concentration.
  • the reaction samples were then applied to digital PCR microfluidic chips.
  • the same set of luL diluted samples are mixed with 19uL of HER2 PCR reaction mix based on droplet digital PCR reaction mix provided by Bio-rad, the reaction samples were applied to Bio-rad’ s droplet digital PCR microfluidic chips for droplet generation, then subsequently transferred to a 96 well plates for separated digital PCR reactions.
  • the results were plotted on a graph to illustrate the relationship between the concentration of the samples and the DNA copy number as tested by the digital PCR system.
  • the linearity of the digital PCR system was assessed by plotting DNA copy number against expected DNA copy number. An ideal linear relationship would yield a straight trend line with a R 2 value close to 1 , indicating a strong linear relationship between DNA copy number and standard concentrations.
  • serial dilution of standard sample to evaluate the linearity of the novel digital PCR system was employed, and the results against a commercially available digital PCR (dPCR) detection system (Bio-rad QX200) were compared.
  • Serial dilution involves successive dilution of a concentrated standard solution to obtain multiple samples with gradually decreasing concentrations.
  • a chip processing instrument for performing PCR comprising:
  • a flow induction unit configured to induce a flowing fluid through a microfluidic channel in a PCR chip engaged with the chip processing instrument
  • a receptacle sized and shaped to engage the PCR chip in a fixed position with respect to: (i) the flow induction unit, (ii) the optical detector, (iii) at least one of the plurality of heat blocks; or (iv) any combination thereof;
  • a signal processor configured to receive signals from the optical detector, wherein the signals comprise information about amplification of nucleic acids in sample droplets of the flowing fluid.
  • the plurality of heat block comprise (i) a high temperature heat block configured to maintain a first region of the PCR chip at a first temperature or a first temperature range, and (ii) a low temperature heat block configured to maintain a second region of the PCR chip at a second temperature or a second temperature range, wherein the first temperature or first temperature range is greater than the second temperature or second temperature range.
  • At least one of the plurality of heat blocks is configured to maintain a temperature that facilitates hot start polymerase activation, nucleic acid denaturing, nucleic acid annealing, nucleic acid extension, or any combination thereof.
  • the plurality of heat blocks comprises a high temperature heat block configured to facilitate hot start polymerase activation and/or nucleic acid denaturing.
  • the high temperature heat block comprises a first region proximate a sample inlet configured to facilitate polymerase activation and a second region configured to facilitate nucleic acid denaturing.
  • the plurality of heat blocks comprises a low temperature heat block configured to facilitate sample inlet heating, nucleic acid annealing, nucleic acid extension, or any combination thereof.
  • the low temperature heat block comprises a section configured to heat a sample reservoir to a temperature that activates a UNG enzyme and/or a reverse transcription reaction.
  • the plurality of heat blocks comprises a polyimide heater or a rod heater.
  • optical detector comprises a plurality of optical submodules, each configured to detect a different wavelength.
  • each of the plurality of optical submodules comprises a light source and a light detector.
  • the signal processor is further configured to computationally process the signals in a manner that conducts a digital PCR analysis.
  • a PCR chip comprising:
  • a nozzle configured to generate sample droplets comprising the sample, wherein the sample droplets are separated from one another by an immiscible phase of a flowing fluid;
  • PCR chip of chip embodiment 1 further comprising a sample reservoir in fluid communication with the sample inlet.
  • PCR chip of any of the preceding chip embodiments further comprising an immiscible phase inlet for receiving a liquid comprising the immiscible phase.
  • PCR chip of chip embodiment 4 further comprising an immiscible phase reservoir in fluid communication with the immiscible phase inlet.
  • sample droplets comprise reagents for performing digital PCR.
  • nozzle comprises a first conduit for delivering the sample toward the fluid flow channel, a second conduit for delivering the immiscible phase toward the fluid flow channel, and a junction of the first conduit and the second conduit where, during operation, the sample droplets in the flowing fluid are produced.
  • nozzle comprises a plurality of second conduits, each for delivering the immiscible phase to the junction.
  • PCR chip of any of the preceding chip embodiments wherein the nozzle is configured to, during operation, produce the flowing fluid under the influence of a vacuum source.
