WO2019144966A1

WO2019144966A1 - Systems and methods for analyzing nucleic acids

Info

Publication number: WO2019144966A1
Application number: PCT/CN2019/073621
Authority: WO
Inventors: Jesus Ching; Phillip You Fai Lee; Alice Wang Yum LEE; Chen Li; Haojie Li; Xiang Li
Original assignee: Coyote Bioscience Co., Ltd.
Priority date: 2018-01-29
Filing date: 2019-01-29
Publication date: 2019-08-01

Abstract

The present invention provides methods and systems for amplifying and analyzing nucleic acid samples. The method may comprise activating a system comprising a fluid flow network, generating partitions, subjecting the partitions to conditions sufficient to perform a nucleic acid amplification reaction, and detecting signals indicative of a presence or absence of amplification products. Systems to perform the method may comprise a fluid flow network, a plurality of thermal zones, and a detector.

Description

SYSTEMS AND METHODS FOR ANALYZING NUCLEIC ACIDS

CROSS-REFERENCE

This application claims priority from International Application No. PCT/CN2018/074473, filed on January 29, 2018, the content of each of which is hereby incorporated by reference in their entirety.

BACKGROUND

Nucleic acid amplification methods may permit selected amplification and identification of nucleic acids of interest from a complex mixture, such as a biological sample. To detect a nucleic acid in a biological sample, the biological sample is typically processed to isolate nucleic acids from other components of the biological sample and other agents that may interfere with the nucleic acid and/or amplification. Following isolation of the nucleic acid of interest from the biological sample, the nucleic acid of interest can be amplified, via, for example, amplification methods such as thermal cycling based approaches (e.g., polymerase chain reaction (PCR) ) . Following amplification of the nucleic acid of interest, the products of amplification can be detected and the results of detection interpreted by an end-user. However, it has been tedious, time consuming and inefficient when multiple or numerous amplification reactions need to be performed.

Droplets have been proposed as containers to perform chemical and biochemical reactions (e.g., nucleic acid amplification) in confined volumes, and various methods have been developed to generate such droplets. However, these techniques often have problems associated with uneven droplet size and composition, relatively low throughput, and/or unable to generate monodisperse droplets.

SUMMARY

Recognized herein is the need for rapid, accurate and high throughput methods and devices for analyzing nucleic acids from complex sample types. Such methods and devices may be useful, for example, in realizing fast sample-to-answer detection and management of diseases detectable via their nucleic acid.

The present disclosure provides methods and systems for efficient amplification of nucleic acids, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules, especially for amplifying and analyzing a large amount of different nucleic acid molecules with high throughput and/or in parallel. Amplified nucleic acid product can be detected rapidly and with high sensitivity.

In an aspect, the present disclosure provides a method for detecting a presence or absence of a target nucleic acid molecule from a raw biological sample. The method comprises (a) activating a system comprising a fluid flow network, wherein the fluid flow network comprises: (i) a first chamber and a second chamber that are in fluid communication at a junction, wherein the first chamber comprises a first liquid phase comprising the raw biological sample, and wherein the second chamber comprises a second liquid phase that is immiscible with the first liquid phase; and (ii) a channel in fluid communication with the junction, wherein the channel is configured to flow a plurality of partitions comprising the first liquid phase segmented by the second liquid phase; (b) subjecting the first liquid phase and the second liquid phase to flow from the first chamber and the second chamber, respectively, to the junction, to generate the plurality of partitions, which plurality of partitions flows along the channel, wherein a given partition of the plurality of partitions comprises a reaction mixture comprising reagents necessary to perform a nucleic acid amplification reaction on the target nucleic acid molecule to yield amplification products of or derived from the target nucleic acid molecule; (c) subjecting the reaction mixture in the given partition to conditions sufficient to perform a plurality of series of primer extension reactions on the target nucleic acid molecule in presence of the reagents to yield the amplification products of the target nucleic acid molecule, wherein an individual series of primer extension reactions differs from at least one other individual series of the plurality of series of primer extension reactions with respect to a denaturing condition and/or an elongation condition; and (d) using a detector to detect a signal indicative of a presence or absence of the amplification product from the given partition, thereby detecting the presence or absence of the target nucleic acid molecule in the raw biological sample.

In some embodiments, the raw biological sample is provided directly from a source of the biological sample to the first chamber without further processing. In some embodiments, the given partition has an aspect ratio greater than 1. In some embodiments, the aspect ratio is greater than 1.5. In some embodiments, the aspect ratio is greater than 2. In some embodiments, (c) is performed in the channel.

In some embodiments, at least one segment of the channel is directed through a plurality of heating and cooling zones. In some embodiments, at least one segment of the channel is in thermal communication with a heating unit. In some embodiments, at least one segment of the channel is in thermal communication with a cooling unit.

In some embodiments, the reagents are provided in the first chamber as part of the first liquid phase. In some embodiments, the method further comprises a third chamber in fluid communication with the junction, wherein the third chamber comprises a third liquid phase comprising the reagents, wherein the third liquid phase is immiscible with the second liquid phase. In some embodiments, the first liquid phase is aqueous. In some embodiments, the second liquid phase comprises an oil. In some embodiments, the oil is a fluorinated oil. In some embodiments, the second liquid phase comprises a non-wetting agent. In some embodiments, the channel comprises a layer of a non-wetting agent. In some embodiments, the activating comprises directing a precursor of the non-wetting agent through the channel prior to generating the partitions.

In some embodiments, the reagents include a polymerizing enzyme (s) and a primer having sequence complementarity to the target nucleic acid molecule. In some embodiments, the target nucleic acid molecule is selected from the group consisting of human immunodeficiency virus I, human immunodeficiency virus II, orthomyxovirus, Ebola virus, Dengue virus, influenza virus, hepatitis A, B, C, D, and E virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile Fever virus, polio virus, measles virus, herpes simplex virus, smallpox virus, adenovirus, Varicella virus, Zika virus, pathogenic bacterium, pathogenic protozoan, and pathogenic parasites. In some embodiments, the polymerizing enzyme (s) include a deoxyribonucleic acid (DNA) polymerase. In some embodiments, the polymerizing enzyme (s) include a reverse transcriptase. In some embodiments, the polymerizing enzymes (s) include a deoxyribonucleic acid (DNA) polymerase and a reverse transcriptase that is separate from the DNA polymerase.

In some embodiments, a segment of the channel downstream of the junction is in sensing communication with the detector. In some embodiments, the detector is an optical detector, and wherein the segment of the channel is in optical communication with the detector. In some embodiments, the channel is in fluid communication with a collection chamber downstream of the junction, and wherein the plurality of partitions is collected in the collection chamber.

In some embodiments, the method further comprises bringing the collection chamber in fluid communication with an additional channel comprising a segment that is in sensing communication with the detector. In some embodiments, the detector is part of the system.

In some embodiments, the nucleic amplification reaction is reverse transcription polymerase chain reaction. In some embodiments, the nucleic acid amplification reaction is polymerase chain reaction (PCR) . In some embodiments, the PCR is isothermal PCR.

In some embodiments, the channel is substantially circular. In some embodiments, the channel has at least one segment in a serpentine configuration. In some embodiments, the system comprises a chip comprising the fluid flow network. In some embodiments, the system further comprises a fluid flow unit in fluid communication with at least one of the first chamber, the second chamber, and the channel. In some embodiments, the fluid flow unit is in fluid communication with the first chamber and the second chamber. In some embodiments, the fluid flow unit is in fluid communication with the channel. In some embodiments, the fluid flow unit is in fluid communication with the first chamber, the second chamber, and the channel. In some embodiments, the fluid flow unit provides positive pressure to the first chamber and the second chamber. In some embodiments, the fluid flow unit provides negative pressure to the channel.

In some embodiments, the method further comprises subjecting the reaction mixture in the given partition to a plurality of series of primer extension reactions to generate the amplification products, wherein each series of the plurality of series of primer extension reactions comprises two or more cycles of (i) incubating the reaction mixture under the denaturing condition characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under the elongation condition characterized by an elongation temperature and an elongation duration. In some embodiments, the target nucleic acid molecule is a ribonucleic acid (RNA) molecule and wherein the amplification products are amplified deoxyribonucleic acid (DNA) molecules generated from the RNA molecule. In some embodiments, the method further comprises subjecting the reaction mixture in the given partition to multiple cycles of a primer extension reaction to reverse transcribe the RNA molecule and generate the amplified DNA molecule in parallel, each cycle comprising (i) incubating the reaction mixture at a denaturing temperature for a denaturing duration that is less than or equal to 60 seconds, followed by (ii) incubating the reaction mixture at an elongation temperature for an elongation duration that is less than or equal to 60 seconds.

An additional aspect of the present disclosure provides a method for detecting a presence or absence of a target nucleic acid molecule from a biological sample. The method comprises (a) activating a system comprising a detector and a fluid flow network, wherein the fluid flow network comprises: (i) a first chamber and a second chamber that are in fluid communication at a junction, wherein the first chamber comprises a first liquid phase comprising the biological sample, and wherein the second chamber comprises a second liquid phase that is immiscible with the first phase; and (ii) a channel in fluid communication with the junction, wherein the channel is configured to flow a plurality of partitions comprising the first liquid phase segmented by the second liquid phase, wherein the channel comprises at least one segment in a plurality of thermal zones for subjecting the plurality of partitions to heating and/or cooling, and a detection segment downstream of the at least one segment, wherein the detection segment is in sensing communication with the detector; (b) subjecting the first liquid phase and the second liquid phase to flow from the first chamber and the second chamber, respectively, to the junction, to generate the plurality of partitions, wherein a given partition of the plurality of partitions comprises a reaction mixture comprising reagents necessary to perform a nucleic acid amplification reaction on the target nucleic acid molecule to yield amplification products of or derived from the target nucleic acid molecule, wherein upon generation, the plurality of partitions flows along the channel; (c) using the plurality of thermal zones to subject the reaction mixture in the given partition flowing through the at least one segment, to conditions sufficient to perform the nucleic acid amplification reaction on the target nucleic acid molecule in presence of the reagents, to yield the amplification products of the target nucleic acid molecule; and (d) using the detector to detect a signal indicative of a presence or absence of the amplification products from the given partition when the given partition is flowing through the detection segment, thereby detecting the presence or absence of the target nucleic acid molecule in the biological sample.

In some embodiments, the given partition has an aspect ratio greater than 1. In some embodiments, the aspect ratio is greater than 1.5. In some embodiments, the aspect ratio is greater than 2.

In some embodiments, the at least one segment is in thermal communication with a heating and/or cooling unit. In some embodiments, the at least one segment comprises a plurality of segments comprising a first segment and a second segment, wherein the first segment is for subjecting the given partition to heating and the second segment is for subjecting the given partition to cooling. In some embodiments, the at least one segment comprises a plurality of segments comprising a first segment and a second segment, wherein the first segment is for incubating the given partition at an elongation temperature or temperature range, and wherein the second segment is for incubating the given partition to a denaturation temperature or temperature range.

In some embodiments, the reagents are provided in the first chamber as part of the first liquid phase. In some embodiments, comprising a third chamber in fluid communication with the junction, wherein the third chamber comprises a third liquid phase comprising the reagents, wherein the third liquid phase is immiscible with the second liquid phase. In some embodiments, the first liquid phase is aqueous. In some embodiments, the second liquid phase comprises an oil. In some embodiments, the oil is a fluorinated oil. In some embodiments, the second liquid phase comprises a non-wetting agent. In some embodiments, the channel comprises a layer of a non-wetting agent. In some embodiments, the activating comprises directing a precursor of the non-wetting agent through the channel prior to generating the partitions.

In some embodiments, the reagents include a polymerizing enzyme (s) and a primer having sequence complementarity to the target nucleic acid molecule. In some embodiments, the target nucleic acid molecule is selected from the group consisting of human immunodeficiency virus I, human immunodeficiency virus II, orthomyxovirus, Ebola virus, Dengue virus, influenza virus, hepatitis virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile Fever virus, polio virus, measles virus, herpes simplex virus, smallpox virus, adenovirus, Varicella virus, Zika virus, pathogenic bacterium, pathogenic protozoan, and pathogenic parasites. In some embodiments, the polymerizing enzyme (s) include a deoxyribonucleic acid (DNA) polymerase. In some embodiments, the polymerizing enzyme (s) include a reverse transcriptase. In some embodiments, the polymerizing enzymes (s) include a deoxyribonucleic acid (DNA) polymerase and a reverse transcriptase that is separate from the DNA polymerase.

In some embodiments, the detector is an optical detector, and wherein the detection segment is in optical communication with the detector. In some embodiments, the channel is in fluid communication with a collection chamber downstream of the detection segment, and wherein the plurality of partitions is collected in the collection chamber.

In some embodiments, the channel is substantially circular. In some embodiments, the channel has at least one segment in a serpentine configuration. In some embodiments, the at least one segment is part of the serpentine configuration. In some embodiments, the system comprises a chip comprising the fluid flow network.

In some embodiments, the system further comprises a fluid flow unit in fluid communication with at least one of the first chamber, the second chamber and the channel. In some embodiments, the fluid flow unit is in fluid communication with the first chamber and the second chamber. In some embodiments, the fluid flow unit is in fluid communication with the channel. In some embodiments, the fluid flow unit is in fluid communication with the first chamber, the second chamber and the channel. In some embodiments, the fluid flow unit provides positive pressure to the first chamber and the second chamber. In some embodiments, the fluid flow unit provides negative pressure to the channel.

In some embodiments, the method further comprises using the plurality of thermal zones to subject the reaction mixture in the given partition to a plurality of series of primer extension reactions to generate the amplification products, each series comprising two or more cycles of (i) incubating the reaction mixture under a denaturing condition characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating the reaction mixture under an elongation condition characterized by an elongation temperature and an elongation duration, wherein an individual series differs from at least one other individual series of the plurality with respect to the denaturing condition and/or the elongation condition. In some embodiments, the target nucleic acid molecule is a ribonucleic acid (RNA) molecule and wherein the amplification products is an amplified deoxyribonucleic acid (DNA) molecule generated from the RNA molecule. In some embodiments, the method further comprises using the plurality of thermal zones to subject the reaction mixture in the given partition to multiple cycles of a primer extension reaction to reverse transcribe the RNA molecule and generate the amplified DNA molecule in parallel, each cycle comprising (i) incubating the reaction mixture at a denaturing temperature for a denaturing duration that is less than or equal to 60 seconds, followed by (ii) incubating the reaction mixture at an elongation temperature for an elongation duration that is less than or equal to 60 seconds.

An additional aspect of the present disclosure provides a system for detecting a presence or absence of a target nucleic acid molecule from a raw biological sample. The system comprises a fluidic network comprising (i) a first chamber and a second chamber that are in fluid communication at a junction, wherein the first chamber is configured to contain a first liquid phase comprising the raw biological sample, and wherein the second chamber is configured to contain a second liquid phase that is immiscible with the first phase; and (ii) a channel in fluid communication with the junction, wherein the channel is configured to flow a plurality of partitions comprising the first liquid phase segmented by the second liquid phase; a fluid flow unit in fluid communication with at least one of the first chamber, the second chamber and the channel; a detector configured to detect a signal indicative of a presence or absence of amplification products generated from the target nucleic acid molecule in a given partition of the plurality of partitions; and one or more computer processors operatively coupled to the fluid flow unit and the detector, wherein the one or more computer processors are individually or collectively programmed to: (a) direct the fluid flow unit to (i) subject the first liquid phase and the second liquid phase to flow from the first chamber and the second chamber, respectively, to the junction, to generate the plurality of partitions, which plurality of partitions flows along the channel, wherein the given partition of the plurality of partitions comprises a reaction mixture comprising reagents necessary to perform a nucleic acid amplification reaction on the target nucleic acid molecule to yield amplification products of or derived from the target nucleic acid molecule; and (ii) subject the plurality of partitions, including the given partition, to flow along the channel, wherein subsequent to generating the plurality of partitions, the reaction mixture in the given partition is subjected to conditions sufficient to perform a plurality of series of primer extension reactions on the target nucleic acid molecule in presence of the reagents, to yield the amplification products, wherein an individual series of primer extension reactions differs from at least one other individual series of the plurality of series of primer extension reactions with respect to a denaturing condition and/or an elongation condition; and (b) direct the detector to detect a signal indicative of a presence or absence of the amplification products from the given partition, thereby detecting the presence or absence of the target nucleic acid molecule in the raw biological sample.

In some embodiments, the system further comprises a third chamber in fluid communication with the junction, wherein the third chamber comprises a third liquid phase comprising the reagents, wherein the third liquid phase is immiscible with the second liquid phase. In some embodiments, the first liquid phase is aqueous. In some embodiments, the second liquid phase comprises an oil. In some embodiments, the oil is a fluorinated oil. In some embodiments, the second liquid phase comprises a non-wetting agent. In some embodiments, the channel comprises a layer of a non-wetting agent.

In some embodiments, the channel is substantially circular. In some embodiments, the channel has at least one segment in a serpentine configuration. In some embodiments, the system comprises a chip comprising the fluid flow network. In some embodiments, the fluid flow unit is in fluid communication with the first chamber and the second chamber. In some embodiments, the fluid flow unit is in fluid communication with the channel. In some embodiments, the fluid flow unit is in fluid communication with the first chamber, the second chamber and the channel. In some embodiments, the fluid flow unit provides positive pressure to the first chamber and the second chamber. In some embodiments, the fluid flow unit provides negative pressure to the channel.

An additional aspect of the present disclosure provides a system for detecting a presence or absence of a target nucleic acid molecule from a biological sample. The system comprises a fluid flow network comprising (i) a first chamber and a second chamber that are in fluid communication at a junction, wherein the first chamber comprises a first liquid phase comprising the biological sample, and wherein the second chamber comprises a second liquid phase that is immiscible with the first phase; and (ii) a channel in fluid communication with the junction, wherein the channel is configured to flow a plurality of partitions comprising the first liquid phase segmented by the second liquid phase at the junction, wherein the channel comprises at least a first segment in a plurality of thermal zones for subjecting the plurality of partitions to heating and/or cooling, and a detection segment downstream of the at least the first segment, wherein the detection segment is in sensing communication with the detector; a fluid flow unit in fluid communication with at least one of the first chamber, the second chamber, and the channel; a detector configured to detect a signal indicative of a presence or absence of an amplification products generated from the target nucleic acid molecule in a given partition of the plurality of partitions; a heating and/or cooling unit in thermal communication with the plurality of thermal zones; and one or more computer processors operatively coupled to the fluid flow unit, the heating and/or cooling unit, and the detector, wherein the one or more computer processors are individually or collectively programmed to: (a) direct the fluid flow unit to subject the first liquid phase and the second liquid phase to flow from the first chamber and the second chamber, respectively, to the junction, to generate the plurality of partitions, wherein the given partition of the plurality of partitions comprises a reaction mixture comprising reagents necessary to perform a nucleic acid amplification reaction on the target nucleic acid molecule to yield amplification products of or derived from the target nucleic acid molecule, wherein upon generation, the plurality of partitions flows along the channel; (b) direct the heating and/or cooling unit to subject the reaction mixture in the given partition flowing through the at least the first segment, to conditions sufficient to perform the nucleic acid amplification reaction on the target nucleic acid molecule in presence of the reagents, to yield the amplification products of the target nucleic acid molecule; and (c) direct the detector to detect a signal indicative of a presence or absence of the amplification products from the given partition when the given partition is flowing through the detection segment, thereby detecting the presence or absence of the target nucleic acid molecule in the biological sample.

In some embodiments, the detector is an optical detector, and wherein the detection segment is in optical communication with the detector. In some embodiments, the channel is in fluid communication with a collection chamber downstream of the detection segment, which collection chamber is for collecting the plurality of partitions. In some embodiments, the channel is substantially circular. In some embodiments, the channel has at least one segment in a serpentine configuration. In some embodiments, the at least one segment is part of the serpentine configuration.

