Methods and systems for thermal control of chemical reactions in droplets

Abstract

Methods and systems for thermally controlling chemical reactions in droplets. In one exemplary method, a first thermal zone and a second thermal zone can be created in a reaction chamber that have different temperatures from one another. An emulsion comprising droplets encapsulated by a carrier liquid is contained in the reaction chamber. The density of the droplets can be mismatched from the carrier liquid, and each droplet can comprise one or more reactants for a chemical reaction. The droplets can be moved from the first thermal zone to the second thermal zone by changing the orientation of the reaction chamber, such that the rate of the chemical reaction is changed in at least a subset of the droplets.

Description

[0001] Prior cross-reference

[0002] This application is based on and claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 994,218, filed March 24, 2020, and is incorporated herein by reference in its entirety for all purposes.

[0003] introduction

[0004] Polymerase chain reaction (PCR) nucleic acid amplification can be advantageously performed in aqueous droplets of emulsion, each droplet forming an independent microreactor. To drive amplification, the emulsion is thermally cycled. This thermal cycling can be performed using a standard thermal cycler with a design in which the emulsion remains stationary while the temperature of the surrounding reaction chamber circulates between different temperatures. However, the temperature cycling throughout the reaction chamber is slow, resulting in long cycle times and reduced yields.

[0005] Figure 1 A thermal cycling device 30 is shown to accelerate the thermal cycling of droplets (see U.S. Patent No. 9,266,104). The thermal cycling device 30 has a reaction chamber 32 for containing an emulsion 33 comprising droplets 34 suspended in an immiscible carrier liquid 36. An inlet channel 38 introduces preheated carrier liquid at different temperatures into the reaction chamber 32, indicated by inflow arrow 40. Simultaneously, an outlet channel 42 removes a corresponding volume of carrier liquid 36 from the reaction chamber 32, indicated by outflow arrow 44. The droplets 34 remain in the reaction chamber 32 and are heated / cooled to various selected temperatures by the incoming preheated carrier liquid. Thus, the thermal cycling device 30 can rapidly thermally cycle the droplets 34 within the reaction chamber 32 to amplify the target sequence contained in the droplets. However, cyclically supplying carrier liquid at different temperatures to the reaction chamber consumes a large amount of carrier liquid, and the rate and accuracy of each temperature change are sensitive to the thermal mass of the reaction chamber and its contents. A different method is needed to thermally control amplification and other chemical reactions. Summary of the Invention

[0006] This invention provides a method and system for thermally controlling chemical reactions in droplets. In one exemplary method, a first hot zone and a second hot zone with different temperatures can be created in a reaction chamber. An emulsion comprising droplets encapsulated by a carrier liquid is contained in the reaction chamber. The density of the droplets may be mismatched with the carrier liquid, and each droplet may include one or more reactants for the chemical reaction. Droplets can be moved from the first hot zone to the second hot zone by changing the orientation of the reaction chamber, such that the rate of the chemical reaction changes in at least one subset of the droplets.

[0007] Brief description of the attached figures

[0008] Figure 1This is a schematic fragment view of a thermal circulation device illustrating a prior art method for achieving thermal circulation of droplets in a reaction chamber by circulating preheated carrier liquids at different temperatures into the reaction chamber.

[0009] Figure 2 This is a schematic fragment of a series of figures (AD) showing an exemplary reaction control device for controlling a chemical reaction within a droplet by moving the droplet between a pair of hot zones within the reaction chamber by changing the direction of the reaction chamber containing the droplet relative to gravity or the G-force vector, thereby altering the temperature of the droplet.

[0010] Figure 3 In a series of diagrams (AD) Figure 2 A schematic fragment of a reaction control device used to expose droplets to a range of different temperatures by varying the temperatures of the various hot zones within the reaction chamber.

[0011] Figure 4 This is a flowchart illustrating exemplary steps of a method for thermally controlling chemical reactions in droplets.

[0012] Figure 5 This is a block diagram of an exemplary system for thermally controlling chemical reactions in droplets.

[0013] Figure 6 This is a side view of another exemplary reaction chamber containing an emulsion, and with curved (gooseneck) droplet traps at opposite ends of the chamber.

[0014] Figure 7 This is a side view of another exemplary reaction chamber containing an emulsion, and having recessed droplet traps at opposite ends of the chamber.

[0015] Figure 8 This is a schematic fragment of an exemplary reaction control device, which contains an emulsion and includes a reaction chamber having a pair of larger-diameter sub-chambers connected to each other by a smaller-diameter channel.

[0016] Figure 9 This is a schematic fragment view of a selected aspect of an exemplary reaction control device, which contains an emulsion and includes a reaction chamber having a pair of sub-chambers connected to each other via droplet channels and liquid carrier channels.

[0017] Figure 10 This is a schematic diagram of an exemplary system for thermally controlled chemical reactions, which includes a reaction control device optically coupled to a detection module.

[0018] Figure 11This is a schematic fragment of an exemplary reaction control device in a series of figures (AD), which contains an emulsion and includes a reaction chamber having a pair of laterally arranged sub-chambers connected at opposite ends by a pair of channels, wherein the reaction chamber is reoriented by rotating a full circle in the plane of the pair of channels, thereby moving droplets of the emulsion between the hot zones of the reaction chamber.

[0019] Figure 12 This is a schematic fragment of an exemplary reaction control device, which contains an emulsion and includes a reaction chamber having a pair of hot zones that are rotated 90 degrees apart from each other.

[0020] Figure 13 This is a schematic fragment of an exemplary reaction control device, which contains an emulsion and includes a reaction chamber having three hot zones that are rotated 120 degrees apart from each other.

[0021] Figure 14 This is a schematic diagram of an exemplary system for thermally controlling chemical reactions in droplets, which includes a pair of pumps for driving a preheated carrier liquid into various hot zones of a reaction chamber containing droplets.

[0022] Figure 15 yes Figure 14 Another view taken after the system was repositioned in the reaction chamber to move droplets between hot zones within the chamber.

[0023] Figure 16 This is a schematic top view of an exemplary centrifugation system having multiple reaction control devices for thermally controlled chemical reactions in droplets.

[0024] Figure 17 yes Figure 16 A schematic diagram of one of the reaction control devices used for a two-step thermal cycle of droplets to promote nucleic acid amplification in the droplets, wherein the reaction chamber of the reaction control device is reversed relative to the centrifugal force G between Figures A and B.

[0025] Figure 18 yes Figure 17 A schematic diagram of the reaction control device, which is used to generate melting curves of the amplified products in the droplets by detecting amplification signals at a series of different temperatures (shown in Figure AD).

[0026] Figure 19 yes Figure 17 A schematic diagram of the reaction control device, which is used for droplet generation, droplet thermal cycling, and detection of droplet amplification signals after thermal cycling.

[0027] Figure 20This is a schematic diagram of any exemplary flow system used for thermally controlling chemical reactions in droplets. Detailed Implementation

[0028] This invention provides methods and systems for thermally controlling chemical reactions in droplets. In one exemplary method, a first and a second hot zone with different temperatures can be created in a reaction chamber. An emulsion comprising droplets encapsulated by a carrier liquid is contained in the reaction chamber. The droplets may have a density mismatch with the carrier liquid, and each droplet may include one or more reactants for the chemical reaction. Droplets can be moved from the first hot zone to the second hot zone by changing the orientation of the reaction chamber, such that the rate of the chemical reaction changes in at least one subset of the droplets. The methods and systems described herein have various advantages, such as greater simplicity, faster and more precise thermal control of the chemical reaction, faster thermal cycling, and / or similar benefits.

[0029] Other aspects of this disclosure are described in the following subsections:

[0030] (I) Definition, (II) Overview of the method and system, (III) Implementation examples, and (IV) Selection aspects.

[0031] I. Definition

[0032] The technical terms used in this disclosure have meanings generally accepted by those skilled in the art. However, the following terms may be further defined as follows.

[0033] Amplicon – a product of an amplification reaction.

[0034] Amplification is the process of preparing multiple copies of an amplicon that matches and / or complements a target sequence. This process is interchangeably referred to as an amplification reaction. For example, amplification can produce an exponential or linear increase in copy number as it proceeds. Typical amplification may result in a copy number of the amplicon greater than 1,000-fold. Exemplary amplification reactions of the methods disclosed herein may include polymerase chain reaction (PCR) or ligase chain reaction (LCR), each driven by thermal cycling (e.g., two-step, three-step, or more than three-step thermal cycling). This method may also use or alternatively use other amplification reactions that can be performed isothermally, such as branched probe DNA detection, cascaded RCA, helicase-dependent amplification, loop-mediated isothermal amplification (LAMP), nucleic acid-based amplification (NASBA), nicking enzyme amplification reaction (NEAR), PAN-AC, Q-β replicase amplification, rolling circle replication (RCA), self-sustaining sequence replication, strand displacement amplification, and / or similar means. Amplification may utilize linear or circular templates.

[0035] Amplification reagents – any reagent that can promote or affect the amplification of a target sequence. Such reagents may include: at least one primer or primer pair for amplifying at least one target sequence, at least one marker for detecting the amplification of said at least one target sequence (e.g., at least one probe comprising a marker and / or a DNA intercalation dye as a marker), at least one polymerase and / or ligase (which may be thermostable), and any combination of nucleoside triphosphates (dNTPs and / or NTPs).