  • serpentine path comprises a plurality of parallel serpentine segments of the fluid flow channel, and wherein each serpentine segment has an upstream portion over a first low temperature heat block, a downstream portion over a second low temperature heat block, and an intermediate portion over a high temperature heat block, wherein the intermediate portion is between the upstream and downstream portions.
  • the optical detection zone is located adjacent to an optical detector configured to obtain optical signals from the sample droplets in the flowing fluid.
  • PCR chip of any of the preceding chip embodiments further comprising a polymerase activation zone configured to perform hot-start polymerase activation in the sample droplets when the sample droplets are flowing in at least one polymerase activation segment of the fluid flow channel within the polymerase activation zone.
  • the polymerase activation zone is located over a high temperature heat block configured to operate at a temperature that facilitates hot start polymerase activation in the sample in the at least one polymerase activation segment of the fluid flow channel.
  • PCR chip of any of the preceding chip embodiments further comprising a first annealing and extension zone for annealing and extending nucleic acids in the sample droplets when flowing in a plurality of first segment portions of the fluid flow channel, wherein the plurality of first segment portions is located in the first annealing and extension zone.
  • the PCR chip of chip embodiment 23 further comprising a denaturing zone for denaturing double- stranded nucleic acids in the sample droplets when flowing in a plurality of second segment portions of the fluid flow channel, wherein the plurality of second segment portions is located in the denaturing zone.
  • PCR chip of chip embodiment 26 wherein, when the PCR chip is engaged with the chip processing instrument, a polymerase activation zone and the denaturing zone are both located over a high temperature heat block of the chip processing instrument.
  • the PCR chip of chip embodiment 26 further comprising a temperature buffer zone disposed between first annealing and extension zone and the denaturing zone.
  • the PCR chip of chip embodiment 26 further comprising a second annealing and extension zone for annealing and extending nucleic acids in the sample droplets when flowing in a plurality of third segment portions of the fluid flow channel, wherein the plurality of third segment portions is located in the second annealing and extension zone.
  • PCR chip of chip embodiment 31 wherein, when the PCR chip engages with the chip processing instrument, the second annealing and extension zone is located over a low temperature heat block configured to operate at a temperature that facilitates annealing and/or extending double-stranded nucleic acids in the sample in the third segment portions.
  • the PCR chip of chip embodiment 31 further comprising a temperature buffer zone disposed between the denaturing zone and the second annealing and extension zone.
  • PCR chip of any of the preceding chip embodiments further comprising a waste chamber coupled to an outlet of the fluid flow channel.
  • PCR chip of any of the preceding chip embodiments wherein the PCR chip has a longest dimension of not greater than about 200 mm or less.
  • a method comprising:
  • sample droplets comprise reagents for performing digital PCR.
  • sample droplets have a diameter of about 160 to 230 micrometers. 4. The method of any of the preceding method embodiments, further comprising producing the fluid by contacting the sample with the carrier fluid at a nozzle upstream of the channel.
  • the nozzle comprises a first conduit for delivering the sample toward the channel, a second conduit for delivering the carrier fluid toward the channel, and a junction of the first conduit and the second conduit where the sample droplets in the fluid are produced.
  • introducing the fluid in a channel comprises applying a vacuum to draw the fluid into the channel.
  • flowing the fluid alternately between the at least one denaturing zone and the at least one annealing and extension zone comprises flowing the fluid through the serpentine path, which repeatedly passes between the at least one denaturing zone and the at least one annealing and extension zone.
  • serpentine path comprises a plurality of parallel serpentine segments of the fluid flow channel, and wherein each serpentine segment has an upstream portion in a first annealing and extension zone, a downstream portion in a second annealing and extension zone, and an intermediate portion in a first denaturing zone, wherein the intermediate portion is between the upstream and downstream portions.
  • detecting an optical signal from the sample droplets comprises detecting a fluorescence associated with the nucleic acid.