In some embodiments, the system comprises a chip comprising the fluid flow network. In some embodiments, the fluid flow unit is in fluid communication with the first chamber and the second chamber. In some embodiments, the fluid flow unit is in fluid communication with the channel. In some embodiments, the fluid flow unit is in fluid communication with the first chamber, the second chamber and the channel. In some embodiments, the fluid flow unit provides positive pressure to the first chamber and the second chamber. In some embodiments, the fluid flow unit provides negative pressure to the channel.

Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIG. 1A illustrates an example circular microfluidic chip with a spiraled microfluidic channel;

FIG. 1B illustrates an example circular microfluidic chip with a spiraled microfluidic channel and multiple heating and cooling zones;

FIG. 2 illustrates an example circular microfluidic chip with a serpentine microfluidic channel and a single heating zone and a single cooling zone;

FIG. 3 illustrates an example rectangular microfluidic chip with a serpentine microfluidic channel and multiple heating zones and a single cooling zone;

FIG. 4 illustrates a rectangular microfluidic chip with a patterned microfluidic channel and multiple heating and cooling zones;

FIG. 5 illustrates an example system comprising a microfluidic chip comprising a fluid flow network, heating and/or cooling units, and a detector; and

FIG. 6 shows a computer control system that is programmed or otherwise configured to implement methods provided herein.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used in the specification and claims, the singular form “a” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “amolecule” includes a plurality of molecules, including mixtures thereof.

As used herein, the terms “amplifying” and “amplification” are used interchangeably and generally refer to generating one or more copies or “amplified product” of a nucleic acid. The term “DNA amplification” generally refers to generating one or more copies of a DNA molecule or “amplified DNA product” . The term “reverse transcription amplification” generally refers to the generation of deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template via the action of a reverse transcriptase.

As used herein, the terms “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. Denaturation may include the inactivation of the cell wall (s) of a pathogen or the shell of a virus, and the inactivation of the protein (s) of inhibitors. 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.

As used herein, the term “elongation” generally refers to the incorporation of nucleotides to a nucleic acid in a template directed fashion. Elongation may occur via the aid of an enzyme, such as, for example, a polymerase or reverse transcriptase. Conditions at which elongation may occur include an “elongation temperature” that generally refers to a temperature at which elongation is permitted to occur and an “elongation duration” that generally refers to an amount of time allotted for elongation to occur.

As used herein, the term “nucleic acid” generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides 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. 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. If present, 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.

As used herein, the term “primer extension reaction” generally refers to the denaturing of a double-stranded nucleic acid, binding of a primer to one or both strands of the denatured nucleic acid, followed by elongation of the primer (s) .

As used herein, the term “reaction mixture” generally refers to a composition comprising reagents 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) . In some cases, reaction mixtures can also comprise one or more reporter agents.

As used herein, a “reporter agent” generally refers to a composition that yields a detectable signal, the presence or absence of which can be used to detect the presence of amplified product.

As used herein, the term “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 to be determined. A target nucleic acid may be any type of nucleic acid, including DNA, RNA, and analog thereof. As used herein, a “target ribonucleic acid (RNA) ” generally refers to a target nucleic acid that is RNA. As used herein, a “target deoxyribonucleic acid (DNA) ” generally refers to a target nucleic acid that is DNA.

As used herein, the term “subject” generally refers to an entity or a medium that has testable or detectable genetic information. A subject can be a person or individual. A subject can be a vertebrate, such as, for example, a mammal. Non-limiting examples of mammals include murines, simians, humans, farm animals, sport animals, and pets. Other examples of subjects include, for example, food, plant, soil, and water.

As used herein, 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 fill the container in which it is put. Thus, 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 fluid (liquids, gases, and the like) .

As used herein, the term “aqueous fluid” generally refers to a fluid that is made with, of, or from water, or a fluid that contains water. For example, an aqueous fluid may be an aqueous solution with water as the solvent. An aqueous fluid of the present disclosure may comprise reagents for conducting a chemical reaction, e.g., polymerase chain reaction (PCR) . Non-limiting examples of aqueous fluid include, but are not limited to, water and other aqueous solutions comprising water, such as cell or biological media, ethanol, salt solutions, etc.

As used herein, the term “non-aqueous fluid” generally refers to a fluid that is made from, with, or using a liquid other than water. Non-limiting examples of non-aqueous fluid include, but are not limited to, oils such as hydrocarbons, silicon oils, fluorocarbon oils, organic solvents etc.

As used herein, the term “partition” generally refers to a division into or distribution in portions or shares. Examples of partitions include segmented fluids, droplets, and wells.

As used herein, the term “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 or equal to about 10 mm, 1 mm, 0.5 mm, or 0.1 mm.

As used herein, a “cross-sectional dimension” of a channel may be measured perpendicularly with respect to the general direction of fluid flow within the channel.

As used herein, the term “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. For example, the channel may have a serpentine or a spiral configuration. In some embodiments, the channel includes one or more branches, with some or all of which connected with one or more other channel (s) . When a channel is curved or bended with a corner or a turning point, 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.

A channel may also have an aspect ratio (length to average cross-sectional dimension) of at least about 2 to 1, 3 to 1, 4 to 1, 5 to 1, 6 to 1, 8 to 1, 10 to 1, 15 to 1, 20 to 1, 30 to 1, 40 to 1, 50 to 1, 60 to 1, 70 to 1, 80 to 1, 90 to 1, 100 to 1 or more. 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. Non-limiting examples of 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) .

As used herein, the term “junction” generally refers to a point or area, where one channel crosses or meets another channel.

The term “sample, ” as used herein, generally refers to any sample containing or suspected of containing a nucleic acid molecule. For example, a subject sample can be a biological sample containing one or more nucleic acid molecules. The biological sample can be obtained (e.g., extracted or isolated) from a bodily sample of a subject that can be selected from blood (e.g., whole blood) , plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears. The bodily sample can be a fluid or tissue sample (e.g., skin sample) of the subject. In some examples, the sample is obtained from a cell-free bodily fluid of the subject, such as whole blood. In such instance, the sample can include cell-free DNA and/or cell-free RNA. In some other examples, the sample is an environmental sample (e.g., soil, waste, ambient air and etc. ) , industrial sample (e.g., samples from any industrial processes) , and food samples (e.g., dairy products, vegetable products, and meat products) .

The term “raw biological sample, ” as used herein, generally refers to any sample containing or suspected of containing a nucleic acid molecule that has not been further processed after the sample has been retrieved. Further processing may include chemical, physical, or biological treatment. For example, a raw biological sample may include a sample derived from the body of a subject and may be selected from whole blood, urine, saliva, mucosal excretions, sputum, stool and tears. In some examples, the sample is obtained from a cell-free bodily fluid of the subject, such as whole blood. In such instance, the sample can include cell-free DNA and/or cell-free RNA. In some other examples, the sample is an environmental sample (e.g., soil, waste, ambient air and etc. ) , industrial sample (e.g., samples from any industrial processes) , and food samples (e.g., dairy products, vegetable products, and meat products) that is further processed.

Methods for analyzing nucleic acid samples

In an aspect, the present disclosure provides methods for detecting the presence or absence of a target nucleic acid molecule from a raw biological sample. The method may comprise activating a system comprising a fluid flow network. The fluid flow network may comprise a first chamber and a second chamber that are in fluid communication at a junction. The first chamber may comprise a first liquid phase comprising a raw biological sample and the second chamber may comprise a second liquid phase that is immiscible with the first liquid phase. The fluid flow network may further comprise a channel in fluid communication with the junction between the first and second chambers. A plurality of partitions may be generated upon bringing the first liquid phase in contact with the second liquid phase at the junction. The first liquid phase may be segmented by the second liquid phase. The channel may be configured to flow the plurality of partitions. The plurality of partitions may not comprise droplets. As an alternative, or in addition to, the first liquid phase may be encompassed by the second liquid phase to yield an emulsion comprising a plurality of droplets.

The method may further comprise subjecting the first and second liquid phases to flow from the first and second chambers, respectively, to the junction. Contacting the first and second liquid phases at the junction may generate a plurality of partitions. The plurality of partitions may comprise a reaction mixture. The reaction mixture may contain reagents for performing a nucleic acid amplification reaction on the target nucleic acid molecule. The nucleic acid amplification reaction may yield amplification products of or derived from the target nucleic acid molecule. The partitions may be subjected to conditions sufficient to perform a plurality of series of primer extension reactions on the target nucleic acid molecule in the presence of the nucleic acid amplification reagents. The sufficient conditions may facilitate the series of primer extension reactions to produce amplification products. An individual series of primer extension reactions may differ from at least one other individual series of the plurality of primer extension reactions with respect to the denaturing condition, the elongation condition, or both the denaturation and elongation condition.

A detector may be used to detect signals indicative of the presence or absence of amplification products in a given partition. Detecting the presence or absence of amplification products in a given partition may thereby detect the presence or absence of one or more target nucleic acid molecule in the raw biological sample.

The fluid flow network may be part of a microfluidic device or a microfluidic chip. The first liquid phase may comprise an aqueous fluid. The second liquid phase may comprise an oil phase.

In another aspect, the present disclosure provides methods for detecting the presence or absence of a target nucleic acid molecule from a biological sample. The method may comprise activating a system comprising a detector and a fluid flow network. The fluid flow network may comprise a first chamber, a second chamber, and a channel. The first chamber and the second chamber may be in fluid communication at a junction. The first chamber may comprise a first liquid phase. The first liquid phase may contain a biological sample. The second chamber may comprise a second liquid phase. The second liquid phase may be immiscible with the first phase. The channel may be in fluid communication with the junction. The channel may be configured to flow a plurality of partitions generated upon contacting the first liquid phase with the second liquid phase at the junction. The channel may comprise at least one segment in a plurality of thermal zones. The plurality of thermal zones may subject the plurality of partitions to heating and/or cooling. The channel may comprise a detection segment downstream of the at least one segment comprising the plurality of thermal zones. The detection segment may be in sensing communication with the detector.

The method may further comprise subjecting the first liquid phase and the second liquid phase to flow from the first and second chambers, respectively, to the junction to generate the plurality of partitions. The plurality of partitions may flow along the channel. A given partition of the plurality of partitions may comprise a reaction mixture containing the reagents for performing a nucleic acid amplification reaction on the target nucleic acid molecule. The nucleic acid amplification reaction may yield amplification products of or derived from the target nucleic acid molecule. The plurality of thermal zones may subject the reaction mixture in the plurality of partitions to conditions sufficient to perform the nucleic acid amplification reaction of the target nucleic acid molecule in the presence of the reagents. The nucleic acid amplification reaction may produce amplification products of the target nucleic acid molecule.

The detector may be used to detect signals indicative of the presence or absence of the amplification produces from a given partition when the partition is flowing through the detection segment. Detecting the presence or absence of amplification products may thereby detect the presence or absence of one or more target nucleic acid molecules in the biological sample.

The fluid flow network may be part of a microfluidic device or a microfluidic chip. A microfluidic chip may comprise more than one fluid flow network. For example, a microfluidic chip may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fluid flow networks. The first liquid phase may comprise an aqueous fluid. The second liquid phase may comprise an oil phase. The first liquid phase may be contained in the first chamber and the second liquid phase may be contained in the second chamber. Contacting the first liquid phase with the second liquid phase may form a partition. The partition may be part of a water-in-oil emulsion. Alternatively, or in addition to, the partition may not be part of a water-in-oil emulsion. The partition may comprise the first liquid phase segmented by the second liquid phase. The first liquid phase may be immiscible with the second liquid phase. The first fluid and the second fluid may be substantially immiscible. The partitions may be completely or substantially isolated from one another. In an example, a channel comprises a plurality of partitions. Each partition may comprise the first liquid phase segmented by the second liquid phase such that the two liquid phases alternate along the length of the channel. Such segments may not be droplets. The individual partitions along the length of the channel may be completely, or substantially, isolated from other individual partitions within the channel.

The two liquid phases may not yield an emulsion comprising droplets. In such a case, the partitions may be generated upon segments of the first liquid phase being separated from one another by the second liquid phase, or vice versa.

The first liquid phase may be segmented by a second liquid phase. The first liquid phase may be segmented by a gas phase or a semi-liquid (e.g., gel) phase. The first liquid phase may be segmented by one or more liquid phases, one or more gas phases, one or more semi-solid phases, or a combination thereof.

The partition may be directed along the length of a channel. The first liquid phase and the second liquid phase may be in contact and form an interface between the two liquid phases. The interface between the first liquid phase and the second liquid phase may have a concave, convex, or planar cross-section normal to, or substantially normal to, the direction of fluid flow. The partitions may have a leading and a tailing interface relative to the direction of fluid flow. The leading and tailing interfaces may be the same shape or may be different shapes. A partition may have an aspect ratio. The aspect ratio may be the length of the partition (e.g., the length parallel to the long dimension of the channel or parallel to the direction of fluid flow) divided by the smallest cross-sectional dimension of the fluid plug (e.g., normal to the direction of fluid flow) . For example, a partition with a length that is greater than the smallest cross-sectional dimension may have an aspect ratio of greater than one.

The partitions may include a skin or a film. The skin or film may be disposed around the partition (e.g., around the first liquid phase) . The skin or film may be disposed at the interface between the first liquid phase and the second liquid phase. Alternatively, or in addition to, the skin or film may encapsulate the partition. The skin or film may isolate the partitions. The skin or film may form upon heating the partition. The skin or film may have a higher viscosity than an interior of the partition. The skin or film may prevent partitions from fusing. The skin or film may prevent the fluid within the partition from mixing with fluid external to the partition.

The partition may contain a reaction mixture comprising reagents to perform a nucleic acid amplification reaction, detectable moieties, and a target nucleic acid derived from the biological sample. The target nucleic acid may be derived from any suitable biological sample of a subject. The biological sample may be a raw biological sample (i.e., sample not chemically, physically, or biologically treated) or a processed biological sample. In an example, the biological sample is a raw biological sample provided directly from a source of the biological sample to the first chamber of the device without further processing. The biological sample may be diluted in an aqueous phase. Dilution of the biological sample may aid in minimizing inhibition of the primer extension reaction. Alternatively, or in addition to, the biological sample may be concentrated. A concentrated biological sample may aid in increasing or otherwise improve sensitivity. The target nucleic acid may be derived from an environmental or food sample. The environmental or food sample may be a raw or treated biological sample. For example, the biological sample may be solid matter (e.g., biological tissue) or may be a fluid (e.g., a biological fluid) . In general, a biological fluid may include any fluid associated with living organisms. Non-limiting examples of a biological sample include blood (or components of blood, e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location (e.g., tissue, circulatory system, bone marrow) of a subject, cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, breath, bone marrow, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, breast, pancreas, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, cavity fluids, sputum, pus, micropiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluid, tears, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cord blood, emphatic fluids, and/or other excretions or body tissues.

The biological sample may be obtained from a subject in a variety of ways. Non-limiting examples of approaches to obtain a nucleic acid sample from a subject include accessing the circulatory system (e.g., intravenously or intra-arterially via a syringe or other needle) , collecting a secreted biological sample (e.g., feces, urine, sputum, saliva, etc. ) , surgically (e.g., biopsy) , swabbing (e.g., buccal swab, oropharyngeal swab) , pipetting, and breathing. Moreover, a nucleic acid sample may be obtained from any anatomical part of a subject where the biological sample is located. The biological sample may be from a genome of the subject. The biological sample may be or contain a cell free nucleic acid. For example, the biological sample may be cell-free DNA.

The biological sample may be obtained directly from the subject. A biological sample obtained directly from a subject may be a biological sample that has not been further processed after being obtained from the subject (e.g., a raw biological sample) , with the exception of any approach used to collect the biological sample from the subject for further processing. For example, blood is obtained directly from a subject by accessing the subject’s circulatory system, removing the blood from the subject (e.g., via a needle) , and entering the removed blood into a receptacle. The receptacle may comprise reagents (e.g., anti-coagulants) such that the blood sample is useful for further analysis. In another example, a swab may be used to access epithelial cells on an oropharyngeal surface of the subject. After obtaining the biological sample from the subject, the swab containing the biological sample can be contacted with a fluid (e.g., a buffer) to collect the biological fluid from the swab. The biological sample may be obtained directly from the subject and provided in the first chamber without sample purification and/or nucleic acid (e.g., DNA or RNA) extraction. For example, the RNA or DNA in a biological sample may not be extracted from the biological sample when providing the sample in the first chamber and/or the first liquid phase. Moreover, in some embodiments, a target nucleic acid (e.g., a target RNA or target DNA) present in a biological sample is not concentrated prior to providing the biological sample to the first liquid phase and/or the first chamber.

The biological sample may be analyzed and nucleic acid target molecules may be detected without further processing. Alternatively, or in addition to, cells contained within the biological sample may be lysed to release the internal nucleic acid molecules (e.g., nuclear DNA) . In an example, the reagents to perform a nucleic acid amplification reaction include a lysing agent. The lysing agent may lyse cells present in the biological sample to release the nucleic acid molecules internal to the cells. Lysing agents may include a detergent, organic solvent, alkali or alkaline earth salts, or a polyhydric alcohol. The detergent may be nonionic, anionic, or cationic. In an example, the reagents do not comprise a lysing agent and the target nucleic acid (s) are extracellular nucleic acid (s) .

A variety of nucleic acid amplification reactions may be used to amplify a target nucleic acid in the biological sample and generate amplification products. Moreover, amplification of a nucleic acid may include linear amplification, exponential amplification, or a combination thereof. Non-limiting examples of nucleic acid amplification methods include reverse transcription, primer extension, polymerase chain reaction, ligase chain reaction, helicase-dependent amplification (e.g., amplification that is preceded by contacting the nucleic acid with a helicase) , asymmetric amplification, rolling circle amplification, and multiple displacement amplification (MDA) . In an example, the nucleic acid amplification reaction is an isothermal reaction. The isothermal reaction may be coupled to an isothermal enzyme cascade to accelerate the reaction. In some embodiments, the amplified product may be DNA. In cases where a target RNA is amplified, DNA can be obtained by reverse transcription of the RNA and subsequent amplification of the DNA can be used to generate an amplified DNA product. The amplified DNA product may be indicative of the presence of the target RNA in the biological sample. In cases where DNA is amplified, any DNA amplification method may be employed. Non-limiting examples of DNA amplification methods include polymerase chain reaction (PCR) , variants of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric interlaced PCR, touchdown PCR, and isothemal PCR) , and ligase chain reaction (LCR) . In an example, the target nucleic acid molecule is amplified by isothermal PCR. In some embodiments, DNA amplification is linear. In some embodiments, DNA amplification is exponential. In some embodiments, DNA amplification is achieved with nested PCR, which can improve sensitivity of detecting amplified DNA products.

In any of the various aspects, more than one nucleic acid amplification reaction described herein may be conducted in each partition. The multiple nucleic acid amplification reactions may be conducted in parallel or sequentially. In general, parallel amplification reactions are amplification reactions that occur in the same reaction partition (e.g., the same partition) and at the same time. Parallel nucleic acid amplification reactions may be conducted, for example, by including reagents for each nucleic acid amplification reaction in a partition to obtain a reaction mixture and subjecting the reaction mixture to conditions for each nucleic amplification reaction. For example, reverse transcription amplification and DNA amplification may be conducted in parallel, by providing reagents for both amplification methods in a partition to obtain a reaction mixture and subjecting the reaction mixture to conditions suitable for conducting both amplification reactions. DNA generated from reverse transcription of the RNA may be amplified in parallel to generate an amplified DNA product. Any suitable number of nucleic acid amplification reactions may be conducted in parallel. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 10,000, or more nucleic acid amplification reactions are conducted in parallel.