[0036] An analyte is a chemical substance or a region thereof that is used to detect, quantify, and / or characterize that chemical substance or region thereof. Exemplary analytes include reactants, catalysts, cofactors, etc., in chemical reactions. Suitable analytes may include nucleic acids, nucleic acid target sequences, proteins (such as enzymes), carbohydrates, lipids, etc.

[0037] A chemical reaction is a process involving the rearrangement of the molecular or ionic structures of one or more substances. Each substance is called a "reactant" in a chemical reaction. Chemical reactions can be unimolecular (only one chemical reacts with itself), bimolecular (two chemical reactants react with each other), tripolecular (three chemical reactants react with each other), and so on. Examples of possible types of chemical reactions include oxidation-reduction, direct binding, decomposition, single substitution / substitution, double substitution / substitution, acid-base reactions, isomerization, racemization, ring opening, cyclization, and hydrolysis. Chemical reactions can occur with or without the presence of an enzyme that catalyzes the reaction.

[0038] Complementarity—This involves rules governing base pairing. A first nucleic acid (or a region thereof) is "complementary" to a second nucleic acid if it hybridizes with it in an antiparallel manner by forming a continuous or nearly continuous series of base pairs. If hybridization of the first nucleic acid (or a region thereof) with the second nucleic acid results in a continuous series of base pairs formed by each nucleotide of the first nucleic acid (or its region thereof), the first nucleic acid (or its region thereof) is said to be "perfectly complementary" to the second nucleic acid. "Complementarity" of the first nucleic acid means that the second nucleic acid is perfectly complementary to the first nucleic acid by at least ten consecutive nucleotides. The "complementarity ratio" between the first nucleic acid (or its region thereof) and the second nucleic acid (or its region thereof) refers to the number or percentage of base pairs that can be formed when the first nucleic acid (or its region thereof) and the second nucleic acid (or its region thereof) hybridize in an optimal antiparallel manner. A first nucleic acid (or its region thereof) complementary to a second nucleic acid (or its region thereof) generally has a complementarity ratio of at least 80% or 90%.

[0039] A droplet is a small volume of liquid encapsulated by an immiscible liquid (e.g., encapsulated by an immiscible liquid that may form a continuous phase of an emulsion). The immiscible liquid may include oil and / or may consist primarily of oil. Droplets used in the methods disclosed herein may, for example, have an average size of less than about 1 μL, 500 nL, 100 nL, 10 nL, or 1 nL. For example, the droplet may be an aqueous droplet.

[0040] Reversal – a reorientation relative to the vertical axis (defined by gravity) or relative to the G-force vector, greater than 90 degrees and less than 270 degrees. The verb "reversal" refers to the process that produces this reorientation.

[0041] A marker is a identifying and / or distinguishing symbol or identifier associated with a structure such as a primer, probe, amplicon, or droplet. Markers can be covalently linked to a structure, for example, by covalent or non-covalent linkage (e.g., through embedding, hydrogen bonding, electrostatic interactions, encapsulation, etc.) to an oligonucleotide. Exemplary markers include optical markers, radioactive markers, magnetic markers, electrical markers, epitopes, enzymes, antibodies, etc. Optical markers can be optically detected through their interaction with light. Applicable exemplary optical markers include photoluminescent groups, quenchers, and embedded dyes.

[0042] Light – light radiation, including ultraviolet, visible, and / or infrared light.

[0043] Nucleic acids are polymers of any length composed of naturally occurring nucleotides (e.g., DNA or RNA) or artificially synthesized substances that can hybridize with DNA or RNA in a sequence-specific manner similar to that of two naturally occurring nucleic acids, for example, by engaging in Watson-Crick base pairing interactions. Nucleic acids can consist of any suitable number of nucleotides (e.g., at least about 5, 10, 100, or 1000). Typically, the length of a nucleic acid chain corresponds to its source; synthetic nucleic acids (such as oligonucleotides) are generally shorter, while biologically / enzymatically produced nucleic acids (such as genomic fragments) are generally longer.

[0044] Nucleic acids can have natural or artificial structures, or a combination of both. Nucleic acids with natural structures, namely deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically have a backbone of alternating pentose sugar groups and phosphate groups. Each pentose sugar group is linked to a nucleotide (e.g., a purine (e.g., adenine (A) or guanine (T))) or a pyrimidine (e.g., cytosine (C), thymine (G), or uracil (U))). Nucleic acids with artificial structures are analogs of natural nucleic acids and can be, for example, produced by altering the pentose sugar and / or phosphate groups and / or one or more nucleotides of the natural backbone. Exemplary artificial nucleic acids include glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs), threonine nucleic acids (TNAs), xenobiotic nucleic acids (XNAs), etc.

[0045] The sequence of a nucleic acid is determined by the order in which the nucleobases are arranged along the backbone. This order typically determines the ability of a nucleic acid to hybridize with another nucleic acid via hydrogen bonds. Specifically, adenine pairs with thymine (or uracil), and guanine pairs with cytosine.

[0046] Oligonucleotides are relatively short and / or chemically synthesized nucleic acids. For example, the length of an oligonucleotide can range from 3 to 1000 nucleotides.

[0047] Partial occupancy refers to the presence of an analyte in only a subset of droplets. Within a set of droplets, partial occupancy means an arrangement where one or more droplets each contain no copy of the analyte, while one or more droplets each contain at least one copy of the analyte. In some cases, one or more droplets each contain exactly one copy of the analyte. The analyte in the droplets may or may not be distributed according to a Poisson distribution.

[0048] Photoluminescence is the emission of light caused by electromagnetic radiation. Any substance can produce photoluminescence when it absorbs photons from electromagnetic radiation (such as light). Exemplary forms of photoluminescence include fluorescence and phosphorescence.

[0049] A primer is an oligonucleotide that, under appropriate reaction conditions (e.g., the presence of an oligonucleotide annealing template, a nucleoside triphosphate, and a polymerization catalyst (such as DNA or RNA polymerase or reverse transcriptase), in an appropriate buffer, and at an appropriate temperature), serves as the starting point for template-guided nucleic acid synthesis. Primers can be of any suitable length, such as 5 to 500 nucleotides. The primer can be a member of a "primer pair," which includes a "forward primer" and a "reverse primer," defining the ends of the amplicon produced in the amplification reaction. (The adjectives "forward" and "reverse" are arbitrary designations relative to each other). The forward primer hybridizes to the 5' complement of the target sequence to be amplified, and the reverse primer hybridizes to the 3' region of the target sequence. The term "primer binding site" is a portion of the template (or its complement) that the primer anneals. The entire sequence of the primer does not need to be perfectly complementary to the primer binding site, as long as there is sufficient complementarity for annealing under the reaction conditions. Therefore, a primer can have a 3' end region that is complementary to the primer binding site and a 5' end region that is not complementary to the primer binding site (and form a "5'-tail").

[0050] A probe is a labeled oligonucleotide used to report the occurrence of an amplification reaction and / or the amplicon formed by the amplification reaction. For example, a probe may be a photoluminescent probe comprising an oligonucleotide labeled with a photoluminescent agent. The probe may be configured to hybridize with at least a portion of the amplicon generated by amplification. A probe (e.g., a hydrolysis probe) may be configured to hybridize with at least a portion of the amplicon during the annealing / extension phase of the amplification cycle of the amplification reaction, or a probe (e.g., a molecular beacon probe) may be configured to hybridize with the amplicon after the amplification reaction has completed, and so on.

[0051] A reaction chamber is a space within a basically or completely enclosed container used for carrying out chemical reactions. A reaction chamber may or may not include one or more channels and / or different sub-chambers. The reaction chamber can be designed to contain emulsions (particularly their droplets) and retain the droplets within it when the chamber is reoriented to control the chemical reaction within them. The reaction chamber may or may not be elongated between its opposite ends, and may or may not have a uniform cross-section or diameter in the middle of the opposite ends. In some cases, it is preferable to have a non-uniform reaction chamber, i.e., a larger volume at each opposite end and a smaller volume in the middle of the opposite ends. This configuration allows for increased separation distance between the opposite ends to better maintain the temperature difference between them. The reaction chamber may or may not be axially symmetrical. In some cases, the reaction chamber may be or include channels with elliptical (e.g., circular) or polygonal (e.g., rectangular) cross-sections. The reaction chamber can be formed and / or clad from one or more metals or plastics through machining, molding, fused, brazing, and other processes. In some cases, it may be advantageous to use a combination of materials with high thermal conductivity and low thermal conductivity to promote heat conduction within each thermal zone while preventing heat conduction between thermal zones.

[0052] Sensing zone – the space where a reaction signal is detected. The term "reaction signal" refers to any detectable signal that is sensitive to a chemical reaction occurring in the droplet.

[0053] Target sequence—a sequence on or within a template that provides a template for the synthesis of complementary sequences.

[0054] Template—The nucleic acid that serves as a template for synthesizing the complementary strand. The template provides primer binding sites, which are extended by sequentially adding complementary nucleotides according to the template.

[0055] A hot zone is an area with a controlled temperature (such as a temperature actively maintained at a selected setpoint). This zone may simply be a portion of the space defined by the reaction chamber. A hot zone may include the ends or recesses of the reaction chamber where droplets can accumulate when the chamber is reoriented.

[0056] II. Overview of Methods and Systems

[0057] This section outlines the method and system of the present invention for thermally controlling chemical reactions in droplets; see [link to relevant documentation]. Figure 2-5 .