  • a digital PCR system comprising:
  • a microfluidic chip comprising: a sample inlet, configured to receive a sample from a sample reservoir; an oil inlet, configured to receive an oil phase from an oil reservoir; a nozzle, configured to be in fluid communication with the sample inlet and the oil inlet to mix the sample and the oil phase to generate a water-in-oil microdroplet; a hot-start polymerase activation chamber, configured to pre-heat the microdroplet from the nozzle at a pre-defined first high temperature to form a pre-heated microdroplet; a microdroplet reaction chamber, configured to perform a pre-defined thermal cycling with the pre-heated microdroplet to form a reacted microdroplet; an optical detection chamber, configured to allow detection of a fluorescence signal from the reacted microdroplet; and a waste chamber, configured to receive the reacted microdroplet after detection; wherein the nozzle, the hot-start polymerase activation chamber, microdroplet reaction chamber, optical detection chamber, and waste chamber are sequentially connected in
  • a chip processing device comprising: a light source, configured to emit a light directed to the optical detection chamber; an optical detection module, configured to detect a fluorescent signal from the optical detection chamber; a temperature control module, configured to thermally regulate temperature of the sample inlet, the hot-start polymerase activation chamber and the microdroplet reaction chamber; a control module, configured to control operation of the device; a pressure control module, configured to control the pressure applied to the microfluidic chip; and optionally a microfluidic chip receiving module, configured to receive the microfluidic chip at a pre-defined receiving position.
  • the device further comprises a signal processing system and a user interface
  • the control module further comprises: a central processing unit configure to operatively connect with the optical detection module, the signal processing system and the user interface; and a microcontroller unit, configured to operatively connect with the light source, the temperature control module, and the pressure control module.
  • the droplet reaction chamber comprises a serpentine microchannel, forming a left droplet reaction portion, a middle droplet reaction portion and a right droplet reaction portion.
  • the temperature control module further comprises a high temperature heat block and a low temperature heat block
  • the high temperature heat block comprises a first high temperature region configured to be in contact with the hot-start polymerase activation chamber and a second high temperature region configured to be in contact with the middle droplet reaction portion, so as to regulate the hot-start polymerase activation zone at a first high temperature and the middle droplet reaction portion at a second high temperature, respectively
  • the low temperature heat block comprises a first low temperature region configured to be in contact with the sample inlet, a second low temperature region configured to be in contact with the left droplet reaction portion, and a third low temperature region configured to be in contact with the right droplet reaction portion, so as to regulate the sample inlet at the first low temperature, the left droplet reaction portion at a second low temperature, and the right droplet reaction portion at a third low temperature.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Un système de PCR numérique consiste (a) en une puce microfluidique possédant une entrée d'échantillon, une entrée d'huile, une buse en communication fluidique avec l'entrée d'échantillon et l'entrée d'huile pour mélanger l'échantillon et la phase huileuse pour générer une microgouttelette d'eau dans l'huile ; une région de réaction de microgouttelettes pour effectuer un cyclage thermique avec la microgouttelette pour former une microgouttelette ayant réagi ; une région de détection optique, configurée pour permettre la détection d'un signal de fluorescence à partir de la microgouttelette ayant réagi ; et (b) en un instrument de traitement de puce possédant un module de détection optique pour détecter un signal fluorescent, et un module de régulation de température pour réguler thermiquement la température de la région de réaction de microgouttelettes.
PCT/US2024/047803 2023-09-22 2024-09-20 Systèmes de pcr numériques intégrés Pending WO2025064904A1 (fr)

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US63/584,502 2023-09-22
US202463617416P 2024-01-03 2024-01-03
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090227007A1 (en) * 2005-02-18 2009-09-10 Canon U.S. Life Sciences, Inc. Devices and methods for monitoring genomic dna of organisms
US20180321170A1 (en) * 2000-11-16 2018-11-08 Canon U.S. Life Sciences, Inc. Method and apparatus for generating thermal melting curves in a microfluidic device
US20200360876A1 (en) * 2006-05-11 2020-11-19 Bio-Rad Laboratories, Inc. Microfluidic devices

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180321170A1 (en) * 2000-11-16 2018-11-08 Canon U.S. Life Sciences, Inc. Method and apparatus for generating thermal melting curves in a microfluidic device
US20090227007A1 (en) * 2005-02-18 2009-09-10 Canon U.S. Life Sciences, Inc. Devices and methods for monitoring genomic dna of organisms
US20200360876A1 (en) * 2006-05-11 2020-11-19 Bio-Rad Laboratories, Inc. Microfluidic devices

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