An advantage of conducting nucleic acid amplification reactions in parallel can include fast transitions between coupled nucleic acid amplification reactions. For example, a target nucleic acid (e.g., target RNA, target DNA) may be extracted or released from a biological sample during heating phases of parallel nucleic acid amplification. In the case of a target RNA, for example, the biological sample comprising the target RNA can be heated and the target RNA released from the biological sample. The released target RNA can immediately begin reverse transcription (via reverse transcription amplification) to produce complementary DNA. The complementary DNA can then be immediately amplified, often on the order of seconds. A short time between release of a target RNA from a biological sample and reverse transcription of the target RNA to complementary DNA may help minimize the effects of inhibitors in the biological sample that may impede reverse transcription and/or DNA amplification.

The reagents for performing nucleic acid amplification reactions may include one or more polymerizing enzymes and one or more primers having sequence complementarity with a target nucleic acid sequence. Primer sets directed to a target nucleic acid may be utilized to conduct a nucleic acid amplification reaction. Primer sets generally comprise one or more primers. For example, a primer set may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more primers. A primer set may comprise primers directed to different amplified products or different nucleic acid amplification reactions. For example, a primer set may comprise a first primer to generate a first strand of nucleic acid product that is complementary to at least a portion of the target nucleic acid and a second primer complementary to the nucleic acid strand product to generate a second strand of nucleic acid product that is complementary to at least a portion of the first strand of nucleic acid product. Any suitable number of primer sets may be used. For example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primer sets may be used. Where multiple primer sets are used, one or more primer sets may each correspond to a particular nucleic acid amplification reaction or amplified product.

In an example, a primer set may be directed to a target RNA. The primer set may comprise a first primer that can be used to generate a first strand of nucleic acid product that is complementary to at least a portion the target RNA. In the case of a reverse transcription reaction, the first strand of nucleic acid product may be DNA. The primer set may also comprise a second primer that can be used to generate a second strand of nucleic acid product that is complementary to at least a portion of the first strand of nucleic acid product. In the case of a reverse transcription reaction conducted in parallel with DNA amplification, the second strand of nucleic acid product may be a strand of nucleic acid (e.g., DNA) product that is complementary to a strand of DNA generated from an RNA template. The amplification product may be an amplified DNA molecule generated from the target RNA.

One or more polymerizing enzymes may be used. Polymerizing enzymes may include DNA polymerases, RNA polymerases, and reverse transcriptases. The polymerizing enzymes may be naturally occurring or synthetically produced. Any suitable polymerase may be used, including commercially available polymerases. A RNA polymerases A DNA polymerase generally refers to an enzyme that is capable of incorporating nucleotides to a strand of DNA in a template bound fashion. Non-limiting examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerases, E. coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase, Φ29 (phi29) DNA polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, and variants, modified products and derivatives thereof. For certain Hot Start Polymerase, a denaturation step at a temperature from about 92℃ to 95℃ (e.g., 94℃ to 95℃) for a time period from about 2 minutes to 10 minutes may be required, which may change the thermal profile based on different polymerases.

The reagent mixture may include a polymerizing enzyme that is a reverse transcriptase and the amplification reaction may be reverse transcription polymerase chain reaction. Any suitable reverse transcriptase may be used. A reverse transcriptase generally refers to an enzyme that is capable of incorporating nucleotides to a strand of DNA, when bound to an RNA template. Non-limiting examples of reverse transcriptases include HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and variants, modified products and derivatives thereof.

In an example, the reagent mixture comprises more than one polymerizing enzyme. The polymerizing enzymes may include a DNA polymerase and a reverse transcriptase. The reverse transcriptase may be separate from the DNA polymerase. A target nucleic acid may be an RNA molecule. The reverse transcriptase may generate a DNA molecule from the RNA molecule. The DNA molecule may be amplified to produce an amplification product.

The target nucleic acid may be a nucleic acid sequence associated with a disease. The disease may be associated with a virus such as for example an RNA virus or a DNA virus. In some embodiments, the virus can be selected from the group consisting of human immunodeficiency virus I (HIV I) , human immunodeficiency virus II (HIV II) , an orthomyxovirus, Ebola virus, Dengue virus, influenza viruses, hepevirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile Fever virus, polio virus, measles virus, herpes simplex virus, smallpox virus, adenovirus, Varicella virus, and Zika virus. In an example, the influenza virus is selected from the group consisting of H1N1 virus, H3N2 virus, H7N9 virus and H5N1 virus. In an example, the adenovirus is adenovirus type 55 (ADV55) or adenovirus type 7 (ADV7) . In an example, the hepatitis C virus is armored RNA-hepatitis C virus (RNA-HCV) . In an example, the disease is associated with a pathogenic bacterium (e.g., Mycobacterium tuberculosis) or a pathogenic protozoan (e.g., Plasmodium) .

The target nucleic acid may be associated with a cancer. Non-limiting examples of the cancers include colorectal cancer, bladder cancer, ovarian cancer, testicular cancer, breast cancer, skin cancer, lung cancer, pancreatic cancer, stomach cancer, esophageal cancer, brain cancer, leukemia, liver cancer, endometrial cancer, prostate cancer, and head and neck cancer.

The target nucleic acid may be associated with food safety. Food safety can be compromised by foodborne illness caused by pathogenic microbes. The pathogenic microbes may be bacteria, viruses, or parasites. The target nucleic acid may be associated with a pathogenic bacterium, a pathogenic virus, or a pathogenic parasite that may compromise food safety.

Food safety may be compromised by a pathogenic bacterium. Non-limiting examples of pathogenic bacteria include Campylobacter jejuni, Clostridium perfringens, Salmonella spp., Escherichia coli O157: H7 enterohemorrhagic (EHEC) , Bacillus cereus, other virulent Escherichia coli such as enteroinvasive (EIEC) , enteropathogenic (EPEC) , enterotoxigenic (ETEC) , enteroaggregative (EAEC or EAgEC) , Listeria monocytogenes, Shigella spp., Staphylococcus aureus, Staphylococcal enteritis, Streptococcus, Vibrio cholerae, including O1 and non-O1, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica and Yersinia pseudotuberculosis, Brucella spp., Corynebacterium ulcerans, Coxiella burnetii or Q fever, Plesiomonas shigelloides, and the like. Sometimes the food safety is compromised by an enterotoxin secreted by a bacterium rather than the bacterium per se. Non-limiting examples of such enterotoxin-secreting bacteria include Staphylococcus aureus, Clostridium botulinum, Clostridium perfringens, Bacillus cereus, Pseudoalteromonas tetraodonis, Pseudomonas spp., Vibrio spp., and the like.

Food safety may be compromised by a pathogenic virus. Non-limiting examples of pathogenic virus include Enterovirus, Hepatitis A, Hepatitis E, Norovirus, Rotavirus, and the like.

Food safety may be compromised by a pathogenic parasite. Non-limiting examples of pathogenic parasite include Diphyllobothrium sp., Nanophyetus sp., Taenia saginata, Taenia solium, Fasciola hepatica, Anisakis sp., Ascaris lumbricoides, Eustrongylides sp., Trichinella spiralis, Trichuris trichiura, Acanthamoeba, Cryptosporidium parvum, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Sarcocystis hominis, Sarcocystis suihominis, and Toxoplasma gondii.

The target nucleic acid may be associated with prenatal testing. Prenatal testing may be conducted during gestation for detecting potential conditions, disorders or diseases associated with fetus. The presence or the amount of the target nucleic acid sequence may be indicative of potential conditions, disorders or diseases in prenatal testing. Non-limiting conditions, disorders and diseases that may be detected in prenatal testing include spina bifida, cleft palate, Tay–Sachs disease, sickle cell anemia, thalassemia, cystic fibrosis, muscular dystrophy, fragile X syndrome, aneuploidy such as Down Syndrome (Trisomy 21) , Edwards Syndrome (Trisomy 18) , and Patau Syndrome (Trisomy 13) , and the like.

The target nucleic acid may be associated with genetic testing. Genetic testing may be conducted for various purposes, including, but not limited to detection of genetic disorders, forensic testing, molecular diagnosis, paternity/maternity testing, and the like. The presence or the amount of the target nucleic acid sequence may be indicative of the result of a genetic testing.

The target nucleic acid may be associated derived from a cancer liquid biopsy. Cancer liquid biopsy may be useful for detecting cancer by analyzing liquid samples from a subject (such as blood or bodily fluid) for indicators of cancers, such as circulating tumor cells or cell-free tumor nucleic acids. The presence or the amount of the target nucleic acid sequence may be indicative of having cancer or being at risk of having cancer in a cancer liquid biopsy. The cancer may be any cancer that can be diagnosed with a cancer liquid biopsy. Non-limiting examples of cancers that can be diagnosed with a cancer liquid biopsy include breast cancer, colon cancer, leukemia, lymphoma, stomach cancer, lung cancer, prostate cancer, and the like.

The nucleic acid amplification reaction may be performed by thermal cycling the reaction mixture within a partition (e.g., partition) . Thermal cycling may comprise a cycle of incubating the reaction mixture at a denaturation temperature for a denaturation duration and incubating a reaction mixture at an elongation temperature for an elongation duration.

Denaturation temperatures may vary depending upon, for example, the particular nucleic acid sample analyzed, the particular source of target nucleic acid (e.g., viral particle, bacteria) in the nucleic acid sample, the reagents used, and/or the reaction conditions. For example, a denaturation temperature may be from about 80℃ to about 110℃. In some examples, a denaturation temperature may be from about 90℃ to about 100℃. In some examples, a denaturation temperature may be from about 90℃ to about 97℃. In some examples, a denaturation temperature may be from about 92℃ to about 95℃. In an example, a denaturation temperature may be greater than or equal to about 80°, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, 100℃, or more.

Denaturation durations may vary depending upon, for example, the particular nucleic acid sample analyzed, the particular source of target nucleic acid (e.g., viral particle, bacteria) in the nucleic acid sample, the reagents used, and/or the reaction conditions. For example, a denaturation duration may be less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, a denaturation duration may be no more than 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

Elongation temperatures may vary depending upon, for example, the particular nucleic acid sample analyzed, the particular source of target nucleic acid (e.g., viral particle, bacteria) in the nucleic acid sample, the reagents used, and/or the reaction conditions. For example, an elongation temperature may be from about 30℃ to about 80℃. In some examples, an elongation temperature may be from about 35℃ to about 72℃. In some examples, an elongation temperature may be from about 45℃ to about 65℃. In some examples, an elongation temperature may be from about 35℃ to about 65℃. In some examples, an elongation temperature may be from about 40℃ to about 60℃. In some examples, an elongation temperature may be from about 50℃ to about 60℃. In still other examples, an elongation temperature may be at least about 35°, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, or 80℃.

Elongation durations may vary depending upon, for example, the particular nucleic acid sample analyzed, the particular source of target nucleic acid (e.g., viral particle, bacteria) in the nucleic acid sample, the reagents used, and/or the reaction conditions. For example, an elongation duration may be less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. For example, an elongation duration may be no more than about 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

The nucleic acid amplification reaction may comprise a primer extension reaction. Multiple cycles of a primer extension reaction may be performed to amplify the target nucleic acid molecule. Any suitable number of cycles may be conducted. For example, the number of cycles conducted may be fewer than or equal to about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 cycles. The number of cycles conducted may depend upon, for example, the number of cycles to obtain a detectable amplified product (e.g., a detectable amount of amplified DNA product that is indicative of the presence of a target RNA in a nucleic acid sample) . For example, the number of cycles to obtain a detectable amplified product (e.g., a detectable amount of DNA product that is indicative of the presence of a target RNA in a nucleic acid sample) may be fewer than about or equal to about 100 cycles, 75 cycles, 70 cycles, 65 cycles, 60 cycles, 55 cycles, 50 cycles, 40 cycles, 35 cycles, 30 cycles, 25 cycles, 20 cycles, 15 cycles, 10 cycles, or 5 cycles. In an example, the number of cycles conducted may be fewer than about 50 cycles.

The time for which amplification yields a detectable amount of amplified product indicative of the presence of a target nucleic acid amplified can vary depending upon the nucleic acid sample from which the target nucleic acid was obtained, the particular nucleic acid amplification reactions to be conducted, and the particular number of cycles of amplification reaction used. For example, amplification of a target nucleic acid may yield a detectable amount of amplified product indicative to the presence of the target nucleic acid at time period of 120 minutes or less; 90 minutes or less; 60 minutes or less; 50 minutes or less; 45 minutes or less; 40 minutes or less; 35 minutes or less; 30 minutes or less; 25 minutes or less; 20 minutes or less; 15 minutes or less; 10 minutes or less; or 5 minutes or less. In an example, a detectable amount of amplified product is obtained in less than or equal to about 30 minutes. In an example, a detectable amount of amplified product is obtained and less than about 30 minutes and fewer than 50 amplification cycles.

The reaction mixture (e.g., within the partitions) may be subjected to a plurality of series of primer extension reactions. An individual series of the plurality may comprise multiple cycles of a particular primer extension reaction, characterized, for example, by particular denaturation and elongation conditions as described elsewhere herein. Generally, each individual series differs from at least one other individual series in the plurality with respect to, for example, a denaturation condition and/or elongation condition. An individual series may differ from another individual series in a plurality of series, for example, with respect to any one, two, three, or all four of denaturing temperature, denaturing duration, elongation temperature, and elongation duration. Moreover, a plurality of series may comprise any number of individual series such as, for example, greater than or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more individual series.

In an example, the partition comprises a reaction mixture and target RNA molecule and the amplification product may be an amplified DNA molecule generated from the RNA molecule. The partition, and therefore the reaction mixture, may be subjected to multiple cycles of a primer extension reaction to reverse transcribe the RNA molecule and generate a DNA molecule in parallel. Each cycle may comprise incubating the reaction mixture at a denaturing temperature for a denaturing duration that is less than or equal to 60 seconds follow by incubating the reaction mixture at an elongation temperature for an elongation duration that is less than or equal to 60 seconds.

The target nucleic acid may be subjected to a denaturing condition prior to initiation of a nucleic acid amplification reaction (e.g., primer extension reaction) . In the case of a plurality of series of primer extension reactions, the target nucleic acid may be subjected to a denaturing condition prior to executing the plurality of series or may be subjected to a denaturing condition between series of the plurality. For example, the target nucleic acid may be subjected to a denaturing condition between a first series and a second series of a plurality of series. Non-limiting examples of such denaturing conditions include a denaturing temperature profile (e.g., one or more denaturing temperatures) and a denaturing agent. In an example, the reaction mixture in a given partition may be subjected to a series of primer extension reactions to generate amplification products. Each series may comprise two or more cycles of incubating the reaction mixture under denaturing conditions for a denaturation duration and temperature followed by incubating the reaction mixture under elongation conditions for an elongation duration and temperature. The individual primer extension reaction may differ from another primer extension reaction with respect to the denaturation conditions and/or the elongation conditions.

The partitions may include one or more detectable moieties that permit detection of the signals. For example, the detectable moieties may yield a detectable signal whose presence or absence is indicative of the presence of an amplified product. The intensity of the detectable signal may be proportional to the amount of amplified product. In some cases, where amplified product is generated of a different type of nucleic acid than the target nucleic acid initially amplified, the intensity of the detectable signal may be proportional to the amount of target nucleic acid initially amplified. For example, in the case of amplifying a target RNA via parallel reverse transcription and amplification of the DNA obtained from reverse transcription, reagents for both reactions may also comprise a detectable moiety that yield a detectable signal indicative of the presence of the amplified DNA product and/or the target RNA amplified. The intensity of the detectable signal may be proportional to the amount of the amplified DNA product and/or the original target RNA amplified. The use of a detectable moiety also enables real-time amplification methods, including real-time PCR for DNA amplification.

Detectable moieties may be linked with nucleic acids, including amplified products, by covalent or non-covalent interactions. Non-limiting examples of non-covalent interactions include ionic interactions, Van der Waals forces, hydrophobic interactions, hydrogen bonding, and combinations thereof. In some embodiments, detectable moieties bind to initial reactants and changes in detectable moiety levels are used to detect amplified product. In some embodiments, detectable moieties are detectable (or non-detectable) as nucleic acid amplification progresses. In some embodiments, an optically-active dye (e.g., a fluorescent dye) is used as a detectable moiety. Non-limiting examples of dyes include SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, phenanthridines and acridines, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue) , SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green) , SYTO-81, -80, -82, -83, -84, -85 (orange) , SYTO-64, -17, -59, -61, -62, -60, -63 (red) , fluorescein, fluorescein isothiocyanate (FITC) , tetramethyl rhodamine isothiocyanate (TRITC) , rhodamine, tetramethyl rhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, , Cy-7, Texas Red, Phar-Red, allophycocyanin (APC) , Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, cascade blue, dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes such as those including europium and terbium, carboxy tetrachloro fluorescein, 5 and/or 6-carboxy fluorescein (FAM) , 5- (or 6-) iodoacetamidofluorescein, 5- { [2 (and 3) -5- (Acetylmercapto) -succinyl] amino} fluorescein (SAMSA-fluorescein) , lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine (ROX) , 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid (AMCA) , BODIPY fluorophores, 8-methoxypyrene-1, 3, 6-trisulfonic acid trisodium salt, 3, 6-Disulfonate-4-amino-naphthalimide, phycobiliproteins, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or other fluorophores.

A detectable moiety may be a sequence-specific oligonucleotide probe that is optically active when hybridized with an amplified product. Due to sequence-specific binding of the probe to the amplified product, use of oligonucleotide probes can increase specificity and sensitivity of detection. A probe may be linked to any of the optically-active detectable moieties (e.g., dyes) described herein and may also include a quencher capable of blocking the optical activity of an associated dye. Non-limiting examples of probes that may be useful as detectable moieties include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes, locked nucleic acid probes, and molecular beacons. Alternatively, or in addition to, the probe maybe any probe that is useful in the context of the methods of the present disclosure.

A detectable moiety may be an RNA oligonucleotide probe that includes an optically-active dye (e.g., fluorescent dye) and a quencher positioned adjacently on the probe. The close proximity of the dye with the quencher can block the optical activity of the dye. The probe may bind to a target sequence to be amplified. Upon the breakdown of the probe with the exonuclease activity of a DNA polymerase during amplification, the quencher and dye are separated, and the free dye regains its optical activity that can subsequently be detected.

A detectable moiety may be a molecular beacon. A molecular beacon includes, for example, a quencher linked at one end of an oligonucleotide in a hairpin conformation. At the other end of the oligonucleotide is an optically active dye, such as, for example, a fluorescent dye. In the hairpin configuration, the optically-active dye and quencher are brought in close enough proximity such that the quencher is capable of blocking the optical activity of the dye. Upon hybridizing with amplified product, however, the oligonucleotide assumes a linear conformation and hybridizes with a target sequence on the amplified product. Linearization of the oligonucleotide results in separation of the optically-active dye and quencher, such that the optical activity is restored and can be detected. The sequence specificity of the molecular beacon for a target sequence on the amplified product can improve specificity and sensitivity of detection.

A detectable moiety may be a radioactive species. Non-limiting examples of radioactive species include ¹⁴C ^, ¹²³I ^, ¹²⁴I ^, ¹²⁵I ^, ¹³¹I, Tc99m, ³⁵S, and ³H.

A detectable moiety may be an enzyme that is capable of generating a detectable signal. Detectable signal may be produced by activity of the enzyme with its substrate or a particular substrate in the case the enzyme has multiple substrates. Non-limiting examples of enzymes that may be used as detectable moieties include alkaline phosphatase, horseradish peroxidase, I2-galactosidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, and luciferase.