[0058] Figure 2An exemplary reaction control device 50 is shown, having a reaction chamber 52 for containing an emulsion 53. The emulsion comprises droplets 54 (see Figure A) containing reactants 55 for a chemical reaction. Each droplet 54 is enveloped by an immiscible carrier liquid 56 (liquid) and has a different density than the carrier liquid. (Droplets may have the same density.) The density mismatch between the droplets and the carrier liquid causes the droplets to move to the top of the reaction chamber 52 (i.e., if the density of the droplets is less than that of the carrier liquid) or to the bottom of the reaction chamber 52 (i.e., if the density of the droplets is greater than that of the carrier liquid). For consistency, in each of the embodiments described in this disclosure, the density of the droplets is lower than that of the carrier liquid, and therefore has buoyancy in the carrier liquid.

[0059] Figure 2 The arrow at the top center indicates the direction of gravity in Figure AD, which is opposite to the buoyancy that causes droplets 54 to move towards the top of reaction chamber 52. In other embodiments, each droplet 54 has a higher density than the carrier liquid 56 and is propelled downwards by gravity in reaction chamber 52. In some embodiments, a force G greater than gravity can be applied to reaction chamber 52 by centrifugal force to increase the buoyancy (or settling force) of the droplets, thereby propelling the droplets to move more quickly in reaction chamber (see Example 5).

[0060] The reaction control device 50 is configured to create two or more hot zones in the reaction chamber 52. Here, a pair of hot zones 58a and 58b are created at opposite ends of the reaction chamber 52 and have different temperatures T1 and T2, respectively. In other embodiments, three or more hot zones are created, each having three or more different temperatures (see Embodiment 3).

[0061] Figure A and D illustrate the change in orientation of reaction chamber 52, indicated by the rotating arrow at 60, thereby moving droplets 54 as a group from hot zone 58a to hot zone 58b. More specifically, the orientation of reaction chamber 52 is changed relative to the direction of gravity (and / or an additional G-force) to facilitate the desired migration of droplets 54. In the depicted embodiment, reaction chamber 52 is reversed to produce this migration. Hot zone 58a is located at the top of reaction chamber 52 in Figure A, while hot zone 58b has this position in Figure D. Accordingly, droplets 54 have a temperature T1 in Figure A and a temperature T2 in Figure D. T2 can be less than T1, thereby reorienting the droplets to cool them (i.e., lower their temperature), which may slow down or stop (or start or accelerate) the chemical reaction of reactants 55 in the droplets (i.e., at least in a subset (one or more) of the droplets). Alternatively, T2 can be greater than T1, thereby redirecting the heating of the droplet (i.e., increasing its temperature), which can initiate or accelerate (or slow down or stop) the chemical reactions in the droplet (i.e., in at least one subset (one or more) of the droplet).

[0062] Once reorientation is complete, rotation of reaction chamber 52 can be paused for any suitable residence time to allow the droplet to incubate at temperature T2 in hot zone 58b. If necessary, reaction chamber 52 can be further reoriented, for example, to move droplet 54 back to hot zone 58a (or a third hot zone within the reaction chamber). When using this process to thermally cycle the droplet, the number of cycles, each temperature, and the residence time of the droplet at each temperature are easily controlled.

[0063] When reaction chamber 52 is reoriented, droplet 54 moves toward hot zone 58b, as indicated by movement arrow 61 in Figures B and C. This movement is accompanied by a net flow of carrier liquid 56 in the opposite direction toward hot zone 58a, indicated by flow arrow 62, which is replaced by droplet 54.

[0064] Droplets 54 and carrier liquid 56 may enter and exit reaction chamber 52 as appropriate through one or more ports, such as inlet 64 and outlet 66. In some embodiments, inlet 64 may serve as both an inlet and outlet for the emulsion, while outlet 66 may serve only as a vent. An upstream gate and a downstream gate may be operatively connected to inlet 64 and outlet 66, respectively, to control when fluid flow is permitted through one or both of the inlet and outlet. In some embodiments, both gates may remain closed when emulsion 53 is being processed in reaction chamber 52 (e.g., when reaction chamber 52 is being reoriented). In some embodiments, both gates may be opened to allow preheated carrier liquid to be added to reaction chamber 52 during emulsion 53 processing (e.g., see Example 4).

[0065] The reaction control device 50 may have gates 68 associated with each port to restrict the movement of droplets 54 out of the reaction chamber 52. For example, the gates 68 may be configured to passively prevent droplets 54 from leaving the reaction chamber 52 until a sufficient pressure differential is generated by a pump to force the droplets through the gates.

[0066] Figure 3 A reaction control device 50 is shown for exposing droplet 54 to a series of different temperatures (T1-T5). For example, the different temperatures could be a series of increasing or decreasing temperatures to raise or lower the droplet temperature, respectively. Raising or lowering the droplet temperature can be used to generate melting or annealing profiles for one or more nucleic acid double strands in the droplet (e.g., see Example 5). Melting / annealing profiles can distinguish the amplification of two or more different target sequences using the same probe and / or label.

[0067] Figures A and D illustrate a series of exemplary configurations. In Figure A, droplet 54 is located in hot zone 58a at temperature T1. In Figure B, reaction chamber 52 has been reoriented so that droplet 54 moves to hot zone 58b at temperature T2. While droplet 54 is held in hot zone 58b, the temperature of another hot zone 58a is being changed to T3. In Figure C, reaction chamber 52 is again reoriented so that droplet 54 moves back to hot zone 58a for holding at T3. While droplet 54 is in hot zone 58a, the temperature of another hot zone 58b is being changed to T4. In Figure D, reaction chamber 52 is again reoriented so that droplet 54 moves back to hot zone 58b for holding at T4. During this holding process, the temperature of another hot zone 58a is changed to T5 to further extend the temperature series of droplet holding. In other embodiments, only the temperature of one hot zone can be changed.

[0068] Figure 4 This is a flowchart illustrating exemplary steps of a method for thermally controlling chemical reactions in droplets. The listed steps can be performed in any suitable order and combination, and can be modified as described in other parts of this document. The steps shown in dashed boxes and those connected by dashed arrows provide exemplary options for modifying the basic method.

[0069] Step 71 represents the generation of emulsion droplets. The droplets can be generated outside the reaction chamber and then introduced into the reaction chamber, or they can be generated inside the reaction chamber (e.g., see Examples 5 and 6). Each droplet, or only a subset of droplets, can contain every reactant required for the chemical reaction. The droplets can be droplets containing a sample, with each droplet containing a portion of the same sample. The droplets can contain an analyte in a partial occupancy, meaning that each droplet in only a subset of the droplets contains at least one copy of the analyte, and optionally, each droplet in only a subset of the droplets does not contain a copy of the analyte. For example, the analyte can be a nucleic acid, a nucleic acid target sequence, a protein, a carbohydrate, a lipid, etc.

[0070] Droplets can be generated by any suitable step and / or apparatus. In some embodiments, droplets can be generated by dividing a bulk phase mixture, which may contain reactants, a sample, and any other suitable reagents for carrying out and / or detecting a chemical reaction. In some embodiments, droplets can be generated by fusing other droplets together.

[0071] Step 72 indicates that two or more hot zones can be created in the reaction chamber. These hot zones can have different, independently selectable, and controllable temperatures. The creation of the hot zones can be performed before or after the droplets appear in the reaction chamber. Each hot zone can occupy any suitable portion of the reaction chamber volume, such as at least about 10%, 20%, 30%, 40%, or 50% of the volume. In some embodiments, the combined hot zones can occupy more than 50%, 60%, 70%, or 80% of the total reaction chamber volume. The temperature of each hot zone can be kept substantially constant until the droplet treatment is complete, or it can be adjusted to a different temperature at any suitable time after the droplets are in the reaction chamber.

[0072] Step 73 indicates that the droplet can be contained in the reaction chamber. Containing the droplet means that the droplet is contained in the reaction chamber and can be mobile, stationary, or a combination thereof when retained.

[0073] Step 74 indicates that the reaction chamber can be reoriented to move droplets between at least two hot zones. Reorientation of the reaction chamber means substantially changing the direction of the reaction chamber relative to gravity or the G-force, causing the droplets to migrate collectively to different hot zones within the reaction chamber. For example, reorientation may include rotating the reaction chamber at least a quarter or a half turn, or about one turn, etc. (see Examples 2-5, for example).

[0074] Step 75 indicates that multiple droplets can be read. Reading means collecting reaction data from multiple droplets, where the reaction data relates to the occurrence of a chemical reaction. Reaction data can be collected by detecting reaction signals that reflect whether and / or the extent of a chemical reaction. Detecting reaction signals can include detecting any suitable signal type, such as optical or other electromagnetic signals, electrical signals, magnetic signals, radioactive decay, etc. In some embodiments, the reaction signal can be an amplification signal of an amplification reaction occurring in the droplets.

[0075] The reading process can be performed any suitable number of times. In some embodiments, multiple droplets may be read only once. For example, droplets may be read inside the reaction chamber (e.g., see Example 5) or removed from the reaction chamber and read outside the reaction chamber (e.g., see Example 6). In other embodiments, droplets may be read multiple times, for example, inside the reaction chamber, with the temperature of the droplets changing between each reading (e.g., see Example 5). If performed inside the reaction chamber, droplets that have moved due to the reorientation of the reaction chamber may be read, such as droplets passing through the sensing area of ​​a channel within the reaction chamber.