The reaction mixture may contain multiple different primers that may amplify multiple different target nucleic acid molecules. The multiple different primers may generate multiple different amplification products. The partitions may contain multiple different detectable moieties. Each different detectable moiety of the multiple detectable moieties may selectively interact with a single amplification product and generate a signal indicative of that amplification product. Each different detectable moiety may generate a unique signal that is distinguishable from the other detectable moieties. For example, fluorescent detectable moieties may emit fluorescence signals of differing wavelengths that may be distinguishable from one another.

The partitions may include one or more additives. Additives may include, but are not limited to, dimethyl sulfoxide, glycerol, betaine monohydrate, bovine serum albumin, surfactants, detergents, formamide, organic solvents, and/or tetramethyl ammonium chloride. The partitions may include nucleic acid molecules and/or nucleic acid analogs. The partitions may include nucleotides and/or nucleotide analogs. The additives may reduce inhibition of the amplification reaction. Alternatively, or in addition to, the additives may reduce interactions between reagents in the partition and the channel wall. The additives may reduce the interaction between the fluid within the partition and the channel wall. Reducing interactions at the channel wall may increase the stability of the partitions.

The fluidic network may comprise a second chamber. The second chamber may comprise a second liquid phase. The second liquid phase may comprise a non-aqueous fluid. The non-aqueous fluid may comprise hydrophobic liquids. Non-limiting examples of the hydrophobic liquids include hydrocarbon solvents (e.g., organic solvents) and oils. Oils may include hydrocarbon oils, silicon oils, and/or fluorocarbon oils. In an example, the oil is a fluorinated oil, such as HFE 7100, HFE 7500, FC-40, FC-43, FC-70, FC-3208, or a combination thereof. In an example, the oil is a mineral oil, such as liquid paraffin, light mineral oil, white oil, refined mineral oil, cycloalkane oil, aromatic oil, or a combination thereof. The oil may also be any oil that is useful for making partitions. Examples of oils and surfactants that may be employed for use are provided in U.S. Patent No. 9,012,390, which is entirely incorporated herein by reference.

The second liquid phase may comprise a non-wetting agent. The non-wetting agent may be a surfactant, detergent, or polymer. The non-wetting agent may reduce the interaction between the partitions and the channel wall. The non-wetting agent may reduce the friction and drag of the partition (e.g., the first liquid phase) moving along the wall. Additionally, or alternatively, the non-wetting agent may reduce the binding of reagent components (e.g., proteins) to the channel wall. The non-wetting agent may comprise a hydrophobic tail and a hydrophilic head group, a polymer-based tail and a hydrophilic head group, a polymer-based tail and a polymer-based head group, a fluorinated tail and a hydrophilic head group, or a fluorinated polymer-based tail and a hydrophilic polymer-based head group. In some embodiments, the non-wetting agent is a di-block copolymer or tri-block copolymer type. For example, the non-wetting agent may be a block copolymer, such as a tri-block copolymer consisting of two perfluoropolyether blocks and one poly (ethylene) glycol block. In an example, the non-wetting agent is selected from the group consisting of PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) , tri-block copolymer EA-non-wetting agent (RainDance Technologies) and DMP (dimorpholino phosphate) -non-wetting agent (Baret, Kleinschmidt, et al., 2009) . The length of PEG in a polymeric species, including a polymeric non-wetting agent, can have any suitable length and may vary between different polymeric species that can be used. In an example, the non-wetting agent is a plant derived surfactant such as sodium lauryl sulfate, ammonium laureth sulfate, disodium lauryl sulfosuccinate, decyl glucoside, glyceryl cocoate, sodium cocoyl isethionate, or any combination thereof. The non-wetting agent may be present in the second liquid phase with a concentration of 0.0001%to 5% (w/w) , e.g., 0.001%to 4% (w/w) , 0.01%to 3% (w/w) , 0.1%to 2% (w/w) , 0.1%to 1% (w/w) . In an example, the non-wetting agent in the second liquid phase has a concentration of at least about 0.1% (w/w) , 0.2% (w/w) , 0.3% (w/w) , 0.4% (w/w) , 0.5% (w/w) , 0.6% (w/w) , 0.7% (w/w) , 0.8% (w/w) , 0.9% (w/w) , 1.0% (w/w) , 1.2% (w/w) , 1.4% (w/w) , 1.6% (w/w) , 1.8% (w/w) , 2.0% (w/w) , 2.5% (w/w) , 3.0% (w/w) , 3.5% (w/w) , 4.0% (w/w) , 4.5% (w/w) , 5.0% (w/w) , 7.0% (w/w) , 10.0% (w/w) , 15.0% (w/w) , 20.0% (w/w) or more. In an example, the non-wetting agent in the second liquid phase has a concentration of less than or equal to about 20.0% (w/w) , 15.0% (w/w) , 10.0% (w/w) , 7.0% (w/w) , 5.0% (w/w) , 4.5% (w/w) , 4.0% (w/w) , 3.5% (w/w) , 3.0% (w/w) , 2.5% (w/w) , 2.0% (w/w) , 1.8% (w/w) , 1.6% (w/w) , 1.4% (w/w) , 1.2% (w/w) , 1.0% (w/w) , 0.9% (w/w) , 0.8% (w/w) , 0.7% (w/w) , 0.6% (w/w) , 0.5% (w/w) , 0.4% (w/w) , 0.3% (w/w) , 0.2% (w/w) , 0.1% (w/w) or less.

The first chamber may be any suitable shape or volume. The first chamber may have a volume that is less than or equal to about 5 milliliters (mL) , 4 mL, 3 mL, 2 mL, 1 mL, 750 microliters (μL) , 500 μL, 250 μL, 100 μL, 50 μL, 40 μL, 30 μL, 20 μL, 10 μL or less. The first chamber may have a volume that is greater than or equal to about 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 100 μL, 250 μL, 500 μL, 750 μL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, or more. The first chamber may be in fluid communication with the junction. Fluid communication between the junction and the first chamber may be provided by the channel. The channel may comprise a segment that branches to form a “Y” or “T” shape at a junction. One branch of the “Y” or “T” shaped segment may be fluidically connected to, or in fluid communication with, the first chamber. The channel may form a junction with another channel at an angle that is less than or equal to about 90°, such as from about 25° to 90°, or 45° to 90°.

The second chamber may be any suitable shape or volume. The second chamber may have a volume that is less than or equal to about 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, 750 μL, 500 μL, 250 μL, 100 μL, 50 μL, 40 μL, 30 μL, 20 μL, 10 μL or less. The second chamber may have a volume that is greater than or equal to about 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 100 μL, 250 μL, 500 μL, 750 μL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, or more. The second chamber may be larger, smaller, or equal in volume to the first chamber. The second chamber may have a volume that is 5 percent, 10 percent, 20 percent, 30 percent, 40, percent, 50 percent, 75 percent, 100 percent, 150 percent, 200 percent, or more larger than the volume of the first chamber. The second chamber may have a volume that is 5 percent, 10 percent, 20 percent, 30 percent, 40, percent, 50 percent, 75 percent, 100 percent, 150 percent, 200 percent, or more smaller than the volume of the first chamber. The second chamber may be in fluid communication with the junction. Fluid communication between the junction and the second chamber may be provided by the channel. The channel may comprise a segment that branches forms a “Y” or “T” shape at a junction. One branch of the “Y” or “T” shaped segment (e.g., a different branch than that fluidically connected to the first chamber) may be fluidically connected to, or in fluid communication with, the second chamber.

The fluid flow network may further comprise a third chamber. The third chamber may comprise a third liquid phase. The third liquid phase may comprise an aqueous fluid. The aqueous fluid may comprise a reaction mixture. The reaction mixture may comprise the reagents for performing a nucleic acid amplification reaction. The third liquid phase may be immiscible with the second liquid phase. The third chamber may be in fluid communication with the junction. Fluid communication between the junction and the third chamber may be provided by the channel. The channel may comprise a segment with multiple branches. One branch the segment (e.g., a different branch than that fluidically connected to the first or second chambers) may be fluidically connected to, or in fluid communication with, the third chamber. The third liquid phase may be added to the first liquid phase during generation of the partitions. Alternatively, or in addition to, the third liquid phase may be segmented by the second liquid phase to form partitions isolated from the partitions containing the first liquid phase. In an example, the first liquid phase and the third liquid phase are combined to form a partition. The first and the third liquid phases may flow to the junction at the same rate or at different rates. The flow rate of the first liquid phase may increase or decrease as a function of time. The flow rate of the third liquid phase may increase or decrease inversely proportional the increase or decrease of the flow rate of the first liquid phase.

The third liquid phase may be used to generate a concentration gradient of a select reagent or target nucleic acid along the length of the channel. For example, the first liquid phase may comprise a nucleic acid target and the third liquid phase may contain the nucleic acid amplification reagents and detectable moieties. The flow rate of the third liquid phase may increase inversely proportional to the decreasing flow rate of the first liquid phase. In this example, the first partition generated may have the highest concentration of the first liquid phase with every subsequent partition having a lower concentration of the first liquid phase, thus, generating a concentration gradient of target nucleic acids that decrease as a function of partition. The concentration gradient of target nucleic acids may be used to collect dilution based kinetic measurements.

The fluid flow network may further comprise a plurality of chambers. Each individual chamber of the plurality of chambers may comprise a different aqueous phases. The different aqueous phases may include different biological samples, target nucleic acids, amplification reagents, detectable moieties, additives, or any combination thereof. The plurality of chamber may allow for multiplexed detection of the target nucleic acids.

The branched segments of the channel may be substantially straight or may comprise bends or curves. In an example, the branched segments are configured to minimize curves or bends. Each branch of the branched segment of the channel may be the same length as one another or a different length. For example, the branch fluidically connected to the first chamber may be longer, shorter, or substantially the same length as the branch fluidically connected to the second chamber. The length of each branch of the branched segment may be greater than or equal to about 50 micrometers (μm) , 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 750 μm, 1000 μm, 1.5 millimeters (mm) , 2 mm, or more. The length of each branch of the branched segment may be less than or equal to about 2 mm, 1.5 mm, 1000 μm, 750 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, or less.

The channel may include a branched segment, a heating and/or cooling segment, a detection segment, and a recycle segment. The channel may be straight, substantially straight, or may comprise one or more curves or bends. The channel may comprise any configuration, including a circular configuration, spiral configuration, serpentine configuration, or any combination thereof. In an example, the channel may comprise both a spiral and one or more serpentine configurations. In an example, the channel comprises a substantially circular configuration. The total length of the channel may be any suitable length. The total length of the channel, containing any secondary channels and branches, may be greater than or equal to about 1 millimeter (mm) , 2 mm, 3 mm, 5 mm, 7 mm, 1 centimeter (cm) , 1.5 cm, 2 cm, 2.5 cm, 3 cm, 5 cm, 7 cm, 10 cm, 15 cm, 20 cm, 30 cm, 40 cm, 50 cm, 75 cm, 100 cm, 150 cm, or more. The total length of the channel, containing any secondary channels and branches, may be less than or equal to about 150 cm, 100 cm, 75 cm, 50 cm, 40 cm, 30 cm, 20 cm, 15 cm, 10 cm, 7 cm, 5 cm, 3 cm, 2.5 cm, 2 cm, 1.5 cm, 1 cm, 7 mm, 5 mm, 3 mm, 2 mm, or less.

The channel may be configured to minimize bending or curving of the channel. The channel may comprise circular configurations, spiral configurations, serpentine configurations, or a combination thereof. The channel may have a minimum bend radius. The minimum bend radius may be greater than or equal to about 2.5 mm, 2.75 mm, 3 mm, 3.25 mm, 3.5 mm, 3.75 mm, 4 mm, 4.5 mm, 5 mm, or greater. The minimum bend radius may be between about 2.5 mm and 3 mm, 2.5 mm and 3.5 mm, 2.5 mm and 4 mm, 2.5 mm and 4.5 mm, or 2.5 mm and 5 mm. In an example, the bend radius is between about 3 mm and 4.5 mm.

The cross-sectional area of the channel may be substantially constant, or may vary. In some embodiments, the cross-sectional area of the channel varies as a function of position in the direction of fluid flow within the channel. The average cross-sectional area of the channel may be greater than or equal to about 1,000 μm ², 2,000 μm ², 3,000 μm ², 5,000 μm ², 10,000 μm ², 20,000 μm ², 30,000 μm ², 50,000 μm ², 100,000 μm ², 200,000 μm ², 300,000 μm ², 500,000 μm ², 1,000,000 μm ², or more. The average cross-sectional area of the channel may be less than or equal to about 1,000,000 μm ², 500,000 μm ², 300,000 μm ², 200,000 μm ², 100,000 μm ², 50,000 μm ², 30,000 μm ², 20,000 μm ², 10,000 μm ², 5,000 μm ², 3,000 μm ², 2,000 μm ² or less.

The cross-sectional area of the channel may vary along with the length of the channel. The channel may have a cross-sectional area that varies by less than or equal to about 25 percent (%) , 20 %, 15 %, 10 %, 5 %, 4 %, 3 %, 2 %, 1 %, or less than the average cross-sectional area. In addition, the channel may have any suitable cross-sectional shape (e.g., circular, oval, triangular, irregular, square, or rectangular etc. ) .

The channel may have any suitable maximal cross-sectional dimension. The maximal cross-sectional dimension generally refers to the largest dimension that can be contained within a cross-section of the first channel, where the cross-section is determined orthogonal to the direction of average fluid flow within the channel. In an example, the cross-section of the channel may be circular and the maximal cross-sectional dimension is the diameter of the circle. The maximum cross-sectional dimension may less than or equal to about 1 mm, 800 μm, 600 μm, 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 100 μm, 75 μm, 50 μm, 25 μm, 10 μm, or less. The maximum cross-sectional dimension may be greater than or equal to about 5 μm, 10 μm, 25 μm, 50 μm, 75 μm, 100 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 600 μm, 800 μm, or more. The cross-sectional dimension may be between about 100 μm and 250 μm, 100 μm and 300 μm, 100 μm and 400 μm, 100 μm and 500 μm, or 100 μm and 750 μm.

The channel may be in fluid communication with one or more additional channel (s) . The both the channel and the one or more additional channels may be microfluidic. The channel may comprise both microfluidic and non-microfluidic segments. In an example, the main segment of the channel may be microfluidic and at least a portion of the branched segments of the channel may not be microfluidic.

The one or more additional channel (s) may be in fluid communication with the first channel. In an example, more than one additional channel may be present and each may be a different distance from the channel. The additional channel (s) may have the same or different lengths, shapes, cross-sectional areas, or other properties. The additional channels may or may not be fluidically connected to one another. An additional channel may be of any suitable length. An additional channel may be substantially straight or may have one or more curves or bends. The shape of the additional channel may substantially the same as the shape of the channel (e.g., such that the additional channel is separated from the first channel by a relatively constant distance of separation) or may be a different shape than the channel.

An additional channel may have any suitable length. The length of the additional channel may be substantially the same as the first channel. The total length of an additional channel may be greater than or equal to about 1 mm, 2 mm, 3 mm, 5 mm, 7 mm, 1 cm, 1.5 cm, 2 cm, 2.5 cm, 3 cm, 5 cm, 7 cm, 10 cm, or more. The total length of an additional channel may be less than or equal to about 10 cm, 7 cm, 5 cm, 3 cm, 2.5 cm, 2 cm, 1.5 cm, 1 cm, 7 mm, 5 mm, 3 mm, 2 mm, or less.

The cross-sectional area of an additional channel may be substantially constant or may vary. The cross-sectional area of the additional channel may vary as a function of position in the direction of fluid flow within the additional channel. The average cross-sectional area of an additional channel may be greater than or equal to about 1,000 μm ², 2,000 μm ², 3,000 μm ², 5,000 μm ², 10,000 μm ², 20,000 μm ², 30,000 μm ², 50,000 μm ², 100,000 μm ², 200,000 μm ², 300,000 μm ², 500,000 μm ², 1,000,000 μm ², or more. The average cross-sectional area of an additional channel may be less than or equal to about 1,000,000 μm ², 500,000 μm ², 300,000 μm ², 200,000 μm ², 100,000 μm ², 50,000 μm ², 30,000 μm ², 20,000 μm ², 10,000 μm ², 5,000 μm ², 3,000 μm ², 2,000 μm ², or less.

The cross-sectional area of an additional channel may vary. For example, the cross-sectional area of an additional channel may vary along with the length of the channel. An additional channel may have a cross-sectional area that varies by less than or equal to about 25 %, 20 %, 15 %, 10 %, 5 %, 4 %, 3 %, 2 %, 1 %, or less than the average cross-sectional area. The cross-sectional area of an additional channel may be the same or different than the cross-sectional area of the channel. An additional channel may have any suitable cross-sectional shape, e.g., circular, oval, triangular, irregular, square, or rectangular. The cross-sectional shape of the additional channel may be the same or different than the cross-sectional shape of the channel.

The additional channel may have any suitable maximal cross-sectional dimension. The maximal cross-sectional dimension may be the largest dimension that can be contained within a cross-section of the additional channel, where the cross-section is determined orthogonal to the direction of average fluid flow within the additional channel. For example, the maximum cross-sectional dimension may be less than or equal to about 1 mm, 800 μm, 600 μm, 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 100 μm, 75 μm, 50 μm, 25 μm, 10 μm, or less. In addition, in some embodiments, the maximum cross-sectional dimension is greater than or equal to about 5 μm, 10 μm, 25 μm, 50 μm, 75 μm, 100 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 600 μm, 800 μm, or more. The maximal cross-sectional dimension of an additional channel may be the same or different from the maximal cross-sectional dimension of the channel.

The channel, and any additional channels, may have any cross-sectional shape including, but not limited to, a circle, square, rectangle, triangle, or polygon. In an example, the channel has a circular cross-sectional shape and the cross-sectional area is constant along the length of the channel. In an example, the cross-sectional shape and area of the channel is the same as the cross-sectional shape and area of any additional channels.

The channel may comprise a layer of non-wetting agent. An additional channel may comprise a layer of non-wetting agent. The layer of non-wetting agent may include any non-wetting agent described herein. The non-wetting agent layer may be applied to the channel prior to generation of the partitions. The non-wetting agent may be the same non-wetting agent as in the second liquid phase or may be a different non-wetting agent. The non-wetting agent may be applied by flowing the second liquid phase from the second chamber through the channel and/or through an additional channel. In an example, the non-wetting agent is applied by flowing a precursor fluid comprising the non-wetting agent through the channel and/or an additional channel. The fluid flow unit may direct the flow of fluid containing the non-wetting agent through the channel. Activating the system may comprise directing a precursor fluid comprising the non-wetting agent through the channel prior to generating the partition.

A plurality of partitions may be formed or generated at the junction between the first chamber and second chamber. In an example, a plurality of partitions may be formed at the junction between a first chamber, second chamber, and third chamber. The partitions may be formed when a portion of the first liquid phase (e.g., aqueous fluid) is substantially segmented by the second liquid phase (e.g., a non-aqueous phase) . . The partition may comprise an aqueous phase segmented by an oil phase. Each partition of the plurality of partitions may have a volume less than or equal to about 50 μL, 40 μL, 30 μL, 20 μL, 15 μL, 10 μL, 5 μL, 2.5 μL, 2 μL, 1 μL, 500 nanoliters (nL) , 400 nL, 300 nL, 200 nL, 100 nL, or less. Each partition of the plurality of partitions may have a volume greater than or equal to about 100 nL, 200 nL, 300 nL, 400 nL, 500 nL, 1 μL, 2 μL, 2.5 μL, 5 μL, 10 μL, 15 μL, 20 μL, 30 μL, 40 μL, 50 μL, or more.

Each partition of the plurality of partitions may have an aspect ratio. The aspect ratio may be the ratio of the largest dimension of the partition (e.g., the length parallel to the long dimension of the channel) to the smallest dimension of the partition (e.g., the cross-sectional diameter of a circular channel) . The aspect ratio of the partition may be modulated by the volume of the aqueous phase in relation to the channel size, external forces on the partition (e.g., fluid flow rate) , and the relative fluid properties of the aqueous and immiscible non-aqueous phases (e.g., density and viscosity) . The aspect ratio of a given partition of the plurality of partitions may be greater than or equal to about 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, or more. The aspect ratio of a given partition of the plurality of partitions may be less than or equal to about 2.5, 2.25, 2, 1.75, 1.5, 1.25, 1, or less.

The partitions may comprise a first liquid phase (e.g., aqueous phase) segmented by a second liquid phase (e.g., non-aqueous phase) . The partitions may be disposed along a channel. The second liquid phase (e.g., the segmenting fluid) may generate a distance between individual partitions. For example, a partition (e.g., comprising an aqueous fluid) may be separated from another partition by an average distance. The average distance may be the length of the segment of the second liquid phase. The average distance between the partitions may stabilize the partitions and/or maintain isolation between the partitions during flow and thermal expansion and contraction of the fluids. The average distance between partitions may be greater than or equal to about 1 millimeters (mm) , 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, or more. The average distance between partitions may be less than or equal to about 6 mm, 5.5 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, or less. The average distance between partitions may be between about 1 mm and 1.5 mm, 1 mm and 2 mm, 1 mm and 2.5 mm, 1 mm and 3 mm, 1 mm and 3.5 mm, 1 mm and 4 mm, between about 1 mm and 4.5 mm, 1 mm and 5 mm, 1 mm and 5.5 mm, or 1 mm and 6 mm.

The average distance between the partitions (e.g., the length of the segmenting fluid) may be less than, greater than, or equal to the length of the partition (e.g., the length of the aqueous phase) . The ratio of the average distance between partitions (e.g., the length of the segmenting fluid) to the length of the partition (e.g., the length of the aqueous fluid) may be greater than or equal to about 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 6, 8, 10, or more. The ratio of the average distance between partitions (e.g., the length of the segmenting fluid) to the length of the partition (e.g., the length of the aqueous fluid) may be less than or equal to about 10, 8, 6, 4, 3, 2.5, 2, 1.75, 1.5, 1.25, 1, or less.

The plurality of partitions may be formed or generated at the junction between the chambers. The plurality of partitions may be driven and/or pulled away from the junction and away from the chambers. For example, a pump, gravity, capillary action, surface tension, electroosmosis, or centrifugal forces may be used to drive and/or pull the partitions away from the junction. In an example, a fluid flow unit is in fluidic communication with at least one of the first chamber, the second chamber, and the channel. In an example, the fluid flow unit may be in fluid communication with the first and the second chambers. The fluid flow unit may apply positive pressure to the chambers (e.g., via a pump or compressor) . In an example, the fluid flow unit is in fluid communication with the channel. The fluid flow unit may apply a negative pressure to the channel (e.g., via vacuum) . The fluid flow unit may be in fluid communication with both the chambers and the channel and may apply both a positive pressure to the chambers and a negative pressure to the channel. The fluid flow unit may drive the partitions away from the junction. The fluid flow unit may comprise a vacuum (e.g., from a vacuum pump or other suitable vacuum source) or pump. Non-limiting examples of pumps include syringe pumps, peristaltic pumps, pressurized fluid sources, or manual pumps.

The fluid flow unit may drive, or pull, the plurality of generated partitions through the channel. The flow of fluid through the channel may be a continuous flow or may be a non-continuous flow. The flow rate of the fluid (e.g., comprising the partitions and the second liquid phase) may be determined by the length of the target nucleic acid molecule, amplification conditions, and fluid flow network configuration. The fluid (e.g., comprising the partitions and second liquid phase) may have a flow rate of greater than or equal to about 0.1 milliliters per hour (mL/h) , 0.2 mL/h, 0.3 mL/h, 0.4 mL/h, 0.5 mL/h, 0.6 mL/h, 0.7 mL/h, 0.8 mL/h, 0.9 mL/h, 1 mL/h, 1.25 mL/h, 1.5 mL/h, or more. The fluid flow rate may be less than or equal to about 1.5 mL/h, 1.25 mL/h, 1 mL/h, 0.9 mL/h, 0.8 mL/h, 0.7 mL/h, 0.6 mL/h, 0.5 mL/h, 0.4 mL/h, 0.3 mL/h, 0.2 mL/h, 0.1 mL/h, or less. In an example, the fluid flow rate may be about 0.3 mL/h. The fluid flow unit may apply a pressure difference (or pressure drop) between the chambers and the channel. The pressure difference may be greater than or equal to about 0.1 pounds per square inch (psi) , 0.5 psi, 1 psi, 5 psi, 10 psi, 15 psi, 20 psi, 30 psi, 40 psi, 50 psi, 60 psi, 70 psi, 80 psi, 90 psi, 100 psi, 150 psi, 200 psi, 250 psi, 300 psi, 350 psi, 400 psi, 450 psi, 500 psi, 750 psi or more.

The channel may comprise a heating and/or cooling segment and a detection segment. Nucleic acid amplification may be performed in the channel. Nucleic acid amplification may be performed in the heating and/or cooling segment of the channel. The heating and/or cooling segment may comprise a plurality of heating segments and a plurality of cooling segments. The heating and/or cooling segment may be in thermal communication with a heating unit and/or with a cooling unit. The heating and/or cooling units may be thermoelectric elements (e.g., Peltier elements) , resistive heating elements, or induction heating elements. The heating and/or cooling segment may have a plurality of heating and cooling zones. In an example, the channel comprises a single heating zone and the single heating zone is thermal cycled. The heating and cooling zones may comprise one or more temperature sensors. The temperature sensors may be thermocouples. The temperature sensors may be coupled to a system controller (e.g., one or more computer processors) . The system controller may monitor and correct temperature fluctuations in real time. The heating and cooling zones may alternate along the fluid flow path. A partition may remain in a heating or cooling zone for a residence time. A residence time may be the time it takes for a partition to enter a zone, flow through the zone, and leave the zone. For example, a partition flowing along the channel may enter a heating zone and remain in that heating zone for a heating zone residence time. The partition may then flow from the heating zone to a cooling zone.

The partition may remain in the cooling zone for a cooling zone residence time. The residence time of a partition in the heating zone may be greater than, equal to, or less than the residence time in a cooling zone. In an example, the residence time of a partition in a heating zone is less than the residence time of a partition in a cooling zone for each heating and cooling (e.g., denaturation and elongation) cycle. The residence time of a partition in a heating or cooling zone may vary as a function of location within the channel. For example, a partition may have a longer residence time in one heating or cooling zone than in another heating or cooling zone. The channel may be configured so that multiple lengths of the channel are disposed in a single heating or cooling zone. For example, the channel may form a serpentine through one or more heating or cooling zones and a partition may have a different residence time in at a different location within a single heating or cooling zone. The heating zone residence time may be less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. The heating zone residence time may be no more than 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. The cooling zone residence time may be less than or equal to about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second. The cooling zone residence time may be no more than about 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

The heating and cooling zones may be at an elevated temperature as compared to the ambient environment. The ambient environment may be room temperature (e.g., approximately 20 ℃) . The heating zone may have a higher temperature than the cooling zone. The heating zone may be at a denaturation temperature and the cooling zone may be at an elongation temperature. The heating zone may incubate the partitions at a denaturation temperature for a denaturation duration. The cooling zone may incubate the partitions at an elongation temperature for an elongation duration. The plurality of heating zones may be at the same operating temperature or may be at different operating temperatures. The plurality of heating zones may be the same temperature as one or more denaturation temperatures. The plurality of heating zones may have a temperature from about 80℃ to about 110℃. The plurality of heating zones may have a temperature from about 90℃ to about 100℃. The plurality of heating zones may have a temperature from about 90℃ to about 97℃. The plurality of heating zones may have a temperature from about 92℃ to about 95℃. The plurality of heating zones may have a temperature of greater than or equal to about 80°, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, 100℃, or more. The temperature across a given heating zone of the plurality of heating zones may be constant or may vary across the heating zone. The temperature across the heating zone may vary by about 5 %, 10 %, 15 %, 20 %, or more. The temperature across the heating zone may vary by less than about 20 %, 15 %, 10 %, 5 %, or less.

The plurality of cooling zones may be at the same operating temperature or may be at different operating temperatures. The plurality of cooling zones may be the same temperature as one or more elongation temperatures. The plurality of cooling zones may have a temperature from about 30℃ to about 80℃. The plurality of cooling zones may have a temperature from about 35℃ to about 72℃. The plurality of cooling zones may have a temperature from about 45℃ to about 65℃. The plurality of cooling zones may have a temperature from about 35℃ to about 65℃. The plurality of cooling zones may have a temperature from about 40℃ to about 60℃. The plurality of cooling zones may have a temperature from about 50℃ to about 60℃. The plurality of cooling zones may have a temperature of at least about or equal to about 35°, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, or 80℃. The temperature across a given cooling zone of the plurality of cooling zones may be constant or may vary across the heating zone. The temperature across the cooling zone may vary by about 5 %, 10 %, 15 %, 20 %, or more. The temperature across the cooling zone may vary by less than about 20 %, 15 %, 10 %, 5 %, or less.

The area of each heating zone of a plurality of heating zones may be the same or may be different. The area of each cooling zone of a plurality of cooling zones may be the same or may be different. The area of a given heating zone may be smaller than the area of a given cooling zone. The area of a given cooling zone may be smaller than the area of a given heating zone. Each microfluidic chip may have greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more heating zones. Each microfluidic chip may have greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more cooling zones. Each heating or cooling zone may have an area that is greater than or equal to about 0.5 square millimeters (mm ²) , 1 mm ², 2 mm ², 3 mm ², 4 mm ², 5 mm ², 7 mm ², 10 mm ², 15 mm ², 20 mm ², 30 mm ², 40 mm ², 50 mm ², 75 mm ², 100 mm ², 150 mm ², 200 mm ², 300 mm ², 400 mm ², 500 mm ², 750 mm ², 1,000 mm ², 1,500 mm ², 2,000 mm ², 3,000 mm ², 4,000 mm ², 5,000 mm ², 7,500 mm ², 10,000 mm ², 15,000 mm ², or greater. Each heating and cooling zone may be any shape. The heating and cooling zones may be the same shape as one another or different shapes. The heating and cooling zones may be rectangular, circular, annular, square, elliptical, wedge, or trapezoidal in shape.

The channel may comprise a detection segment or a plurality of detection segments. The detection segment may include or be in sensing communication (e.g., optical communication) with one or more sensors for sensing or detection of partitions or contents of the partitions along the detection segment. The detection segment may be downstream of the junction and downstream of the heating and/or cooling segment. Alternatively, or in addition to, the detection segment and the heating and/or cooling segments may be the same segment. For example, the one or more sensors may be in sensing communication with the entire channel and may detect signals in real-time. The detection segment (s) may be in sensing communication with one or more detectors. A detector may be integrated with and a part of the system. The detector may sense signals indicative of the presence or absence of an amplification product. The detector may be an optical or electronic detector. The detector may be an electronic detector and the channel may be in electrically communication with the detector. The electronic detector may detect impedance, conductivity, or charge signals. The electronic detector may detect changes in impedance, conductivity, or charge signals. The detector may be an optical detector and the channel may be in optical communication with the detector. The optical detector may detect fluorescence, absorbance, refractive index, or luminescence signals. The detector may comprise a light source and a detector. The light source may generate a single wavelength (or frequency) of light or multiple wavelengths of light. The light source may excite a single detectable moiety or may excite multiple detectable moieties. The detected signals may be photon emissions from the detectable moieties. The detector may be integrated with the chip or external to the chip. The detector may comprise a charge-coupled device (CCD) camera. The CCD camera may be capable of detecting multiple wavelengths of emitted light. Each wavelength or wavelength range (or frequency or frequency range) of light may be associated with a single detectable signal. A single detectable signal may be indicative of the presence of a single target molecule. A sample may include multiple target molecules, each associated with a different wavelength or wavelength range of light. The detector may detect multiple wavelengths of light and, therefore, detect multiple target molecules during a single detection cycle or over multiple detection cycles. The excitation energy may be provided by a source of excitation energy that is integrated with the chip. In some cases, the excitation energy may be provided by a source of excitation energy that is external to the chip. For example, the excitation energy may be provided by a light-emitting diode or a laser. The signals may be optical signals (e.g., fluorescent signals) , electrochemical signals, and/or electrostatic signals. In some embodiments, on one side of the chip (e.g., above the collection chamber) , an optical image acquisition device (e.g., a CCD camera) and accompanying fluorescence excitation light sources are provided.

Prior to detection, the detector may collect a background signal (e.g., background light) to establish a reference intensity profile. Subsequently, the detector may collect a signal to detect a target. This may include comparing a detected signal against the background signal. In some example, the detector detects light and compares the detected light to a reference profile to identify differences. The differences may be indicative of a presence or absence of a target molecule.

The amplification product may be detected at a sensitivity of at least about 90 %. For example, the amplification product may be detected at a sensitivity of at least about 60 %, 70 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 %or higher. As used herein, sensitivity generally refers to a measure of the proportion of positive signals that are correctly identified as such.

The amplification product may be detected at a specificity of at least about 90%. For example, the amplification product may be detected at a specificity of at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or higher. As used herein, specificity generally refers to a measure of the proportion of negatives signals that are correctly identified as such.

The channel may comprise or be in fluid communication with an additional channel to recycle the partition to the detection segment of the channel. The partitions may be recycled to the channel at an additional junction disposed between the channel and the recycle channel. The additional junction may be upstream of the detection segment of the channel. The partitions may be recycled to the detection segment after the signals from the partitions have been detected at least once. Recycling, or returning, the partitions back to the detector may enable the presence or absence of amplification products to be detected more than once.

The channel may be in fluid communication with a collection chamber. The collection chamber may be downstream of the junction, the heating and/or cooling segment, and/or the detection segments of the channel. The collection chamber may include a detector or may be a portion of the detection segment. The collection chamber may be in thermal communication with one or more heating and/or cooling zones. The collection chamber may collect the plurality of partitions. The collection chamber may be in fluid communication with the detection segment of the channel. The collection chamber may be in communication (e.g., optical or electrical communication) with the detector. The collection chamber may be in fluid communication with the fluid flow unit. The fluid flow unit may direct partitions in the collection chamber to the segment of the channel that is in sensing communication with the detector (e.g., the detection segment) . The collection chamber may be fluidically connected to the channel by an additional channel. The additional channel may be a recycle, or return, channel.

The collection chamber may include a planar array and may be dimensioned to accommodate the plurality of partitions in a single layer. For example, the collection chamber may be dimensioned in a manner to avoid or have little to no stacking of the plurality of partitions. The collection chamber, including the planar array, may be as described in PCT/CN2017/075955, filed March 8, 2017, which is entirely incorporated herein by reference. The collection chamber may be enclosed by two parallel planar surfaces and the average distance between the two parallel planar surfaces may define a height of the collection chamber. The height of the collection chamber may be about or less than about an average diameter of the partitions generated. For example, the height of the collection chamber may be less than or equal to about 2000 μm, 1000 μm, 750 μm, 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15 μm, 10 μm, 5 μm, 1 μm, 0.1 μm, 0.01 μm, or less. The collection chamber (or, when applicable, a planar surface comprised by the collection chamber) may have a diameter of greater than or equal to about 0.01μm, 0.1 μm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or more. In an example, the collection chamber may be configured to have a dimension to accommodate the plurality of partitions in multiple layers.

As an alternative or in addition to, the collection chamber may comprise wells that are dimensioned to hold a single partition (e.g., droplet) of the plurality of partitions. Each of the wells may have a dimension (e.g., width, length, depth) that is less than an average diameter of a given partition of the plurality of partitions. For example, each of the wells may have a dimension that is less than or equal to about 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15 μm, 10 μm, or less. The collection chamber, including the wells, may be as described in PCT/CN2017/075955, filed March 8, 2017, which is entirely incorporated herein by reference.

The partitions may be located in an individually addressable area within the collection chamber. For example, each of the plurality of partitions may be directed to a confined structure or space that is coded, arranged, or arrayed in a way to enable identification of a partition present in the collection chamber. For example, the confined structure or space may be a well and may be dimensioned to accommodate a single partition. The single partition may be stably maintained in the confined structure or space of the collection chamber.

The detection segment may include the collection chamber. The detection segment may be the collection chamber. The detection segment may include one or more detectors. A detector may detect targets within a single or multiple partitions simultaneously. In an example, the detector is an optical detector and the collection chamber is in optical communication with detector. The detector may image the entire collection chamber and all of the partitions within the chamber at once. Alternatively, or in addition to, the detector may image portions or sections of the collection chamber (e.g., the planar array comprising droplets) and a portion of the partitions within the collection chamber at one time. The detector may image portions or section of the collection chamber sequentially.

The chip or a component thereof (e.g., the channels, the chambers, etc. ) may be made with a variety of materials and methods. For example, the chip or a component thereof may be formed from solid materials, in which the channels may be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, physical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, electrodeposition etc. Various fabrication processes (e.g., soft lithography, hot embossing, injection molding, and laser ablation) may be used to produce the chips or components thereof.

In some embodiments, the chip or a component thereof is formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane ( "PDMS" ) , polytetrafluoroethylene ( "PTFE" or

) , etc. For example, a channel (e.g., a microfluidic channel) may be implemented by fabricating the fluidic system separately using PDMS or other soft lithography techniques. Other examples of potentially suitable polymers include, but are not limited to, polyethylene terephthalate (PET) , polyacrylate, polymethacrylate, polycarbonate, polystyrene, polyethylene, polypropylene, polyvinylchloride, cyclic olefin copolymer (COC) , polytetrafluoroethylene, a fluorinated polymer, a silicone such as polydimethylsiloxane, polyvinylidene chloride, bis-benzocyclobutene (BCB) , a polyimide, a fluorinated derivative of a polyimide, etc. . Combinations, copolymers, or blends involving polymers including those described above are also envisioned.

In some embodiments, the chip or a component thereof is made from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, extruding, etc. ) . The hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with a fluidic network. In one embodiment, the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a "prepolymer" ) . Suitable polymeric liquids include, for example, thermoplastic polymers, thermoset polymers, waxes, metals, or mixtures or composites thereof heated above their melting point. In some embodiments, a suitable polymeric liquid includes a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials can be solidified from, for example, a melt state or by solvent evaporation. A variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material. Non-limiting examples of such polymers include polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three-membered cyclic ether group commonly referred to as an epoxy group, 1, 2-epoxide, or oxirane. For example, diglycidyl ethers of bisphenol A may be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones. Another example is Novolac polymers. Non-limiting examples of silicone elastomers suitable for use herein include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc. In some embodiments, silicone polymers (e.g., the silicone elastomer polydimethylsiloxane) are used. Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, MI, e.g., Sylgard 182, Sylgard 184, and Sylgard 186.

One advantage of forming structures such as microfluidic structures or channels from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non-polymeric materials. Thus, structures can be fabricated and then oxidized and essentially irreversibly sealed to other silicone polymer surfaces or to the surfaces of other substrates reactive with the oxidized silicone polymer surfaces, without the use of separate adhesives or other sealing approaches. In most cases, sealing can be completed simply by contacting an oxidized silicone surface to another surface without the application of auxiliary pressure to form the seal. That is, the pre-oxidized silicone surface acts as a contact adhesive against suitable mating surfaces. Specifically, in addition to being irreversibly sealable to itself, oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma) .