[0076] Step 76 indicates that the temperature of one or more thermal zones can be changed. In other words, the droplet can be continuously exposed to two or more different selected temperatures within the same thermal zone. Changing the temperature of the thermal zone allows, for example, altering the thermal cycling profile between different thermal cycles of the amplification reaction, or the melting or annealing profile of the amplicon generated in an amplification reaction involving chemical reactions.

[0077] The return arrow at 77 indicates that the reaction chamber can be reoriented multiple times. For example, multiple reorientations of the reaction chamber allow for thermal cycling of the droplet to facilitate polymerase chain reaction (PCR) or ligase chain reaction (LCR), etc. The droplet can be thermally cycled any suitable number of times, such as at least 10, 20, 25, or 30 times. Thermal cycling can be a two-step thermal cycle, where each cycle has only two temperature steps (e.g., denaturation temperature and annealing / extension temperature). In other embodiments, thermal cycling can be at least a three-step thermal cycle, where each thermal cycle has three or more temperature steps (e.g., denaturation temperature, annealing temperature, and extension temperature).

[0078] Figure 5 This is a block diagram of an exemplary system 80 for thermally controlling chemical reactions in droplets. System 80 may include an encapsulation portion 81, a reaction portion 82, and a detection portion 83. Here, portions 81-83 are shown as operating independently and in series (see also Example 6). However, portions 81-83 may have any suitable overlap. For example, the encapsulation portion 81 may be incorporated into the reaction portion 82 (e.g., see Example 5), and / or the detection portion 83 may include a portion of the reaction portion 82 (e.g., see Example 5).

[0079] The encapsulation portion 81 generates droplets of the emulsion. The encapsulation portion may include at least one droplet generator 84. The droplet generator 84 can receive the reaction mixture and the carrier liquid and form droplets of the reaction mixture surrounded by the carrier liquid. The droplet generator can operate through any suitable mechanism, such as crossflow, co-flow, flow-focusing, or confinement gradient. The mechanism can operate in any suitable mode, such as dripping, squeezing, jetting, tip-streaming, and tip-multi-breaking.

[0080] The reaction section 82 includes a reaction chamber 52, which is operatively connected to a thermal control system 86, a directional drive 87, a pump 88, and / or a centrifuge (e.g., see Example 5), etc. The reaction section 82 is exemplified by various reaction control devices disclosed herein, such as Examples 1-6.

[0081] A thermal control system 86 provides temperature control of the reaction chamber 52 to create two or more hot zones therein. The thermal control system may include any suitable number of heaters 89 thermally connected to each hot zone of the reaction chamber 52. Exemplary heaters (also referred to as heating devices) may be conductive, convective, and / or radiant heaters and may be located outside (or inside) the reaction chamber. At least one temperature sensor 90 may be operatively associated with each hot zone of the reaction chamber 52. At least one controller 91 may be communicated with the heaters 89 and the sensors 90 to form a feedback loop to maintain the temperature of each hot zone according to a setpoint.

[0082] The directional drive device 87 is operatively connected to the reaction chamber 52 and is configured to rotate the reaction chamber 52 relative to the gravity vector or G-force vector to change the orientation of the reaction chamber 52. The motor 92 of the directional drive device can generate torque, which can drive the reaction chamber 52 to rotate about a rotation axis. The directional drive device 87 can rotate the reaction chamber 52 in only one rotational direction or in the opposite rotational direction.

[0083] At least one pump 88 can be operatively connected to the reaction chamber 52. Operating the pump can drive fluid (such as a preheated carrier liquid) into and / or out of the reaction chamber. The reaction chamber can be isolated from the pump 88 by a gate that can be opened and closed at any suitable time.

[0084] The detection section 83 can be configured to detect any suitable light from the droplet, such as emitted light, scattered light, polarized light, and / or similar. For example, the detected light may include light emitted from a luminescent marker present in the droplet. The marker may be photoluminescent or chemiluminescent, etc. The light source 93 can generate light to illuminate the droplet located in the sensing region 94. Light from the light source 93 can be propagated to the sensing region 94 through any suitable irradiation optics. The sensing region 94 may be formed by a channel or chamber, etc. In some embodiments, the sensing region 94 may be at least a portion of a channel or chamber irradiated by the light source 93 and optically coupled to at least one photosensor 95. The sensing region 94 may be located inside or outside the reaction chamber 52. Reaction control devices having an internal sensing region within the reaction chamber are described elsewhere herein (e.g., in Examples 2 and 5).

[0085] III. Examples

[0086] This section describes additional aspects and features of methods and systems for thermally controlling chemical reactions in droplets. Any suitable aspects and features of this section may be combined with each other and with any suitable aspects and features described in other parts of this disclosure, such as Sections 1, 2, and 4, in any suitable combination. The embodiments in this section are for illustrative purposes and should not limit the full scope of this disclosure.

[0087] Example 1. Droplet trapping gate for reaction chamber

[0088] This embodiment describes an exemplary droplet trapping gate for a reaction chamber; see Figure 6 and 7 .

[0089] Figure 6 An exemplary aspect of a reaction control device 150 is shown, which is one embodiment of the reaction control device 50 described in Section 2. The reaction control device 150 includes a reaction chamber 152 containing droplets 154 encapsulated by a carrier liquid 156. A pair of hot zones 158a, 158b are located at opposite ends of the reaction chamber. The reaction chamber 152 has a curved inlet 164 and a curved outlet 166, each inlet or outlet having a radial curvature forming a droplet trapping gate 168 shaped like a swan's neck. The curvature of the inlet 164 and outlet 166 prevents droplets 154 from becoming trapped at either end of the reaction chamber 152, such that when the reaction chamber is properly reoriented, substantially all droplets 154 move as a group between the hot zones of the reaction chamber 152.

[0090] Figure 7 An exemplary aspect of a reaction control device 250 is shown, which is one embodiment of the reaction control device 50 described in Section 2. The reaction control device 250 includes a reaction chamber 252 that contains droplets 254 encapsulated by a carrier liquid 256. The reaction chamber has an inlet 264 and an outlet 266, each inlet or outlet having a recessed droplet trap 268. The width of the droplet trap 268 is smaller than the diameter of each droplet 254. Therefore, droplets 254 will not pass through the trap 268 when migrating between hot zones within the reaction chamber, but can be forced through the trap 268 by a connected pump to enter and / or exit the reaction chamber 252.

[0091] Example 2. Implementation of the Apparatus and System

[0092] This embodiment describes implementations of exemplary devices and systems for controlling chemical reactions in droplets; see Figure 8-11 .

[0093] Figure 8An exemplary aspect of a reaction control device 350 is shown, which is one embodiment of the reaction control device 50 described in Section 2. The reaction control device 350 includes a reaction chamber 352 containing droplets 354 encapsulated by a carrier liquid 356. The reaction chamber 352 has a pair of hot zones 358a and 358b, maintained at different temperatures (T1 and T2), located at opposite ends of the reaction chamber. The extent of each hot zone is generally indicated by a dashed box. The reaction chamber 352 forms a pair of wider sub-chambers 367a and 367b, connected to each other by a narrower channel 370. The hot zones 358a and 358b generally correspond to the sub-chambers 367a and 367b, respectively.

[0094] Figure 9 An exemplary reaction control device 450 is shown as an option, which is one embodiment of the reaction control device 50 described in Section 2. The reaction control device 450 includes a reaction chamber 452 containing droplets 454 encapsulated by a carrier liquid 456. The reaction chamber 452 has a pair of hot zones 458a, 458b, maintained at different temperatures (T1 and T2) and located at opposite ends of the reaction chamber. The extent of each hot zone is generally indicated by a dashed box. The reaction chamber 452 forms a pair of sub-chambers 467a, 467b, which are connected to each other via a droplet channel 470a and a carrier liquid channel 470b, respectively. The carrier liquid channel 470b has a pair of bends 471a, 471b, connected to the sub-chambers 467a, 467b, respectively. The reaction chamber 452 can be reoriented to move droplets 454 primarily or entirely through the droplet channel 470a from sub-chamber 467a to sub-chamber 467b (or vice versa). Simultaneously, a corresponding volume of liquid carrier 456 flows from sub-chamber 467b to sub-chamber 467a (or vice versa) through liquid carrier channel 470b. Each bend 471a, 471b is located in the same hot zone 458a or 458b as sub-chamber 467a or 467b. Therefore, the corresponding volume of liquid carrier 456 entering the sub-chamber has been preheated to the correct temperature, which helps reduce temperature fluctuations in the sub-chamber and allows droplets 454 to reach their respective desired temperatures more quickly.

[0095] Figure 10 Aspects of an exemplary system 580 for thermally controlling chemical reactions in droplets are shown. System 580 is one embodiment of system 80 in Section 2, which includes a reaction control device 550 optically coupled to a detection module 578. The reaction control device 550 includes a reaction chamber 552 containing a droplet 554 encapsulated by a carrier liquid 556. The reaction chamber 552 has a pair of hot zones 558a and 558b, which are maintained at different temperatures (T1 and T2) and are located at opposite ends of the reaction chamber. The extent of each hot zone is generally indicated by a dashed box. The reaction chamber 552 forms a pair of sub-chambers 567a and 567b, which are connected to each other via a droplet channel 570a and a carrier liquid channel 570b, respectively.

[0096] The detection section 583 of system 580 includes a detection module 578 optically coupled to each other and a sensing region 594 within the droplet channel 570a. The detection module 578 is configured to detect light, such as photoluminescence, from the droplet 554 as the droplet moves along the droplet channel 570a and passes through the sensing region 594, in response to the reorientation of the reaction chamber 552. The detection module 578 includes a light source 593, a photosensor 595, a beam splitter 596, and an objective lens 597. The light source 593 generates light radiation that propagates through the beam splitter 596 and the objective lens 597 to the sensing region 594 and illuminates it. This illumination can induce photoluminescent markers in the droplet 554 to emit photoluminescence. The photoluminescence is collected by the objective lens 597, propagates through the beam splitter 596, and is incident on the photosensor 595 for detection. For example, the detection module 578 can be used to collect amplification data during or after each of the multiple thermal cycles that the droplet 554 undergoes in the reaction chamber 552.

[0097] Figure 11 An exemplary reaction control device 650 is shown, which is one embodiment of the reaction control device 50 in Section 2. The reaction control device 650 includes a reaction chamber 652 containing droplets 654 encapsulated by a carrier liquid 656. The reaction chamber 652 has a pair of hot zones 658a, 658b, maintained at different temperatures (T1 and T2), and are generally demarcated by dashed lines. The reaction chamber 652 includes a pair of sub-chambers 667a, 667b, which are connected to each other by a pair of channels 670a, 670b. Figure AD shows the reaction chamber 652 being reoriented in a plane parallel to the channels 670a, 670b by rotating a full circle in the direction indicated by the turning arrow 660, thereby moving droplets 654 as a group from sub-chamber 667a to 667b. Droplets 654 can return to sub-chamber 667a by rotating another full circle in the same or opposite direction of rotation.

[0098] Example 3. Hot zone offset less than 180 degrees

[0099] This example describes an exemplary reaction control device having thermal zones that are rotated less than 180 degrees apart; see [link to example]. Figure 12 and 13 .

[0100] Figure 12An exemplary reaction control device 750 is shown, comprising a rectangular reaction chamber 752 containing an emulsion of droplets 754 encapsulated by a carrier liquid 756. The reaction chamber 752 has a pair of hot zones 758a and 758b, which are rotated 90 degrees relative to each other in the image plane. In other words, rotating the reaction chamber 752 by 90 degrees about an axis orthogonal to the image plane can move droplets 754 from hot zone 758a to hot zone 758b, or vice versa. Therefore, the reaction chamber 752 can be configured to have up to four hot zones, each located approximately at a different corner of the reaction chamber. An inlet 764 and an outlet 766 are in fluid communication with hot zones 758a and 758b, respectively; however, in other embodiments, one or both of the inlet and outlet may be placed at different corners of the reaction chamber 752.

[0101] Figure 13 An exemplary reaction control device 850 is shown, comprising a triangular reaction chamber 852 containing an emulsion of droplets 854 encapsulated by a carrier liquid 856. The reaction chamber 852 has three hot zones 858a-c, each at a temperature T1-T3, and rotated 120 degrees relative to each other in the image plane. In other words, rotating the reaction chamber 852 120 degrees about an axis orthogonal to the image plane moves droplets 854 from hot zone 858a to hot zone 858b or hot zone 858c. By rotating the reaction chamber one full revolution or each hot zone 120 degrees, droplets 854 can be continuously moved to the respective hot zones 858a-c. An inlet 864 and an outlet 866 are fluidly connected to hot zones 858a and 858b, respectively; however, in other embodiments, one or both of the inlet and outlet may be placed at different corners of the reaction chamber 852. In other embodiments, the reaction chamber can be any suitable polygonal shape, such as a pentagon (with up to five hot zones), a hexagon (with up to six hot zones), and so on.

[0102] Example 4. Addition of preheated carrier fluid

[0103] This embodiment describes an exemplary system 980, which includes... Figure 6 The reaction control device 150 and a pair of pumps (T1 pump 999a and T2 pump 999b) are used to drive the preheated carrier liquid 156 into the reaction chamber 152 containing droplets 154; see Figure 14 and 15 .

[0104] System 980 has a pair of hot zones 958a and 958b maintained at different temperatures T1 and T2. The hot zones are generally delineated by dashed boxes. Hot zones 958a and 958b include the hot zones 158a and 158b of reaction chamber 152 and the lengths 1001a and 1001b of pipes connected to the inlet 164 and outlet 166 of reaction chamber 152, respectively. Therefore, the lengths 1001a and 1001b of pipes act as reservoirs to store preheated carrier fluid 156. In other embodiments, hot zones 958a and 958b may include carrier fluid 156 contained in the chambers of pumps 999a and 999b and / or other chambers located in the flow path between pumps 999a and 999b and reaction chamber 152.

[0105] Figure 14 and 15 A pair of opposite configurations of reaction chamber 152 are shown. Figure 14 In this process, droplet 154, after migrating from hot zone 158b (where temperature T2), has reached hot zone 158a. To accelerate the heating or cooling of droplet 154 to temperature T1, a certain volume of carrier liquid 156, preheated to T1, is driven from the length 1001a of the tube into reaction chamber 152 by pump T1 999a. Pump T2 999b can promote the flow of carrier liquid 156, actively causing a corresponding volume of carrier liquid 156 to leave reaction chamber 152 through outlet 166. Alternatively, pump T2 999b can be replaced by a chamber that passively expands and contracts in response to the action of pump T1 999a. Figure 15 In the image, droplet 154 has just reached the hot zone 158b in response to the reversal of the reaction chamber 152 around a horizontal axis located in the image plane. To accelerate the heating or cooling of the droplet to T2, a certain volume of carrier liquid 156 preheated to T2 is driven into the reaction chamber 152 from the length 1001b of the tube by pump T2 999b. Pump T1 999a can promote the flow of carrier liquid 156, which can actively cause a corresponding volume of carrier liquid 156 to leave the reaction chamber 152 through inlet 164.

[0106] Example 5. Centrifuge system for driving droplet movement

[0107] This embodiment describes a centrifugation system 1080 comprising multiple reaction control devices 1050a-d, each reaction control device having a hot-zone reaction chamber 1052 for containing droplets containing samples (e.g., samples 1-4); see Figure 16-19 Centrifuge system 1080 is Figure 5 One implementation of system 80.

[0108] To increase the thermal cycling rate, the droplets need to move more rapidly between different hot zones. This can be achieved by using a centrifuge to generate a G-force to drive the quasi-droplet fluid to one end first. To drive it back to the other end, the reaction chamber is flipped over during rotation. However, system 1080 can also operate without centrifugal force, using gravity instead of a G-force to drive the droplets between hot zones.

[0109] The reaction control devices 1050a-d are supported by a rotor 1110, which rotates about an axis denoted as 1111 (see...). Figure 16 This rotation applies a force 1112 (G) to the emulsion contained in each reaction chamber 1052. Figure 16 Only the G-force of the reaction control device 1050a is shown in the figure. The G-force 1112 can be at least 2, 5, 10, 25, 50 or 100 times the force of gravity, so that the droplets can move more rapidly between hot zones relative to gravity-driven migration.

[0110] Each reaction control unit 1050a-d has a pair of heaters 1089a, 1089b to create hot zones 1058a, 1058b of different temperatures in each reaction chamber 1052 (e.g., T1 and T2, respectively) (see See Figure 16 and 17 Each directional actuator 1087 and each reaction control device 1050a-d (see...) Figure 16 The components are operatively connected together. The directional drive is configured to change the orientation of the reaction control device (indicated by the rotating arrow 1060 of reaction control device 1050a) to move droplets between pairs of hot zones as rotor 1110 rotates. This change of orientation is relative to the G-force 1112 applied to the reaction control device. Each directional drive 1087 can rotate the corresponding reaction control device 1050a-d by any suitable angle, for example, by flipping each device half a turn about its respective axis of rotation in the described embodiment, which is transverse to or parallel to the axis of rotation of rotor 1110. Reorientation of each reaction chamber 1052 causes droplets to move from one hot zone 1058a or 1058b to another hot zone 1058b or 1058a.

[0111] Figure 17The reaction control device 1050a of system 1080 is shown to be used for a two-step thermal cycle to facilitate nucleic acid amplification in droplets (e.g., only a subset of the droplets in chamber 1052 contains the target sequence). As shown, an amplification signal can be detected from droplet 1054 during or after each cycle (e.g., each thermal cycle) of any suitable number of thermal cycles. Reaction chamber 1052 has a pair of channels 1070a, 1070b that connect a pair of sub-chambers 1067a, 1067b to each other. Sub-chamber 1067a is maintained at temperature T1 by heater 1089a, and sub-chamber 1067b is maintained at temperature T2 by heater 1089b. In each thermal cycle, droplet 1054 migrates to hot zones 1058a (temperature T1) and hot zones 1058b (temperature T2).

[0112] Figure 17 Figure A shows the process of droplet 1054 migrating from sub-chamber 1067a (hot zone 1058a at temperature T1) to sub-chamber 1067b (hot zone 1058b at temperature T2) through channel 1070b. The corresponding volume of carrier liquid 1056 is moving in the opposite direction through channel 1070a.