In some embodiments, the chip or a component thereof is produced using more than one layer or substrate, e.g., more than one layer of PDMS. For instance, chips having channels with multiple heights and/or chips having interfaces positioned such as described herein may be produced using more than one layer or substrate, which may then be assembled or bonded together, e.g., using plasma bonding, to produce the chip. For example, a chip of the present disclosure may be molded from masters comprising two or more layers of photoresists, e.g., where two PDMS molds are then bonded together by activating the PDMS surfaces using O ₂ plasma or other suitable techniques. For example, the masters from which the PDMS chip is cast may contain one or more layers of photoresist, e.g., to form a 3D chip. In some embodiments, one or more of the layers has one or more mating protrusions and/or indentations which are aligned to properly align the layers, e.g., in a lock-and-key fashion. For example, a first layer may have a protrusion (having any suitable shape) and a second layer may have a corresponding indentation which can receive the protrusion, thereby causing the two layers to become properly aligned with respect to each other.

The chip and/or one or more of the chambers may comprise a filter or an enrichment device to remove some substances from the nucleic acid sample, and/or to enrich other components in the nucleic acid sample. Non-limiting examples of the filter or enrichment device include filtration membranes, e.g., nitrocellulose, cellulose acetate, polycarbonate, polypropylene and polyvinylidene fluoride microporous membranes, and ultrafiltration membranes (e.g., those made from polysulfone, polyvinylidene fluoride, cellulose etc. ) . The aqueous and/or non-aqueous fluid flowed from one or more of the chambers may be driven through one or more of the filters to enter the channel. The filtrates may be collected in a separate chamber.

In an example, the channel is a continuous material with a circular cross-section. The channel may be formed by micromachining, injection molding, extruding, or any other method resulting in a smooth, low friction interior surface. The channel may comprise a hydrophobic material. Alternatively, or in addition to, the interior surface of the channel may comprise hierarchical microstructures or nanostructures that generate a superhydrophobic surface. The hydrophobic surface may facilitate low friction flow of the partition along the length of the channel. The hydrophobic surface may have a contact angle with water that is greater than or equal to about 100°, 105°, 110°, 115°, 120°, 125°, 130°, 140°, or greater. The hydrophobic surface may have a contact angle with water that is between about 100° and 105°, 100° and 110°, 100° and 115°, 100° and 120°, 100° and 125°, 100° and 130°, 100° and 140°, 115° and 120°, 115° and 125°, 115° and 130°, or 115° and 140°.

FIG. 1A illustrates an example circular microfluidic chip with a spiraled microfluidic channel. The microfluidic chip 101 may be substantially circular. The diameter of the microfluidic chip may be less than or equal to about 500 mm, 400 mm, 300 mm, 200 mm, 100 mm, 50 mm, or less. The diameter of the microfluidic chip may be greater than or equal to about 50 mm, 100 mm, 200 mm, 300 mm, 400 mm, 500 mm, or greater. In an example, the diameter of the microfluidic chip is about 120 mm. The channel 102 may be in a spiral configuration. The spiral configuration may have a junction 103 and branch at the inner portion of the spiral.

FIG. 1B illustrates an example circular microfluidic chip with a spiraled microfluidic channel and multiple heating and/or cooling zones. The microfluidic chip 101 may be substantially circular and comprise a spiral channel. The spiral channel may form an annular shape. One end of the channel may branch and be in fluid communication with a first chamber 104 and a second chamber 105. The first chamber and the second chamber may comprise a first liquid phase and a second liquid phase. The first liquid phase may be aqueous and comprise a target nucleic acid provided as a raw or processed biological sample. The first liquid phase may additionally comprise a reaction mixture containing reagents for a nucleic acid amplification reaction. The second liquid phase may be an oil phase and may comprise a non-wetting agent. The microfluidic chip may be integrated with a fluid flow unit. The fluid flow unit may drive the first and second liquid phases from the first and second chambers to the junction to generate partitions. The partitions may comprise the first liquid phase and may be surrounded by the second liquid phase. The partitions may flow from the junction through the channel.

The channel may comprise a heating and/or cooling segment 106, a detection segment 109, and a waste outlet 110 downstream of the junction. The heating and/or cooling segment 106 may comprise heating zones 107 and cooling zones 108. The heating zone 107 may be higher in temperature than the cooling zone 108. The heating zones 107 and cooling zones 108 may alternate along the annular shape defined by the channel. For example, a partition flowing along the channel may flow through a heating zone 107 followed by a cooling zone 108 followed by a subsequent heating zone 107. Both the heating and cooling zones may be higher in temperature than the ambient temperature. In an example, the heating zones 107 have a temperature of about 95 ℃ and the cooling zones 108 have a temperature of about 55 ℃. The heating zone 107 may be at temperature for nucleic acid denaturation. The cooling zone 108 may be at a temperature for nucleic acid elongation. The heating zones 107 and the cooling zones 108 may be in thermal communication with heating units and cooling units, respectively. The partitions may flow from the junction through the heating 107 and cooling 108 zones. Flowing the partition through the alternating thermal zones may thermal cycle the partitions and subject the partitions to multiple cycles (e.g., denaturation and elongation) of a primer extension reaction. The partitions may be subjected to a flow rate of about 0.3 mL/h and about 50 primer extension reaction cycles.

Partitions that have passed through the heating and/or cooling segment 106 of the channel may subsequently flow through the detection segment 109 of the channel. The partitions containing a target nucleic acid molecule may contain amplification products. Each partition of the plurality of partitions may further comprise detectable moieties. The detectable moieties may produce a detectable signal in partitions that contain amplification products. The detectable signals may be detected by a detector in communication with the detection segment of the channel. The detector may be an optical detector. The detector may detect partitions that pass by a single point in the detection segment or may detect partitions at multiple points of the detection segment. The detector may further comprise a light source. The detectable moiety may be a fluorophore. The light source may be of a suitable wavelength to excite the fluorophore. The excited fluorophore may emit a wavelength of light. The detector may detect the wavelength of emitted light. The detector may have one or more light sources that have wavelengths suitable to excite one or more fluorophores. The detector may be able to detect one or more wavelengths of emitted light. After detection of the presence or absence of amplification products, the partitions may flow to a waste 110 area. Partitions in the waste 110 area may be disposed of. Alternatively, or in addition, the partitions may flow to a collection chamber. Partitions in the collection chamber may be recycled back to the detection segment 109 through an additional channel. The partitions may be flows through detection segment 109 one or more additional times to detect the presence or absence of the amplification products.

FIG. 2 illustrates an example circular microfluidic chip 201 with a serpentine microfluidic channel 202 with a single heating 204 and single cooling 205 zone. The microfluidic channel 202 may be branched at one end and have a junction 203 at the branch point. The microfluidic channel 202 may serpentine in a radial direction. The microfluidic channel 202 may form and annular shape. The microfluidic chip 201 may comprise one heating zone 204 and one cooling zone 205. The heating zone 204 may be in a circular or annular shape. The heating zone 204 may be disposed towards the center of the microfluidic chip 201. The cooling zone 205 may have an annular shape with an inner circumference that is less than the outer circumference. The cooling zone 205 may be disposed adjacent to the outer edge of the microfluidic chip 201. The microfluidic chip 201 may further comprise a first and a second chamber. The first and second chambers may be in fluid communication with the junction 203. The microfluidic chip 201 may additionally comprise a segment of the channel 202 that is in single communication with a detector.

FIG. 3 illustrates an example rectangular microfluidic chip with a serpentine microfluidic channel, multiple heating zones, and a single cooling zone. The microfluidic chip 301 may have a rectangular or square shape. The microfluidic chip 301 may have one dimension (e.g., length) that is greater than another dimension (e.g., width) . One dimension (e.g., length) may be greater than or equal to about 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 75 mm, 100 mm, 125 mm, 150 mm, 200 mm, 250 mm, 300 mm, 400 mm, 500 mm, or more. One dimension (e.g., length) may be less than or equal to about 500 mm, 400 mm, 300 mm, 250 mm, 200 mm, 150 mm, 125 mm, 100 mm, 75 mm, 50 mm, 40 mm, 30 mm, 20 mm, 10 mm, or less. Another dimension (e.g., width) may be greater than or equal to about 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 75 mm, 100 mm, 125 mm, 150 mm, 200 mm, 250 mm, 300 mm, 400 mm, 500 mm, or more. Another dimension (e.g., width) may be less than or equal to about 500 mm, 400 mm, 300 mm, 250 mm, 200 mm, 150 mm, 125 mm, 100 mm, 75 mm, 50 mm, 40 mm, 30 mm, 20 mm, 10 mm, 5 mm, or less. In an example, the microfluidic chip 301 has a length of 200 mm and a width of 140 mm. The channel 302 may be in a serpentine configuration. The channel 302 may serpentine in the direction of either one dimension (e.g., the length) or in the direction of another dimension (e.g., the width) . In an example, the channel 302 may serpentine across the width of the microfluidic chip 301. The microfluidic chip 301 may comprise one or more heating zones 303 and one or more cooling zones 304. In an example, the microfluidic chip has two heating zones 303 and a single cooling zone 304. The cooling zone 304 may be disposed between the two heating zones 303. As a partition progresses from one end of the channel to the other, the partition may enter a heating zone 303 to denature any nucleic acid molecules present in the partition. The partition may then progress across a cooling zone 304 and an elongation reaction may be performed if a target nucleic acid is present in the partition. The partition may then enter the other heating zone 303. The heating 303 and cooling 304 zones may be disposed to have a long axis perpendicular to the direction of fluid flow (e.g., partition movement) . The heating 303 and cooling 304 zones may be disposed to have a long axis that is parallel to the long axis of the microfluidic chip 301. In an example, the heating 303 and cooling 304 zones may be disposed with a long axis perpendicular to the long axis of the microfluidic chip 301 and the direction of fluid flow may be parallel to a long axis of heating 303 and cooling 304 zones.

FIG. 4 illustrates an example rectangular microfluidic chip with a patterned microfluidic channel and multiple heating and cooling zones. The microfluidic chip 401 may have a rectangular or square shape. The microfluidic chip 401 may have one dimension (e.g., length) that is greater than another dimension (e.g., width) . One dimension (e.g., length) may be greater than or equal to about 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 75 mm, 100 mm, 125 mm, 150 mm, 200 mm, 250 mm, 300 mm, 400 mm, 500 mm, or more. One dimension (e.g., length) may be less than about 500 mm, 400 mm, 300 mm, 250 mm, 200 mm, 150 mm, 125 mm, 100 mm, 75 mm, 50 mm, 40 mm, 30 mm, 20 mm, 10 mm, or less. Another dimension (e.g., width) may be greater than or equal to about 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 75 mm, 100 mm, 125 mm, 150 mm, 200 mm, 250 mm, 300 mm, 400 mm, 500 mm, or more. Another dimension (e.g., width) may be less than or equal to about 500 mm, 400 mm, 300 mm, 250 mm, 200 mm, 150 mm, 125 mm, 100 mm, 75 mm, 50 mm, 40 mm, 30 mm, 20 mm, 10 mm, 5 mm, or less. In an example, the microfluidic chip 401 has a length of 300 mm and a width of 70 mm. The channel 402 may be in a patterned configuration. The patterned configuration may be patterned to increase or decrease the residence time in a heating or cooling zone. For example, the channel 402 may be patterned so that the residence time in a heating channel is less than the residence time in the cooling channel. The microfluidic chip 401 may comprise one or more heating zones 403 and one or more cooling zones 404. In an example, the microfluidic chip has two heating zones 403 and two cooling zones 404. The heating 403 and cooling 404 zones may alternate across a short dimension (e.g., width) of the microfluidic chip 401. The heating 403 and cooling 404 zones may be disposed to have a long axis that is parallel to the long axis of the microfluidic chip 401. The heating 403 and cooling 404 zones may be disposed with a long axis perpendicular to the long axis of the microfluidic chip 301.

Systems for analyzing nucleic acid samples

In another aspect, the present disclosure provides systems for detecting the presence or absence of a target nucleic acid molecule from a raw biological sample. The system may comprise a fluidic network, a fluid flow unit, a detector, and one or more computer processors. The fluid network may comprise a first chamber and a second chamber that are in fluid communication at a junction. The first chamber may contain a first liquid phase. The first liquid phase may contain a raw biological sample. The second chamber may contain a second liquid phase. The second liquid phase may be immiscible with the first liquid phase. The fluidic network may further comprise a channel in fluid communication with the junction. The channel may be configured to flow a plurality of partitions. The partitions may comprise the first liquid phase segmented by the second liquid phase. The fluid flow unit may be in fluid communication with at least one of the first chamber, the second chamber, and the channel. The detector may be configured to detect a signal indicative of a presence or absence of an amplification product generated from the target nucleic acid molecule in a given partition of the plurality of partitions.

The one or more computer processors may be operatively coupled to the fluid flow unit and the detectors. The one or more computer processors may be individually or collectively programed to direct the fluid flow unit to subject the first liquid phase and the second liquid phase to flow from the first chamber to the second chamber, respectively, to the junction. The flow of liquid from the first chamber and the second chamber may generate a plurality of partitions. The plurality of partitions may be directed to flow along the channel. A given partition of the plurality of partitions may contain a reaction mixture comprising reagents for performing a nucleic acid amplification reaction on the target nucleic acid molecule. The nucleic acid amplification reaction may generate and yield amplification products of or derived from the target nucleic acid molecule. The one or more computer processors may further direct the flow unit to subject the plurality of partitions to flow along the channel. Subsequent to generation of the partitions, the reaction mixture in a given partition may be subjected to conditions sufficient to perform a plurality of series of primer extension reactions on the target nucleic acid molecule to yield amplification products of the target nucleic acid molecule. An individual series of primer extension reactions may differ from at least one other individual series of the plurality of series of primer extension reactions with respect to the denaturing condition, elongation condition, or both the denaturing and elongation condition. The one or more computer processors may direct the detector to detect a signal indicative of a presence or absence of the amplification product from a given partition. The presence or absence of an amplification product in a given partition may thereby detect the presence or absence of the one or more target nucleic acid molecules in the raw biological sample.

In another aspect, the present disclosure provides systems for detecting the presence or absence of a target nucleic acid molecule from a biological sample. The system may comprise a fluid flow network, a fluid flow unit, a detector, a heating and/or cooling unit, and one or more computer processors. The fluid flow unit may comprise a first and second chamber and a channel. The first chamber and the second chamber may be in fluid communication at a junction. The first chamber may comprise a first liquid phase containing biological sample and the second chamber may comprise a second liquid phase immiscible with the first liquid phase. The channel may be in fluid communication with the junction. The channel may be configured to flow plurality of partitions generated upon bringing the first liquid phase in contact with the second liquid phase at the junction. The channel may comprise at least a first segment. The first segment may be disposed in a plurality of thermal zones. The plurality of thermal zones may subject the plurality of partitions to heating and/or cooling. The channel may further comprise a detection segment downstream of the first segment. The detection segment may be in sensing communication with the detector. The fluid flow unit may be in fluid communication with at least one of the first chamber, the second chamber, and the channel. The detector may be configured to detect a signal indicative of a presence or absence of an amplification product generated from the target nucleic acid molecule in a given partition of the plurality of partitions. A heating and/or cooling unit may be in thermal communication with the plurality of thermal zones.

The one or more computer processors may be operatively coupled to the fluid flow unit, the heating and/or cooling unit, and the detector. The one or more computer processors may be individually or collectively programed to direct the fluid flow unit, direct the heating and/or cooling unit, and direct the detector. The one or more computer processors may direct the fluid flow unit to subject the first liquid phase and the second liquid phase to flow from the first chamber and the second chamber, respectively, to the junction to generate the plurality of partitions. Upon generation, the partitions may be directed to flow along the channel. A given partition of the plurality of partitions may comprise a reaction mixture that contains the reagents to perform a nucleic acid amplification reaction on the target nucleic acid molecule. The nucleic acid amplification reaction may generate amplification products of or derived from the target nucleic acid molecule. The one or more computer processors may direct the heating and/or cooling unit to subject the reaction mixture in a given partition flowing through the first segment to conditions sufficient to perform the nucleic acid amplification reaction of the target nucleic acid molecule. The nucleic acid amplification reaction may yield amplification products of the target nucleic acid molecule. The one or more computer processors may direct the detector to detect signals indicative of the presence or absence of the amplification products form a given partition. The presence or absence of amplification products may be detected when the given partition is flowing through the detection segment of the channel. Detection of the presence or absence of amplification products may thereby detect the presence or absence of the one or more target molecules in the biological sample.

The system may comprise a chip (e.g., a microfluidic chip) . The chip may comprise the fluid flow network. The fluid flow network may be a portion of a microfluidic device or a microfluidic chip. The one or more computer processors may be individually or collectively programmed to subject the nucleic acid sample or portion thereof in each of the plurality of partitions to the nucleic acid amplification reaction on the chip. The amplification process is as described elsewhere in the present disclosure.

The system may comprise one or more actuators, chips, detectors, heating units, cooling units, fluid flow units, and computer processors. The one or more actuators may be integrated with the system and may move the chip from one location to another location, may move the detector from one location to another location, and/or may move the heating and cooling units. The detector may be integrated with the system or integrated with the chip. The heating and cooling units may be integrated with the system or integrated with the chip. The chip may comprise a thermally conductive material that may contact the heating or cooling units to heat or cool portions of the chip.

The first liquid phase may comprise an aqueous fluid. The aqueous fluid may comprise a nucleic acid sample and reagents to perform a nucleic acid amplification reaction. The biological sample and reagents to perform a nucleic acid amplification reaction are as described elsewhere in the present disclosure.

The second liquid phase in the second chamber may comprise hydrophobic liquids. Non-limiting examples of the hydrophobic liquids include hydrocarbon solvents (e.g., organic solvents and oils. Oils may include hydrocarbon oils, silicon oils, and fluorocarbon oils. The oil may be a fluorinated oil, such as HFE 7100, HFE 7500, FC-40, FC-43, FC-70, FC-3208, or a combination thereof. The oil may be a mineral oil, such as liquid paraffin, light mineral oil, white oil, refined mineral oil, cycloalkane oil, aromatic oil, or a combination thereof. The oil may also be any oil that is useful for making partitions. Examples of oils and surfactants that may be employed for use are provided in U.S. Patent No. 9,012,390, which is entirely incorporated herein by reference.

The second liquid phase may comprise a non-wetting agent. The non-wetting agent may be a surfactant, detergent, or polymer. The non-wetting agent may reduce the interaction between the partitions and the channel wall. The non-wetting agent may reduce the friction and drag of the partition (e.g., the first liquid phase) moving along the wall. Additionally, or alternatively, the non-wetting agent may reduce the binding of reagent components (e.g., proteins) to the channel wall. The non-wetting agent may comprise a hydrophobic tail and a hydrophilic head group, a polymer-based tail and a hydrophilic head group, a polymer-based tail and a polymer-based head group, a fluorinated tail and a hydrophilic head group, or a fluorinated polymer-based tail and a hydrophilic polymer-based head group. In some embodiments, the non-wetting agent is a di-block copolymer or tri-block copolymer type. For example, the non-wetting agent may be a block copolymer, such as a tri-block copolymer consisting of two perfluoropolyether blocks and one poly (ethylene) glycol block. In an example, the non-wetting agent is selected from the group consisting of PFPE-PEG-PFPE (perfluoropolyether-polyethylene glycol-perfluoropolyether) , tri-block copolymer EA-non-wetting agent (RainDance Technologies) and DMP (dimorpholino phosphate) -non-wetting agent (Baret, Kleinschmidt, et al., 2009) . The length of PEG in a polymeric species, including a polymeric non-wetting agent, can have any suitable length and may vary between different polymeric species that can be used. In an example, the non-wetting agent is a plant derived surfactant such as sodium lauryl sulfate, ammonium laureth sulfate, disodium lauryl sulfosuccinate, decyl glucoside, glyceryl cocoate, sodium cocoyl isethionate, or any combination thereof. The non-wetting agent may be present in the second liquid phase with a concentration of 0.0001%to 5% (w/w) , e.g., 0.001%to 4% (w/w) , 0.01%to 3% (w/w) , 0.1%to 2% (w/w) , 0.1%to 1% (w/w) . In an example, the non-wetting agent in the second liquid phase has a concentration of at least about 0.1% (w/w) , 0.2% (w/w) , 0.3% (w/w) , 0.4% (w/w) , 0.5% (w/w) , 0.6% (w/w) , 0.7% (w/w) , 0.8% (w/w) , 0.9% (w/w) , 1.0% (w/w) , 1.2% (w/w) , 1.4% (w/w) , 1.6% (w/w) , 1.8% (w/w) , 2.0% (w/w) , 2.5% (w/w) , 3.0% (w/w) , 3.5% (w/w) , 4.0% (w/w) , 4.5% (w/w) , 5.0% (w/w) , 7.0% (w/w) , 10.0% (w/w) , 15.0% (w/w) , 20.0% (w/w) or more. In an example, the non-wetting agent in the second liquid phase has a concentration of at most about 0.1% (w/w) , 0.2% (w/w) , 0.3% (w/w) , 0.4% (w/w) , 0.5% (w/w) , 0.6% (w/w) , 0.7% (w/w) , 0.8% (w/w) , 0.9% (w/w) , 1.0% (w/w) , 1.2% (w/w) , 1.4% (w/w) , 1.6% (w/w) , 1.8% (w/w) , 2.0% (w/w) , 2.5% (w/w) , 3.0% (w/w) , 3.5% (w/w) , 4.0% (w/w) , 4.5% (w/w) , 5.0% (w/w) , 7.0% (w/w) , 10.0% (w/w) , 15.0% (w/w) , 20.0% (w/w) or less.