[0113] Figure 17 Figure B shows the reaction control device 1050a rotated half a turn relative to Figure A (to change the orientation of reaction chamber 1052 relative to force G 1112). Droplet 1054 is migrating from sub-chamber 1067b (hot zone 1058b at temperature T2) to sub-chamber 1067a (hot zone 1058a at temperature T1) through channel 1070a. In other words, droplet 1054 can migrate alternately through two channels 1070a, 1070b (compare Figures A and B). As each droplet passes through the sensing area 1094a of channel 1070a, detection module 1078 of system 1080 collects amplification data from droplet 1054. In other embodiments, reaction chamber 1052 may have only one channel connecting sub-chambers 1067a and 1067b to each other (e.g., see Section 2). In other embodiments, reaction chamber 1052 may be configured such that droplets 1054 migrate back and forth between hot zones primarily or entirely through the same channel (a pair of channels).

[0114] Figure 17 This illustrates the case where amplification signals are detected at only one temperature. However, by detecting the amplification signals of droplet 1054 at two, three, or more different temperatures, the amount of information in the amplification assay can be greatly increased, allowing for the differentiation of amplified products with different melting temperatures. For example, Figure 18Figure AD shows the reaction control device 1050a used to generate the melting / annealing curves of the amplified product in the droplet. The amplification signal is detected using the detection module 1078 from the droplet, which alternately passes through sensing regions 1094a and 1094b in response to each reversal of the reaction chamber 1052. The droplet exhibits a series of increasing or decreasing temperatures T3-T6 in Figure AD (see also...). Figure 3 The amplified signal can be detected before and / or after the droplet's thermal cycling is complete. In Figure A, droplet 1054 moves from hot zone 1058a at temperature T3 through sensing zone 1094b to hot zone 1058b at temperature T4. In Figure B, droplet 1054 moves from hot zone 1058b at temperature T4 through sensing zone 1094a to hot zone 1058a at its current temperature T5. In Figure C, droplet 1054 moves from hot zone 1058a at temperature T5 through sensing zone 1094b to hot zone 1058b at its current temperature T6. In Figure D, droplet 1054 moves from hot zone 1058a at temperature T6 through sensing zone 1094a to hot zone 1058b (still at temperature T5).

[0115] Figure 19 Showing Figure 17 The reaction control device 1050a is used to generate droplets (Figures A and B), thermally cycle the droplets (Figures C and D), and detect the amplification signal of the droplets (Figure E). In Figure A, the reaction chamber 1052 contains a carrier liquid 1056 that is immiscible with each other and a (non-separated) reaction mixture 1114. In Figure B, the reaction chamber 1052 is flipped relative to Figure A. In response, the reaction mixture 1114 moves from hot zone 1058a to hot zone 1058b through channel 1070a. When the reaction mixture leaves the outlet of channel 1070a, the reaction mixture 1114 is separated into droplets 1054. In Figures C and D, the droplets 1054 move back and forth between hot zones 1058a and 1058b by repeatedly flipping the reaction chamber 1052, performing a two-step thermal cycle. In Figure E, the detection module 1078 detects the amplification signal from the droplets 1054 passing through the sensing zone 1094a.

[0116] Example 6. Flow system for thermal control

[0117] This embodiment describes a circulation system 1180, which includes an encapsulation assembly 1181, a reaction control device 1150, and sensing zones 1194 arranged in series; see Figure 20 Distribution system 1180 is Figure 5 One implementation of system 80.

[0118] Encapsulation component 1181 has a droplet generator 1184. The droplet generator receives a reaction mixture 1214 containing a sample through sample inlet 1216 and a carrier liquid 1156, such as oil, through carrier inlet 1218. The reaction mixture 1214 and the carrier liquid 1156 flow in different channels to a channel intersection 1220, where a droplet emulsion is generated in the carrier liquid 1156. The emulsion flows through inflow channel 1222 to reaction control device 1150. Reaction control device 1150 has a reaction chamber 1152, which includes at least two thermal zones 1158a, 1158b. Droplets of the emulsion can be heated to different temperatures in reaction chamber 1152 by changing their orientation relative to gravity, as described elsewhere herein, for example, through thermal cycling. Changing the orientation can control chemical reactions, such as the amplification of target sequences in the droplets. The emulsion then flows out of reaction chamber 1152 through outflow channel 1224 and through sensing area 1194, which is optically coupled to detection module 1178. The detection module may have a light source 1193 to illuminate the sensing area 1194, and a photosensor 1195 to detect the light from the sensing area 1194. The emulsion then flows downstream of the sensing area 1194 to the waste.

[0119] IV. Aspects of Choice

[0120] This section describes the aspects selected in this disclosure as a series of indexed paragraphs.

[0121] A1. A method for controlling a chemical reaction, the method comprising: (i) creating a first hot zone and a second hot zone in a reaction chamber, the temperatures of the first hot zone and the second hot zone being different from each other; (ii) containing an emulsion in the reaction chamber, the emulsion comprising droplets encapsulated by a carrier liquid and having a density mismatch with the carrier liquid, each droplet comprising one or more reactants for the chemical reaction; and (iii) changing the orientation of the reaction chamber to move the droplets from the first hot zone to the second hot zone such that the rate of the chemical reaction changes in at least one subset of the droplets.

[0122] A2. The method described in paragraph A1, wherein changing direction initiates or accelerates a chemical reaction in at least one subset of the droplets.

[0123] A3. The method described in paragraphs A1 or A2 further includes reorienting the reaction chamber to return the droplets to the first hot zone.

[0124] A4. The method as described in paragraph A3 further includes changing the temperature of the first hot zone while the droplet is in the second hot zone before reorientation.

[0125] A5. The method as described in paragraphs A3 or A4, wherein the reorientation again alters the rate of chemical reactions in at least one subset of the droplets.

[0126] A6. The method as described in paragraph A5, wherein reorientation slows down or stops the chemical reaction in at least a subset of the droplets.

[0127] A7. The method as described in any one of paragraphs A3 to A6, wherein the plurality of droplets are redirected to pass through a sensing zone within a reaction chamber, the method further comprising detecting a signal relating to a chemical reaction from the plurality of droplets passing through the sensing zone.

[0128] A8. The method as described in paragraph A7, wherein the detection signal includes detecting photoluminescence from a plurality of droplets.

[0129] A9. The method as described in any of paragraphs A1 to A8, wherein the reaction chamber has at least three hot zones, including a first hot zone and a second hot zone, wherein the at least three hot zones are respectively maintained at selected, different temperatures from each other, the method further comprising rotating the reaction chamber to move droplets from the first hot zone to each of the other at least three hot zones.

[0130] A10. The method as described in any of paragraphs A1 to A9, wherein the chemical reaction is catalyzed by an enzyme, and the enzyme exists only in a subset of the droplets, optionally having a Poisson distribution in the droplets.

[0131] A11. The method as described in paragraph A10, wherein one or more reactants include reactants having photoluminescence, the photoluminescence being altered by a chemical reaction, the method further comprising detecting photoluminescence from a plurality of droplets.

[0132] A12. The method as described in any of paragraphs A1 to A9, wherein changing direction facilitates the generation of amplicons corresponding to target sequences present in at least one subset of the droplets, wherein, optionally, the reactant in one or more reactants is an oligonucleotide that hybridizes with the amplicon and / or target sequence at one or two of the different temperatures.

[0133] A13. As described in paragraph A12, the oligonucleotide hybridizes with the amplicon and / or target sequence only at one of the different temperatures.

[0134] A14. The method as described in paragraphs A12 or A13, wherein at least one of the droplets does not contain a target sequence.

[0135] A15. The method as described in any of paragraphs A12 to A14, wherein at least one of the droplets contains only one copy of the target sequence before changing direction.

[0136] A16. The method described in any of paragraphs A12 to A15, wherein each droplet contains polymerase, ligase and / or reverse transcriptase.

[0137] A17. The method described in any of paragraphs A12 through A16, wherein each droplet comprises one or more mononucleotides as reactants in a chemical reaction.

[0138] A18. The method described in any of paragraphs A12 to A17, wherein the chemical reaction adds one or more nucleotides to an oligonucleotide.

[0139] A19. The method described in any of paragraphs A1 to A9 and A11 to A18 further comprises, when the droplet is located in the second hot zone, performing an isothermal amplification reaction in at least one subset of the droplet, wherein the isothermal amplification reaction comprises a chemical reaction.

[0140] A20. The method described in paragraph A19 further comprises reorienting the reaction chamber to move droplets from the second hot zone to the first hot zone to slow down or stop the isothermal amplification reaction.

[0141] A21. The method as described in any of paragraphs A1 to A9 and A11 to A18, further comprising performing PCR, said PCR comprising a chemical reaction in at least one subset of the droplets while the droplets are retained in the reaction chamber.

[0142] A22. The method described in paragraph A21, wherein performing PCR includes thermally cycling the droplet by moving the droplet multiple times to each of the first and second hot zones by reorienting the reaction chamber multiple times.

[0143] A23. The method described in paragraph A22 further comprises collecting amplification data from the droplet while the droplet remains in the reaction chamber.

[0144] A24. The method as described in paragraph 23, wherein the amplified data is collected from multiple droplets passing through the sensing zone in the reaction chamber.

[0145] A25. The method as described in paragraphs A23 or A24, wherein collecting amplification data includes detecting photoluminescence from multiple droplets.

[0146] A26. The method as described in any of paragraphs A23 to A25, wherein the thermal cycling includes passing the droplet through a plurality of thermal cycles, wherein collecting amplification data includes collecting amplification data from at least one subset of the droplet during or after each of two or more of the plurality of thermal cycles, optionally after each of the plurality of thermal cycles.

[0147] A27. The method as described in any of paragraphs A23 to A26, wherein collecting amplification data comprises collecting amplification data from at least one subset of droplets at each of a series of increasing or decreasing temperatures, and using the amplification data to generate a melting curve or an annealing curve.

[0148] A28. The method as described in paragraph A27 further includes changing the temperature of at least one of the first and second hot zones between a pair of temperatures in the series of temperatures.

[0149] A29. The method described in any of paragraphs A21 to A28, wherein PCR is driven by a two-step thermal cycle using a first thermal zone and a second thermal zone.

[0150] A30. The method as described in any of paragraphs A21 to A28, wherein PCR is performed by driving a droplet through a series of thermal cycles, wherein the reaction chamber includes a third thermal zone having a selected temperature different from that of the first and second thermal zones, and the method further comprises moving the droplet to each of the first, second, and third thermal zones in each thermal cycle.

[0151] A31. The method described in any of paragraphs A19 to A30, wherein the droplet contains a probe having a label, and the method further includes detecting an amplified signal from the label.

[0152] A32. The method as described in paragraph A31, wherein each droplet includes a polymerase with exonuclease activity, thereby degrading copies of the probe during isothermal amplification or PCR.

[0153] A33. The method as described in paragraph A31, wherein the probe is not subjected to isothermal amplification or PCR degradation.

[0154] A34. The method as described in any of paragraphs 18 to A33, wherein the droplet contains an embedded dye, the method further comprising detecting an amplified signal from the embedded dye.

[0155] A35. The method described in any of paragraphs A18 to A34, wherein the target sequence or its complementary sequence is amplified by isothermal amplification or PCR, and wherein only a subset of the droplets contains the target sequence.

[0156] A36. The method as described in any of paragraphs A1 to A35, wherein the first hot zone and the second hot zone are interconnected by a channel, and wherein a change of direction causes at least one subset of the droplets to move from the first hot zone to the second hot zone through the channel.

[0157] A37. As described in paragraph A36, wherein a pair of channels respectively connect the first hot zone and the second hot zone to each other.

[0158] A38. The method as described in paragraph A37, wherein the pair of channels is a droplet channel and a liquid-carrying channel, and wherein changing direction causes the droplet to move primarily or entirely through the droplet channel between the first and second hot zones.

[0159] A39. The method as described in paragraph A38, wherein the liquid-carrying channel has a first end located in a first hot zone and a second end located in a second hot zone.

[0160] A40. The method as described in paragraph A39, wherein the first end portion of the liquid-carrying channel adjacent to the first end and / or the second end portion of the liquid-carrying channel adjacent to the second end has a tortuous path.

[0161] A41. The method described in any of paragraphs A1 to A40 further includes forming droplets outside the reaction chamber; and introducing the formed droplets into the reaction chamber.

[0162] A42. The method described in any of paragraphs A1 to A40 further includes forming droplets in the reaction chamber.

[0163] A43. The method described in any of paragraphs A1 to A42, wherein the density of each droplet is lower than that of the carrier liquid.

[0164] A44. The method described in any of paragraphs A1 to A43, wherein the density of each droplet is higher than that of the carrier liquid.

[0165] A45. The method described in any of paragraphs A1 to A44 further comprises rotating the reaction chamber multiple full revolutions, causing the droplets to move alternately as a group between a first hot zone and a second hot zone.

[0166] A46. The method described in any of paragraphs A1 to A45 further comprises alternating rotation of the reaction chamber in opposite directions of rotation, causing the droplets to move alternately as a group between the first and second hot zones.

[0167] A47. The method as described in any of paragraphs A1 to A46 further includes rotating the reaction chamber in a series of rotation intervals, each rotation interval causing droplets to move as a group from one of the first and second hot zones to the other, and wherein said rotating reaction chamber further includes pausing the rotation of the reaction chamber between successive rotation intervals of the series of rotation intervals.

[0168] A48. The method as described in paragraph A47, wherein the rotating reaction chamber includes pausing the rotation of the container during a series of pause intervals of at least two different durations.

[0169] A49. The method of any one of paragraphs A1 to A48 further includes using a pump to drive a preheated carrier liquid into the reaction chamber of the second hot zone as the droplet moves from the first hot zone to the second hot zone.

[0170] A50. The method described in paragraph A49, wherein the preheated carrier fluid is preheated to the temperature of the second hot zone.

[0171] A51. The method as described in paragraphs A49 or A50 further comprises reorienting the reaction chamber to move the droplet from the second hot zone to the first hot zone, and using a pump to drive the preheated carrier liquid into the reaction chamber of the first hot zone as the droplet moves from the second hot zone to the first hot zone.

[0172] A52. The method as described in paragraph A51, wherein the preheated carrier fluid driven into the first hot zone is preheated to the temperature of the first hot zone.

[0173] A53. The method described in any of paragraphs A1 to A52, wherein the change of direction is performed while the reaction chamber is being rotated in a centrifuge.

[0174] A54. The method described in any of paragraphs A1 to A53 further includes detecting a reaction signal from a plurality of droplets.

[0175] A55. The method as described in paragraph A54, wherein detecting the reaction signal includes detecting the reaction signal from each of a plurality of droplets passing through the sensing zone of the reaction chamber.

[0176] A56. The method as described in paragraphs A54 or A55, wherein detecting the reaction signal includes detecting an amplified signal from multiple droplets after each of multiple thermal cycles.

[0177] A57. The method described in any of paragraphs A54 to A56, wherein detecting the reaction signal comprises detecting the amplified signal from multiple droplets at each of a series of rising or falling temperatures in the reaction chamber to generate a melting curve or an annealing curve.

[0178] A58. The method as described in any of paragraphs A1 to A57, wherein the first heating device and the second heating device remain associated with the first and second hot zones, respectively, when the orientation of the reaction chamber changes.

[0179] A59. The method described in any of paragraphs A1 to A58, wherein if the density of the droplet is less than that of the carrier liquid, the direction is changed so that the first hot zone moves from a height higher than that of the second hot zone to a height lower than that of the second hot zone, and vice versa if the density of the droplet is greater than that of the carrier liquid.

[0180] B1. A system for controlling a chemical reaction, the system comprising: (i) a reaction chamber for containing an emulsion, the emulsion comprising droplets encapsulated by a carrier liquid and having a density mismatch with the carrier liquid, each droplet containing one or more reactants for the chemical reaction; (ii) a thermal control system configured to create a first hot zone and a second hot zone in the reaction chamber having different temperatures from each other; and (iii) a directional drive device configured to change the orientation of the reaction chamber to move the droplets as a group between the first hot zone and the second hot zone.

[0181] B2. The system as described in paragraph B1 further includes a droplet generator configured to generate droplets, the droplet generator being connected to or connectable to a reaction chamber so that droplets move from the droplet generator to the reaction chamber.

[0182] B3. The system as described in paragraphs B1 or B2 further includes a detection module configured to detect reaction signals from a plurality of droplets, each of the plurality of droplets being located in a sensing area inside or downstream of the reaction chamber.

[0183] B4. The system as described in paragraph B3, wherein the sensing zone is a region of the channel, and the detection module includes a light source for illuminating each of a plurality of droplets passing through the sensing zone, and a detector for detecting light from the sensing zone.

[0184] As used in this disclosure, the term "exemplary" means "illustrative" or "used as an example." Similarly, the term "example" means "illustrative." Neither of these words implies desirability or superiority.

[0185] The above disclosure may include several different inventions with independent uses. While various of these inventions have been disclosed in their preferred forms, the specific embodiments of the invention disclosed and illustrated herein should not be considered limiting, as many variations are possible. The subject matter of this invention includes all novel and non-obvious combinations and sub-combinations of the various elements, features, functions, and / or characteristics disclosed herein. Certain novel and non-obvious combinations and sub-combinations are specifically pointed out in the following paragraphs. Inventions embodied in other combinations and sub-combinations of features, functions, elements, and / or characteristics may be claimed in applications claiming priority to this application or related applications. Such paragraphs, whether referring to different or identical inventions, and whether broader, narrower, identical, or different from the scope of the original paragraph, are also considered to be included within the subject matter of the invention disclosed herein. Furthermore, ordinal designations used to identify elements, such as first, second, or third, are used to distinguish elements and do not indicate a specific position or order of these elements unless otherwise specifically stated.

Claims

1. A method for controlling a chemical reaction, the method comprising: A first and a second thermal zone with different temperatures are created in the reaction chamber; The reaction chamber contains an emulsion comprising droplets encapsulated by a carrier liquid and having a density mismatch with the carrier liquid, each droplet comprising one or more reactants for the chemical reaction; Changing the orientation of the reaction chamber, moving the droplets from the first hot zone to the second hot zone, causes a change in the rate of the chemical reaction in at least one subset of the droplets; and The preheated carrier liquid is driven into the reaction chamber; The change in direction facilitated the generation of amplicones, which corresponded to target sequences present in at least one subset of the droplets; and At least one of the droplets does not contain a target sequence.

2. The method of claim 1, wherein changing the direction initiates or accelerates a chemical reaction in at least a subset of the droplets.

3. The method of claim 1, further comprising reorienting the reaction chamber to return the droplet to the first hot zone.

4. The method of claim 3, further comprising changing the temperature of the first hot zone while the droplet is located in the second hot zone before reorientation.

5. The method of claim 3, wherein, The reorientation again alters the rate of chemical reactions in at least one sub-group of the droplet.

6. The method of claim 5, wherein, The reorientation slows down or stops the chemical reaction in at least one subset of the droplets.