The system may further comprise a third chamber in fluid communication with the junction. The third chamber may comprise a third liquid phase. The third liquid phase may comprise the reagents for the nucleic acid amplification reaction. The third liquid phase may be an aqueous phase. The third liquid phase may be immiscible with the second liquid phase.

The channel may include a branched segment, a heating and/or cooling segment, a detection segment, and a recycle segment. The channel may be straight, substantially straight, or may comprise one or more curves or bends. In an example, the branched segment is configured to minimize curves or bends. The channel may comprise any configuration, including a circular configuration, spiral configuration, serpentine configuration, or any combination thereof. In an example, the channel may comprise both a spiral and one or more serpentine configurations. In an example, the channel comprises a substantially circular configuration. The channel may be as described elsewhere in the present disclosure.

The one or more computer processors may be collectively or individually programmed to form a plurality of partitions by directing the fluid flow unit. A plurality of partitions may be formed or generated at the junction between the first chamber and second chamber. In an example, a plurality of partitions may be formed at the junction between a first chamber, second chamber, and third chamber. The partitions may be generated as a water-in-oil emulsion. Each partition of the plurality of partitions may have an aspect ratio. The aspect ratio may be the ratio of the largest dimension of the partition to the smallest dimension of the partition. The aspect ratio of the partition may be modulated by the volume of the aqueous phase in relation to the channel size, external forces on the partition (e.g., fluid flow rate) , and the relative fluid properties of the aqueous and oil phases (e.g., density and viscosity) . The aspect ratio of a given partition of the plurality of partitions may be greater than or equal to about 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, or more. The aspect ratio of a given partition of the plurality of partitions may be less than or equal to about 2.5, 2.25, 2, 1.75, 1.5, 1.25, 1, or less.

The one or more computer processors may be programmed, collectively or individually, to direct the fluid flow unit to drive, or pull, the plurality of generated partitions through the channel. The fluid flow may be continuous flow or non-continuous flow. The flow rate of the fluid (e.g., comprising the partitions and the second liquid phase) may be determined by the length of the target nucleic acid molecule, amplification conditions, and fluid flow network configuration. The fluid (e.g., comprising the partitions and second liquid phase) may have a flow rate of greater than or equal to about 0.1 milliliters per hour (mL/h) , 0.2 mL/h, 0.3 mL/h, 0.4 mL/h, 0.5 mL/h, 0.6 mL/h, 0.7 mL/h, 0.8 mL/h, 0.9 mL/h, 1 mL/h, 1.25 mL/h, 1.5 mL/h, or more. The fluid flow rate may be less than or equal to about 1.5 mL/h, 1.25 mL/h, 1 mL/h, 0.9 mL/h, 0.8 mL/h, 0.7 mL/h, 0.6 mL/h, 0.5 mL/h, 0.4 mL/h, 0.3 mL/h, 0.2 mL/h, 0.1 mL/h, or less. In an example, the fluid flow rate may be about 0.3 mL/h. The fluid flow unit may apply a pressure difference between the chambers and the channel. The pressure difference may be greater than or equal to about 0.1 psi, 0.5 psi, 1 psi, 5 psi, 10 psi, 15 psi, 20 psi, 30 psi, 40 psi, 50 psi, 60 psi, 70 psi, 80 psi, 90 psi, 100 psi, 150 psi, 200 psi, 250 psi, 300 psi, 350 psi, 400 psi, 450 psi, 500 psi, 750 psi or more.

The one or more computer processors may be individually or collectively programmed to subject each of the plurality of partitions to thermal cycling to subject the nucleic acid sample or portion thereof in each of the plurality of partitions to the nucleic acid amplification reaction. The thermal cycling may comprise cycling a temperature of each of the plurality of partitions between a first temperature and a second temperature that is greater than the first temperature. In some cases, the thermal cycling may comprise cycling a temperature of each of the plurality of partitions between more than two different temperatures. Thermal cycling may be performed by the computer processor directing the partitions down the channel.

The channel may comprise a heating and/or cooling segment and a detection segment. Nucleic acid amplification may be performed in the channel. Nucleic acid amplification may be performed in the heating and/or cooling segment of the channel. The heating and/or cooling segment may comprise a plurality of heating segments and a plurality of cooling segments. The heating and/or cooling segment may be in thermal communication with a heating unit and/or with a cooling unit. The heating and/or cooling units may be thermoelectric elements (e.g., Peltier elements) , resistive heating elements, or induction heating elements. The thermal units (e.g., heating or cooling units) may be integrated with the system or integrated with the chip. The heating and/or cooling segment may have a plurality of heating and cooling zones. In an example, the channel comprises a single heating and cooling zone that thermal cycles the partitions within the channel. The heating and cooling zones may comprise one or more temperature sensors. The temperature sensors may be thermocouples. The temperature sensors may be coupled to a system controller (e.g., one or more computer processors) . The system controller may monitor and correct temperature fluctuations in real time. The heating and cooling zones may alternate along the fluid flow path.

The one or more computer processors may be individually or collectively programmed to direct or control the temperature of the heating and cooling zones. The heating and cooling zones may be at an elevated temperature as compared to the ambient environment. The ambient environment may be room temperature (e.g., approximately 20 ℃) . The heating zone may have a higher temperature than the cooling zone.

The one or more computer processors may be individually or collectively programmed to control the heating zone at a denaturation temperature and the cooling zone at an elongation temperature. The heating zone may incubate the partitions at a denaturation temperature for a denaturation duration. The cooling zone may incubate the partitions at an elongation temperature for an elongation duration. The one or more computer processors may be programmed so that the plurality of heating zones is at the same operating temperature or are at different operating temperatures. The plurality of heating zones may be the same temperature as one or more denaturation temperatures. The heating zones may have a temperature of any described herein.

The plurality of cooling zones may be at the same operating temperature or may be at different operating temperatures. The plurality of cooling zones may be the same temperature as one or more elongation temperatures. The cooling zones may have a temperature of any described herein.

The area of each heating zone of a plurality of heating zones may be the same or may be different. The area of each cooling zone of a plurality of cooling zones may be the same or may be different. The area of a given heating zone may be smaller than the area of a given cooling zone. The area of a given cooling zone may be smaller than the area of a given heating zone. Each microfluidic chip may have greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more heating zones. Each microfluidic chip may have greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more cooling zones. The heating and cooling zones may be shaped, sized, and positioned on the microfluidic chip as described elsewhere in the present disclosure.

The channel may comprise a detection segment. The detection segment may be downstream of the junction and downstream of the heating and/or cooling segment. Alternatively, or in addition to, the detection segment and the heating and/or cooling segments may be the same segment. For example, the detection segment may be in sensing communication with the entire channel and may detect signals in real-time. The detection segment may be in sensing communication with a detector. The detector may be integrated with and a part of the system. The detector may sense signals indicative of the presence or absence of an amplification product. The detector may be an optical detector and the channel may be in optical communication with the detector. The detector may comprise a light source and a detector. The light source may generate a single wavelength of light or multiple wavelengths of light. The light source may excite a single detectable moiety or may excite multiple detectable moieties. The detected signals may be photon emissions from the detectable moieties. The signals may be integrated with the chip or external to the chip. The detector may comprise a charge-coupled device (CCD) camera. The CCD camera may be capable of detecting multiple wavelengths of emitted light. The excitation energy may be provided by a source of excitation energy that is integrated with the chip. In some cases, the excitation energy may be provided by a source of excitation energy that is external to the chip. For example, the excitation energy may be provided by a light-emitting diode or a laser. The signals may be optical signals (e.g., fluorescent signals) , electrochemical signals, and/or electrostatic signals. In some embodiments, on one side of the chip (e.g., above the collection chamber) , an optical image acquisition device (e.g., a CCD camera) and accompanying fluorescence excitation light sources are provided.

The one or more computer processors may be individually or collectively programmed to direct excitation energy to the plurality of partitions and detect the signals as emissions from the plurality of partitions. The signals may be detected using a detector that is integrated with the chip. In some cases, the signals may be detected using a detector that is external to the chip (e.g., CCD camera) .

The excitation energy may be provided by a source of excitation energy that is integrated with the chip. In some cases, the excitation energy may be provided by a source of excitation energy that is external to the chip. For example, the excitation energy may be provided by a light-emitting diode or a laser. The signals may be optical signals, fluorescent signals and/or electrostatic signals.

The one or more computer processors may be individually or collectively programmed to simultaneously detect the signals while the plurality of partitions is flowing at a flow rate less than about 5ml/h through the detection segment. The plurality of partitions may be detected while flowing at a flow rate of less than about 4 ml/h, less than about 3 ml/h, less than about 2 ml/h, less than about 1 ml/h, less than about 0.5 ml/h, less than about 0.1 ml/h, or less through the detection segment. The the one or more computer processors may be individually or collectively programmed to simultaneously detect the signals while the plurality of partitions is substantially stationary/not moving.

The amplification product may be detected at a sensitivity of at least about 90%. For example, the amplification product may be detected at a sensitivity of at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or higher. As used herein, sensitivity generally refers to a measure of the proportion of positive signals that are correctly identified as such.

The channel may comprise an additional channel to recycle the partition to the detection segment of the channel. The partitions may be incorporated into the channel at an additional junction. The additional junction may be upstream of the detection segment of the channel. The partitions may be incorporated to the detection segment after the signals from the partitions have been detected at least once. The one or more computer processors may be programmed to recycle, or return, the partitions to the detector after a first detection cycle has been completed.

The channel may be in fluid communication with a collection chamber. The collection chamber may be downstream of the junction, the heating and/or cooling segment, and/or the detection segments of the channel. The collection chamber may collect the plurality of partitions. The collection chamber may be in fluid communication with the detection segment of the channel. The collection chamber may be in fluid communication with the fluid flow unit. The fluid flow unit may direct partitions in the collection chamber the segment of the channel that is in sensing communication with the detector (e.g., the detection segment) . The collection chamber may be fluidically connected to the channel by an additional channel. The additional channel may be a recycle, or return, channel.

The channel may be in fluid communication with a collection chamber. The collection chamber may be downstream of the junction, the heating and/or cooling segment, and/or the detection segments of the channel. The collection chamber may collect the plurality of partitions. The collection chamber may be in fluid communication with the detection segment of the channel. The collection chamber may be in fluid communication with the fluid flow unit. The fluid flow unit may direct partitions in the collection chamber the segment of the channel that is in sensing communication with the detector (e.g., the detection segment) . The collection chamber may be fluidically connected to the channel by an additional channel. The additional channel may be a recycle, or return, channel. The additional channel and collection chamber may be as described elsewhere in the present disclosure.

FIG. 5 illustrates an example system 501 comprising a microfluidic chip comprising a fluid flow network 502, one or more heating and/or cooling units 507, and a detector 508. The fluid flow network 502 may comprise multiple chambers and at least one channel 506. A first chamber 504 and a second chamber 505 may be fluidically connected to a junction 503. The first 504 and second 505 chambers may be upstream of the junction 503. The junction 503 may be fluidically connected to a channel 506. The first chamber 504 may comprise a first liquid phase. The first liquid phase may be an aqueous fluid. The aqueous fluid may comprise a biological sample. The biological sample may contain one or more target nucleic acid molecules. The aqueous fluid may further comprise a reaction mixture. The reaction mixture may comprise reagents to perform a nucleic acid amplification reaction. The second chamber 505 may comprise a second liquid phase. The second liquid phase may comprise a non-aqueous fluid, such as an oil. The non-aqueous fluid may further comprise one or more non-wetting agents. The second liquid phase may be immiscible with the first liquid phase. The fluid flow network may be in fluid communication with a fluid flow unit. The fluid flow unit may be in fluid communication with the first 504 and second 505 chambers, the channel 506, or both the first and second chamber s and the channel 506. The fluid flow unit may apply a positive pressure to the first 504 and the second 505 chambers to drive the first and second liquid phase downstream to the junction 503. Alternatively, or in addition to, the fluid flow unit may apply a negative pressure to the channel 506 to pull the first liquid phase and the second liquid phase from the first 504 and second 505 chambers to the junction 503.

The first liquid phase and the second liquid phase may contact one another at the junction 503. Contacting the first and second liquid phases may form a partition 507. The partitions 507 may contain a portion of the biological sample and the reaction mixture. The partitions 507 may serve as an individual reaction vessel to allow for an amplification reaction independent of the amplification reactions that may or may not occur in the other partitions 507. The fluid flow unit may drive, pull, or both drive and pull the partitions 507 through the channel 506. The channel 506 may comprise one or more segments. The channel 506 may comprise a heating and/or cooling segment and a detection segment. Both the heating and/or cooling segments and the detection segment may be downstream from the junction 503. The heating and/or cooling segment may comprise a plurality of heating and/or cooling units 508. The heating and/or cooling units 508 may be in thermal communication with the channel 506. The heating and/or cooling units 508 may have different operating temperatures. For example, a heating unit may have a higher operating temperature than a cooling unit. The heating unit may have an operating temperature suitable to denature nucleic acid molecules within the partitions 507. The cooling unit may have an operating temperature suitable to conduct an elongation reaction within the partitions 507. The partitions 508 may flow through segments of the channel 506 that are in thermal communication with the heating and/or cooling units 508 and, therefore, may be in thermal communication with the heating and/or cooling units 508. The partitions may alternate thermal communication with the heating units and the cooling units. For example, the partitions 507 may be driven, or pulled, through the channel and may contact a heating zone, followed by a cooling zone, followed by another heating zone. This sequence of contact with the heating and cooling zones may thermal cycle the partitions 507 and provide suitable conditions for a nucleic acid amplification reaction.

The channel 506 may be in signal communication with a detector 509. The channel 506 may have a plurality of detector segments in signal communication with one or more detectors 509. The detector segment (s) may be downstream of the heating and/or cooling segment. The detector (s) may be an optical detector. The optical detector may detect signals indicative of the presence or absence of amplifications products within a given partition 507. The optical detector may detect fluorescence, luminescence, or colorimetric signals. Signals from the partitions 507 may be detected when the partitions 507 are stationary, substantially stationary, or when the partitions 507 are flowing through the channel 506. The partitions may flow through the detection segment once or more than once. The channel 506 may further comprise a recycle or return channel that returns the partitions 507 back to the detection segment to enable signal detection more than once. The fluid flow network may further comprise a chamber downstream 510 of the detector 509. The chamber downstream 510 of the detector 509 may be a waste chamber or a collection chamber. A waste chamber may collect the first and second liquid phase at the end of the channel 506 for disposal. A collection chamber may collect the partitions 507 after they have passed the detector 509 for return to the detector segment or for storage and later use. The system may further comprise one or more computer processors individually or collectively programmed implement the processes described herein.

The system 501 may include a plurality of channels for sample processing. For example, the system 501 may include greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more channels.

The plurality of channels may be used to process portions from the same sample at the same time. Each channel may be the same as or similar to channel 506. In some examples, the sample may be divided into the plurality of channels such that each channel comprises a portion of the sample. The portion of the sample in each channel may be divided into a plurality of partitions within the channel. The portions of the sample divided into the different channels may be processed simultaneously or sequentially. The portions of the sample in the different channels may be processed under the same, similar, or different reaction conditions. For example, the different portions or fractions of the sample may be processed under different heating conditions (e.g., different heating and cooling cycles) or with different reagents. The different reagents may be different sets of primers or different primer concentrations. For example, the sample may be processed in three channels: a first channel may process a first portion of the sample using a first primer set, a second channel may process a second portion of the sample using a second primer set, and a third channel may process a third portion of the sample using a third primer set. The first primer set, second primer set, and third primer set may include the same primers but at different concentrations. This may enable a quantitative assessment of the sample under the different conditions. Each of the plurality of channels may be in communication with one or more detectors. Target molecules (e.g., amplification products) may be detected in each partition within one of the plurality of channels. Alternatively, or in addition to, the plurality of channels may combine into a single channel and target molecules within the plurality of partitions may be detected in the single channel.

Computer control systems

The present disclosure provides computer control systems that are programmed to implement methods of the disclosure. FIG. 6 shows a computer system 601 that is programmed or otherwise configured to nucleic acid sample processing and analysis, including formation of nucleic acid containing partitions, amplification, and detection. The computer system601 can regulate various aspects of methods and systems of the present disclosure.

The computer system 601 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 601 also includes memory or memory location 610 (e.g., random-access memory, read-only memory, flash memory) , electronic storage unit 615 (e.g., hard disk) , communication interface 620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 625, such as cache, other memory, data storage and/or electronic display adapters. The memory 610, storage unit 615, interface 620 and peripheral devices 625 are in communication with the CPU 605 through a communication bus (solid lines) , such as a motherboard. The storage unit 615 can be a data storage unit (or data repository) for storing data. The computer system 601 can be operatively coupled to a computer network ( “network” ) 630 with the aid of the communication interface 620. The network 630 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 630 in some cases is a telecommunication and/or data network. The network 630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 630, in some cases with the aid of the computer system 601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 601 to behave as a client or a server.

The CPU 605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 610. The instructions can be directed to the CPU 605, which can subsequently program or otherwise configure the CPU 605 to implement methods of the present disclosure. Examples of operations performed by the CPU 605 can include fetch, decode, execute, and writeback.

The CPU 605 can be part of a circuit, such as an integrated circuit. One or more other components of the system 601 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC) .

The storage unit 615 can store files, such as drivers, libraries and saved programs. The storage unit 615 can store user data, e.g., user preferences and user programs. The computer system 601 in some cases can include one or more additional data storage units that are external to the computer system 601, such as located on a remote server that is in communication with the computer system 601 through an intranet or the Internet.

The computer system 601 can communicate with one or more remote computer systems through the network 630. For instance, the computer system 601 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC) , slate or tablet PC’s (e.g.,

iPad,

Galaxy Tab) , telephones, Smart phones (e.g.,

iPhone, Android-enabled device,

) , or personal digital assistants. The user can access the computer system 601 via the network 630.

Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 601, such as, for example, on the memory 610 or electronic storage unit 615. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 605. In some cases, the code can be retrieved from the storage unit 615 and stored on the memory 610 for ready access by the processor 605. In some situations, the electronic storage unit 615 can be precluded, and machine-executable instructions are stored on memory 610.