7. The method of claim 3, wherein, The method further includes reorienting multiple droplets through a sensing zone within a reaction chamber, and detecting signals related to the chemical reaction from the multiple droplets passing through the sensing zone.

8. The method of claim 7, wherein, The detection signal includes the detection of photoluminescence from multiple droplets.

9. The method of claim 1, wherein the reaction chamber has at least three hot zones, including a first hot zone and a second hot zone, and wherein the at least three hot zones are respectively maintained at selected, different temperatures from each other, the method further comprising rotating the reaction chamber to move droplets from the first hot zone to each of the other at least three hot zones.

10. The method of claim 1, wherein the chemical reaction is catalyzed by an enzyme, and the enzyme exists only in a subset of the droplets.

11. The method of claim 10, wherein the one or more reactants include reactants having photoluminescence, the photoluminescence being altered by a chemical reaction, the method further comprising detecting photoluminescence from a plurality of droplets.

12. The method of claim 1, wherein the reactant in one or more reactants is an oligonucleotide, the oligonucleotide hybridizing with an amplicon and / or a target sequence at one or two of the different temperatures.

13. The method of claim 12, wherein the reactant in one or more of the reactants is an oligonucleotide that hybridizes with the amplicon and / or target sequence at one or two of the different temperatures, and the oligonucleotide hybridizes with the amplicon and / or target sequence only at one of the different temperatures.

14. The method of claim 1, wherein at least one of the droplets contains only one copy of the target sequence before changing direction.

15. The method of claim 1, wherein each droplet contains polymerase, ligase and / or reverse transcriptase.

16. The method of claim 1, wherein each droplet comprises one or more mononucleotides as reactants in a chemical reaction.

17. The method of claim 1, wherein, The chemical reaction involves adding one or more nucleotides to an oligonucleotide.

18. The method of claim 1, further comprising, when the droplet is located in the second hot zone, performing an isothermal amplification reaction in at least one subset of the droplet, wherein, The isothermal amplification reaction includes a chemical reaction.

19. The method of claim 18, further comprising reorienting the reaction chamber to move droplets from the second hot zone to the first hot zone to slow down or stop the isothermal amplification reaction.

20. The method of claim 1, further comprising performing PCR, said PCR comprising a chemical reaction in at least a subset of the droplets while the droplets are retained in the reaction chamber.

21. The method of claim 20, wherein performing PCR comprises thermally cycling the droplet by repeatedly moving the droplet to each of the first and second hot zones by repeatedly reorienting the reaction chamber.

22. The method of claim 21, further comprising collecting amplification data from the droplet while the droplet remains in the reaction chamber.

23. The method of claim 22, wherein, The amplified data is collected from multiple droplets passing through the sensing zone in the reaction chamber.

24. The method of claim 22, wherein collecting amplified data includes detecting photoluminescence from a plurality of droplets.

25. The method of claim 22, wherein the thermal cycling comprises subjecting the droplet to multiple thermal cycles, wherein, Collecting amplification data includes collecting amplification data from at least one subset of droplets during or after each of two or more of the plurality of thermal cycles.

26. The method of claim 22, wherein collecting amplification data comprises collecting amplification data from at least one subset of droplets at each of a series of increasing or decreasing temperatures, and using the amplification data to generate a melting curve or an annealing curve.

27. The method of claim 26, further comprising changing the temperature of at least one of the first and second hot zones between a pair of temperatures in the series of temperatures.

28. The method of claim 20, wherein PCR is driven by a two-step thermal cycle using a first thermal zone and a second thermal zone.

29. The method of claim 20, wherein PCR is performed by driving a droplet through a series of thermal cycles, wherein the reaction chamber includes a third thermal zone having a selected temperature different from that of the first and second thermal zones, the method further comprising moving the droplet to each of the first, second and third thermal zones in each thermal cycle.

30. The method of claim 18, wherein the droplet comprises a probe having a label, the method further comprising detecting an amplified signal from the label.

31. The method of claim 30, wherein each droplet comprises a polymerase having exonuclease activity, thereby degrading copies of the probe during isothermal amplification or PCR.

32. The method of claim 30, wherein the probe is not subjected to isothermal amplification or PCR degradation.

33. The method of claim 17, wherein the droplet contains an embedded dye, the method further comprising detecting an amplified signal from the embedded dye.

34. The method of claim 17, wherein the target sequence or its complementary sequence is amplified by isothermal amplification or PCR, and wherein only a subset of the droplets contains the target sequence.

35. The method of claim 1, wherein the first hot zone and the second hot zone are interconnected via a channel, and wherein, The change of direction causes at least a subset of the droplets to move through the channel from the first hot zone to the second hot zone.

36. The method of claim 35, wherein a pair of channels respectively connect the first hot zone and the second hot zone to each other.

37. The method of claim 36, wherein the pair of channels is a droplet channel and a liquid-carrying channel, and wherein changing direction causes the droplet to move primarily or entirely through the droplet channel between the first and second hot zones.

38. The method of claim 37, wherein the liquid-carrying channel has a first end located in a first hot zone and a second end located in a second hot zone.

39. The method of claim 38, wherein the first end portion of the liquid-carrying channel adjacent to the first end and / or the second end portion of the liquid-carrying channel adjacent to the second end have a tortuous path.

40. The method of claim 1, further comprising forming droplets outside the reaction chamber; and introducing the formed droplets into the reaction chamber.

41. The method of claim 1, further comprising forming droplets within the reaction chamber.

42. The method of claim 1, wherein the density of each droplet is lower than that of the carrier liquid.

43. The method of claim 1, wherein the density of each droplet is higher than that of the carrier liquid.

44. The method of claim 1, further comprising rotating the reaction chamber a plurality of full revolutions, causing the droplets to move alternately as a group between a first hot zone and a second hot zone.

45. The method of claim 1, further comprising alternatingly rotating the reaction chamber in opposite directions of rotation, causing the droplets to move alternately as a group between the first and second hot zones.

46. ​​The method of claim 1, further comprising rotating the reaction chamber in a series of rotation intervals, each rotation interval causing droplets to move as a group from one of the first hot zone and the second hot zone to the other, and wherein said rotating reaction chamber further comprises pausing the rotation of the reaction chamber between successive rotation intervals of the series of rotation intervals.

47. The method of claim 46, wherein the rotating reaction chamber includes pausing the rotation of the container during a series of pause intervals of at least two different durations.

48. The method of claim 1, further comprising pumping preheated carrier liquid into the second hot zone as the droplet moves from the first hot zone to the second hot zone.

49. The method of claim 48, wherein the preheated carrier fluid is preheated to the temperature of the second hot zone.

50. The method of claim 48, further comprising reorienting the reaction chamber to move the droplet from the second hot zone to the first hot zone, and using a pump to drive the preheated carrier liquid into the reaction chamber of the first hot zone as the droplet moves from the second hot zone to the first hot zone.

51. The method of claim 50, wherein the preheated carrier fluid driven into the first hot zone is preheated to the temperature of the first hot zone.

52. The method of claim 1, wherein the change of direction is performed while the reaction chamber is rotated in the centrifuge.

53. The method of claim 1, further comprising detecting reaction signals from a plurality of droplets.

54. The method of claim 53, wherein detecting the reaction signal comprises detecting the reaction signal from each of a plurality of droplets passing through the sensing zone of the reaction chamber.

55. The method of claim 53, wherein detecting the reaction signal comprises detecting an amplified signal from a plurality of droplets after each of a plurality of thermal cycles.

56. The method of claim 53, wherein detecting the reaction signal comprises detecting the amplified signal from the plurality of droplets at each of a series of increasing or decreasing temperatures in the reaction chamber to generate a melting curve or an annealing curve.

57. The method of claim 1, wherein the first heating device and the second heating device remain associated with the first and second hot zones, respectively, when the orientation of the reaction chamber changes.

58. The method of claim 1, wherein if the density of the droplet is less than that of the carrier liquid, the direction is changed so that the first hot zone moves from a height higher than that of the second hot zone to a height lower than that of the second hot zone, and vice versa if the density of the droplet is greater than that of the carrier liquid.

59. A system for controlling a chemical reaction, the system comprising: A reaction chamber for containing an emulsion comprising droplets encapsulated by a carrier liquid and having a density mismatch with the carrier liquid, each droplet containing one or more reactants for a chemical reaction; The thermal control system is configured to create a first hot zone and a second hot zone with different temperatures in the reaction chamber; as well as A directional drive device is configured to change the direction of the reaction chamber, causing droplets to move as a group between a first hot zone and a second hot zone; and The pump is configured to drive the preheated carrier liquid into the second hot zone as the droplets move from the first hot zone to the second hot zone; The change in direction facilitated the generation of amplicones, which corresponded to target sequences present in at least one subset of the droplets; and At least one of the droplets does not contain a target sequence.

60. The system of claim 59, further comprising a droplet generator configured to generate droplets, the droplet generator being connected to or connectable to a reaction chamber such that droplets move from the droplet generator to the reaction chamber.

61. The system of claim 59, further comprising a detection module configured to detect reaction signals from a plurality of droplets, each of the plurality of droplets being located in a sensing area inside or downstream of the reaction chamber.

62. The system of claim 61, wherein the sensing zone is a region of the channel, and the detection module includes a light source for illuminating each of a plurality of droplets passing through the sensing zone, and a detector for detecting light from the sensing zone.

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