The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

Aspects of the systems and methods provided herein, such as the computer system601, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer (s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The computer system 601 can include or be in communication with an electronic display 635 that comprises a user interface (UI) 640 for providing, for example, nucleic acid sequence information. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 605. The algorithm can, for example, regulate systems or implement methods provided herein.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A method for detecting a presence or absence of a target nucleic acid molecule from a raw biological sample, comprising:

(a) activating a system comprising a fluid flow network, wherein said fluid flow network comprises:

i. a first chamber and a second chamber that are in fluid communication at a junction, wherein said first chamber comprises a first liquid phase comprising said raw biological sample, and wherein said second chamber comprises a second liquid phase that is immiscible with said first liquid phase; and

ii. a channel in fluid communication with said junction, wherein said channel is configured to flow a plurality of partitions comprising said first liquid phase segmented by said second liquid phase;

(b) subjecting said first liquid phase and said second liquid phase to flow from said first chamber and said second chamber, respectively, to said junction, to generate said plurality of partitions, which plurality of partitions flows along said channel, wherein a given partition of said plurality of partitions comprises a reaction mixture comprising reagents necessary to perform a nucleic acid amplification reaction on said target nucleic acid molecule to yield amplification products of or derived from said target nucleic acid molecule;

(c) subjecting said reaction mixture in said given partition to conditions sufficient to perform a plurality of series of primer extension reactions on said target nucleic acid molecule in presence of said reagents to yield said amplification products of said target nucleic acid molecule, wherein an individual series of primer extension reactions differs from at least one other individual series of said plurality of series of primer extension reactions with respect to a denaturing condition and/or an elongation condition; and

(d) using a detector to detect a signal indicative of a presence or absence of said amplification product from said given partition, thereby detecting said presence or absence of said target nucleic acid molecule in said raw biological sample.
The method of claim 1, wherein said raw biological sample is provided directly from a source of said biological sample to said first chamber without further processing.
The method of claim 1, wherein said given partition has an aspect ratio greater than 1.
The method of claim 1, wherein (c) is performed in said channel.
The method of claim 4, wherein at least one segment of said channel is directed through a plurality of heating and cooling zones.
The method of claim 4, wherein at least one segment of said channel is in thermal communication with a heating unit.
The method of claim 4, wherein at least one segment of said channel is in thermal communication with a cooling unit.
The method of claim 1, wherein said reagents are provided in said first chamber as part of said first liquid phase.
The method of claim 1, further comprising a third chamber in fluid communication with said junction, wherein said third chamber comprises a third liquid phase comprising said reagents, wherein said third liquid phase is immiscible with said second liquid phase.
The method of claim 1, wherein said first liquid phase is aqueous.
The method of claim 1, wherein said second liquid phase comprises an oil.
The method of claim 1, wherein said second liquid phase comprises a non-wetting agent.
The method of claim 1, wherein said channel comprises a layer of a non-wetting agent.
The method of claim 13, wherein said activating comprises directing a precursor of said non-wetting agent through said channel prior to generating said partitions.
The method of claim 1, wherein said reagents include a polymerizing enzyme (s) and a primer having sequence complementarity to said target nucleic acid molecule.
The method of claim 15, wherein said target nucleic acid molecule is selected from the group consisting of human immunodeficiency virus I, human immunodeficiency virus II, orthomyxovirus, Ebola virus, Dengue virus, influenza virus, hepatitis A, B, C, D, and E virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile Fever virus, polio virus, measles virus, herpes simplex virus, smallpox virus, adenovirus, Varicella virus, Zika virus, pathogenic bacterium, pathogenic protozoan, and pathogenic parasites.
The method of claim 15, wherein said polymerizing enzyme (s) include a deoxyribonucleic acid (DNA) polymerase and/or a reverse transcriptase.
The method of claim 15, wherein said polymerizing enzymes (s) include a deoxyribonucleic acid (DNA) polymerase and a reverse transcriptase that is separate from said DNA polymerase.
The method of claim 1, wherein a segment of said channel downstream of said junction is in sensing communication with said detector.
The method of claim 19, wherein said detector is an optical detector, and wherein said segment of said channel is in optical communication with said detector.
The method of claim 1, wherein said channel is in fluid communication with a collection chamber downstream of said junction, and wherein said plurality of partitions is collected in said collection chamber.
The method of claim 21, further comprising bringing said collection chamber in fluid communication with an additional channel comprising a segment that is in sensing communication with said detector.
The method of claim 1, wherein said detector is part of said system.
The method of claim 1, wherein said nucleic amplification reaction is reverse transcription polymerase chain reaction or polymerase chain reaction (PCR) .
The method of claim 1, wherein said channel is substantially circular.
The method of claim 1, wherein said channel has at least one segment in a serpentine configuration.
The method of claim 1, wherein said system comprises a chip comprising said fluid flow network.
The method of claim 1, wherein said system further comprises a fluid flow unit in fluid communication with at least one of said first chamber, said second chamber, and said channel.
The method of claim 28, wherein said fluid flow unit is in fluid communication with said first chamber and said second chamber.
The method of claim 28, wherein said fluid flow unit is in fluid communication with said channel.
The method of claim 28, wherein said fluid flow unit is in fluid communication with said first chamber, said second chamber, and said channel.
The method of claim 28, wherein said fluid flow unit provides positive pressure to said first chamber and said second chamber.
The method of claim 28, wherein said fluid flow unit provides negative pressure to said channel.
The method of claim 1, further comprising subjecting said reaction mixture in said given partition to a plurality of series of primer extension reactions to generate said amplification products, wherein each series of said plurality of series of primer extension reactions comprises two or more cycles of (i) incubating said reaction mixture under said denaturing condition characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating said reaction mixture under said elongation condition characterized by an elongation temperature and an elongation duration.
The method of claim 1, wherein said target nucleic acid molecule is a ribonucleic acid (RNA) molecule and wherein said amplification products are amplified deoxyribonucleic acid (DNA) molecules generated from said RNA molecule.
The method of claim 35, further comprising subjecting said reaction mixture in said given partition to multiple cycles of a primer extension reaction to reverse transcribe said RNA molecule and generate said amplified DNA molecule in parallel, each cycle comprising (i) incubating said reaction mixture at a denaturing temperature for a denaturing duration that is less than or equal to 60 seconds, followed by (ii) incubating said reaction mixture at an elongation temperature for an elongation duration that is less than or equal to 60 seconds.
A method for detecting a presence or absence of a target nucleic acid molecule from a biological sample, comprising:

(a) activating a system comprising a detector and a fluid flow network, wherein said fluid flow network comprises:

i. a first chamber and a second chamber that are in fluid communication at a junction, wherein said first chamber comprises a first liquid phase comprising said biological sample, and wherein said second chamber comprises a second liquid phase that is immiscible with said first phase; and

ii. a channel in fluid communication with said junction, wherein said channel is configured to flow a plurality of partitions comprising said first liquid phase segmented by said second liquid phase, wherein said channel comprises at least one segment in a plurality of thermal zones for subjecting said plurality of partitions to heating and/or cooling, and a detection segment downstream of said at least one segment, wherein said detection segment is in sensing communication with said detector;

(b) subjecting said first liquid phase and said second liquid phase to flow from said first chamber and said second chamber, respectively, to said junction, to generate said plurality of partitions, wherein a given partition of said plurality of partitions comprises a reaction mixture comprising reagents necessary to perform a nucleic acid amplification reaction on said target nucleic acid molecule to yield amplification products of or derived from said target nucleic acid molecule, wherein upon generation, said plurality of partitions flows along said channel;

(c) using said plurality of thermal zones to subject said reaction mixture in said given partition flowing through said at least one segment, to conditions sufficient to perform said nucleic acid amplification reaction on said target nucleic acid molecule in presence of said reagents, to yield said amplification products of said target nucleic acid molecule; and

(d) using said detector to detect a signal indicative of a presence or absence of said amplification products from said given partition when said given partition is flowing through said detection segment, thereby detecting said presence or absence of said target nucleic acid molecule in said biological sample.
The method of claim 37, wherein said given partition has an aspect ratio greater than 1.
The method of claim 37, wherein said at least one segment is in thermal communication with a heating and/or cooling unit.
The method of claim 39, wherein said at least one segment comprises a plurality of segments comprising a first segment and a second segment, wherein said first segment is for subjecting said given partition to heating and said second segment is for subjecting said given partition to cooling.
The method of claim 39, wherein said at least one segment comprises a plurality of segments comprising a first segment and a second segment, wherein said first segment is for incubating said given partition at an elongation temperature or temperature range, and wherein said second segment is for incubating said given partition to a denaturation temperature or temperature range.
The method of claim 37, wherein said reagents are provided in said first chamber as part of said first liquid phase.
The method of claim 37, further comprising a third chamber in fluid communication with said junction, wherein said third chamber comprises a third liquid phase comprising said reagents, wherein said third liquid phase is immiscible with said second liquid phase.
The method of claim 37, wherein said first liquid phase is aqueous and/or said second liquid phase comprises an oil.
The method of claim 37, wherein said second liquid phase comprises a non-wetting agent.
The method of claim 37, wherein said channel comprises a layer of a non-wetting agent.
The method of claim 46, wherein said activating comprises directing a precursor of said non-wetting agent through said channel prior to generating said partitions.
The method of claim 37, wherein said reagents include a polymerizing enzyme (s) and a primer having sequence complementarity to said target nucleic acid molecule.
The method of claim 48, wherein said target nucleic acid molecule is selected from the group consisting of human immunodeficiency virus I, human immunodeficiency virus II, orthomyxovirus, Ebola virus, Dengue virus, influenza virus, hepatitis virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile Fever virus, polio virus, measles virus, herpes simplex virus, smallpox virus, adenovirus, Varicella virus, Zika virus, pathogenic bacterium, pathogenic protozoan, and pathogenic parasites.
The method of claim 48, wherein said polymerizing enzyme (s) include a deoxyribonucleic acid (DNA) polymerase and/or a reverse transcriptase.
The method of claim 48, wherein said polymerizing enzymes (s) include a deoxyribonucleic acid (DNA) polymerase and a reverse transcriptase that is separate from said DNA polymerase.
The method of claim 37, wherein said detector is an optical detector, and wherein said detection segment is in optical communication with said detector.
The method of claim 37, wherein said channel is in fluid communication with a collection chamber downstream of said detection segment, and wherein said plurality of partitions is collected in said collection chamber.
The method of claim 37, wherein said nucleic amplification reaction is reverse transcription polymerase chain reaction or polymerase chain reaction (PCR) .
The method of claim 37, wherein said channel is substantially circular.
The method of claim 37, wherein said channel has at least one segment in a serpentine configuration.
The method of claim 56, wherein said at least one segment is part of said serpentine configuration.
The method of claim 37, wherein said system comprises a chip comprising said fluid flow network.
The method of claim 37, wherein said system further comprises a fluid flow unit in fluid communication with at least one of said first chamber, said second chamber and said channel.
The method of claim 59, wherein said fluid flow unit is in fluid communication with said first chamber and said second chamber.
The method of claim 59, wherein said fluid flow unit is in fluid communication with said channel.
The method of claim 59, wherein said fluid flow unit is in fluid communication with said first chamber, said second chamber and said channel.
The method of claim 59, wherein said fluid flow unit provides positive pressure to said first chamber and said second chamber.
The method of claim 59, wherein said fluid flow unit provides negative pressure to said channel.
The method of claim 37, further comprising using said plurality of thermal zones to subject said reaction mixture in said given partition to a plurality of series of primer extension reactions to generate said amplification products, each series comprising two or more cycles of (i) incubating said reaction mixture under a denaturing condition characterized by a denaturing temperature and a denaturing duration, followed by (ii) incubating said reaction mixture under an elongation condition characterized by an elongation temperature and an elongation duration, wherein an individual series differs from at least one other individual series of said plurality with respect to said denaturing condition and/or said elongation condition.
The method of claim 37, wherein said target nucleic acid molecule is a ribonucleic acid (RNA) molecule and wherein said amplification products is an amplified deoxyribonucleic acid (DNA) molecule generated from said RNA molecule.
The method of claim 66, further comprising using said plurality of thermal zones to subject said reaction mixture in said given partition to multiple cycles of a primer extension reaction to reverse transcribe said RNA molecule and generate said amplified DNA molecule in parallel, each cycle comprising (i) incubating said reaction mixture at a denaturing temperature for a denaturing duration that is less than or equal to 60 seconds, followed by (ii) incubating said reaction mixture at an elongation temperature for an elongation duration that is less than or equal to 60 seconds.
A system for detecting a presence or absence of a target nucleic acid molecule from a raw biological sample, comprising:

a fluidic network comprising (i) a first chamber and a second chamber that are in fluid communication at a junction, wherein said first chamber is configured to contain a first liquid phase comprising said raw biological sample, and wherein said second chamber is configured to contain a second liquid phase that is immiscible with said first phase; and (ii) a channel in fluid communication with said junction, wherein said channel is configured to flow a plurality of partitions comprising said first liquid phase segmented by said second liquid phase;

a fluid flow unit in fluid communication with at least one of said first chamber, said second chamber and said channel;

a detector configured to detect a signal indicative of a presence or absence of amplification products generated from said target nucleic acid molecule in a given partition of said plurality of partitions; and

one or more computer processors operatively coupled to said fluid flow unit and said detector, wherein said one or more computer processors are individually or collectively programmed to:

(a) direct said fluid flow unit to (i) subject said first liquid phase and said second liquid phase to flow from said first chamber and said second chamber, respectively, to said junction, to generate said plurality of partitions, which plurality of partitions flows along said channel, wherein said given partition of said plurality of partitions comprises a reaction mixture comprising reagents necessary to perform a nucleic acid amplification reaction on said target nucleic acid molecule to yield amplification products of or derived from said target nucleic acid molecule; and (ii) subject said plurality of partitions, including said given partition, to flow along said channel, wherein subsequent to generating said plurality of partitions, said reaction mixture in said given partition is subjected to conditions sufficient to perform a plurality of series of primer extension reactions on said target nucleic acid molecule in presence of said reagents, to yield said amplification products, wherein an individual series of primer extension reactions differs from at least one other individual series of said plurality of series of primer extension reactions with respect to a denaturing condition and/or an elongation condition; and

(b) direct said detector to detect a signal indicative of a presence or absence of said amplification products from said given partition, thereby detecting said presence or absence of said target nucleic acid molecule in said raw biological sample.
The system of claim 68, wherein said given partition has an aspect ratio greater than 1.
The system of claim 68, further comprising a third chamber in fluid communication with said junction, wherein said third chamber comprises a third liquid phase comprising said reagents, wherein said third liquid phase is immiscible with said second liquid phase.
The system of claim 68, wherein said first liquid phase is aqueous and/or said second liquid phase comprises an oil.
The system of claim 68, wherein said second liquid phase comprises a non-wetting agent.
The system of claim 68, wherein said channel comprises a layer of a non-wetting agent.
The system of claim 68, wherein a segment of said channel downstream of said junction is in sensing communication with said detector.
The system of claim 74, wherein said detector is an optical detector, and wherein said segment of said channel is in optical communication with said detector.
The system of claim 68, wherein said channel is in fluid communication with a collection chamber downstream of said junction, and wherein said plurality of partitions is collected in said collection chamber.
The system of claim 68, wherein said channel is substantially circular.
The system of claim 68, wherein said channel has at least one segment in a serpentine configuration.
The system of claim 68, wherein said system comprises a chip comprising said fluid flow network.
The system of claim 68, wherein said fluid flow unit is in fluid communication with said first chamber and said second chamber.
The system of claim 68, wherein said fluid flow unit is in fluid communication with said channel.
The system of claim 68, wherein said fluid flow unit is in fluid communication with said first chamber, said second chamber and said channel.
The system of claim 68, wherein said fluid flow unit provides positive pressure to said first chamber and said second chamber.
The system of claim 68, wherein said fluid flow unit provides negative pressure to said channel.
A system for detecting a presence or absence of a target nucleic acid molecule from a biological sample, comprising:

a fluid flow network comprising (i) a first chamber and a second chamber that are in fluid communication at a junction, wherein said first chamber comprises a first liquid phase comprising said biological sample, and wherein said second chamber comprises a second liquid phase that is immiscible with said first phase; and (ii) a channel in fluid communication with said junction, wherein said channel is configured to flow a plurality of partitions comprising said first liquid phase segmented by said second liquid phase at said junction, wherein said channel comprises at least a first segment in a plurality of thermal zones for subjecting said plurality of partitions to heating and/or cooling, and a detection segment downstream of said at least said first segment, wherein said detection segment is in sensing communication with said detector;

a fluid flow unit in fluid communication with at least one of said first chamber, said second chamber, and said channel;

a detector configured to detect a signal indicative of a presence or absence of an amplification products generated from said target nucleic acid molecule in a given partition of said plurality of partitions;

a heating and/or cooling unit in thermal communication with said plurality of thermal zones; and

one or more computer processors operatively coupled to said fluid flow unit, said heating and/or cooling unit, and said detector, wherein said one or more computer processors are individually or collectively programmed to:

(a) direct said fluid flow unit to subject said first liquid phase and said second liquid phase to flow from said first chamber and said second chamber, respectively, to said junction, to generate said plurality of partitions, wherein said given partition of said plurality of partitions comprises a reaction mixture comprising reagents necessary to perform a nucleic acid amplification reaction on said target nucleic acid molecule to yield amplification products of or derived from said target nucleic acid molecule, wherein upon generation, said plurality of partitions flows along said channel;

(b) direct said heating and/or cooling unit to subject said reaction mixture in said given partition flowing through said at least said first segment, to conditions sufficient to perform said nucleic acid amplification reaction on said target nucleic acid molecule in presence of said reagents, to yield said amplification products of said target nucleic acid molecule; and

(c) direct said detector to detect a signal indicative of a presence or absence of said amplification products from said given partition when said given partition is flowing through said detection segment, thereby detecting said presence or absence of said target nucleic acid molecule in said biological sample.
The system of claim 85, wherein said given partition has an aspect ratio greater than 1.
The system of claim 85, wherein said at least one segment is in thermal communication with a heating and/or cooling unit.
The system of claim 87, wherein said at least one segment comprises a plurality of segments comprising a first segment and a second segment, wherein said first segment is for subjecting said given partition to heating and said second segment is for subjecting said given partition to cooling.
The system of claim 87, wherein said at least one segment comprises a plurality of segments comprising a first segment and a second segment, wherein said first segment is for incubating said given partition at an elongation temperature or temperature range, and wherein said second segment is for incubating said given partition to a denaturation temperature or temperature range.
The system of claim 85, further comprising a third chamber in fluid communication with said junction, wherein said third chamber comprises a third liquid phase comprising said reagents, wherein said third liquid phase is immiscible with said second liquid phase.
The system of claim 85, wherein said first liquid phase is aqueous and/or said second liquid phase comprises an oil.
The system of claim 85, wherein said second liquid phase comprises a non-wetting agent.
The system of claim 85, wherein said channel comprises a layer of a non-wetting agent.
The system of claim 85, wherein said detector is an optical detector, and wherein said detection segment is in optical communication with said detector.
The system of claim 85, wherein said channel is in fluid communication with a collection chamber downstream of said detection segment, which collection chamber is for collecting said plurality of partitions.
The system of claim 85, wherein said channel is substantially circular.
The system of claim 85, wherein said channel has at least one segment in a serpentine configuration.
The system of claim 97, wherein said at least one segment is part of said serpentine configuration.
The system of claim 85, wherein said system comprises a chip comprising said fluid flow network.
The system of claim 85, wherein said fluid flow unit is in fluid communication with said first chamber and said second chamber.
The system of claim 85, wherein said fluid flow unit is in fluid communication with said channel.
The system of claim 85, wherein said fluid flow unit is in fluid communication with said first chamber, said second chamber and said channel.
The system of claim 85, wherein said fluid flow unit provides positive pressure to said first chamber and said second chamber.
The system of claim 85, wherein said fluid flow unit provides negative pressure to said channel.