Systems and methods involving continuous flow droplet reactions
阅读说明：本技术 涉及连续流动液滴反应的系统和方法 (Systems and methods involving continuous flow droplet reactions ) 是由 科迪·扬布尔 安德鲁·海奇 安德鲁·拉森 于 2017-11-27 设计创作，主要内容包括：本文描述了涉及连续流动仪器的系统,其包括用于数字液滴定量的所有必要组件,无需在仪器操作的各阶段之间引入关键试剂或收集和转移液滴。数字定量可以在没有任何额外的流体或消耗品处理并且不会使流体暴露于外部污染的风险的情况下进行。(Described herein are systems involving continuous flow instruments that include all the necessary components for digital droplet quantification without the need to introduce critical reagents or collect and transfer droplets between stages of instrument operation. Digital quantification can be performed without any additional fluid or consumable handling and without exposing the fluid to the risk of external contamination.)
1, a system for conducting assays in continuous flow, the system comprising:
a. a flow path for the continuous phase comprising an th inlet and a th outlet;
b. , a zero dead volume syringe configured to provide dispersed phase;
c. a second zero dead volume injector configured to provide a second dispersed phase;
d. a coalescer;
e. a reactor;
f. a detector; and
g. and a controller.
2. The system of claim 1, wherein the continuous phase comprises a hydrophobic agent.
3. The system of claim 1, wherein the continuous phase comprises oil.
4. The system of claim 1, wherein the continuous phase comprises a hydrophobic fluorinated oil.
5. The system of claim 4, wherein the hydrophobic fluorinated oil comprises an agent selected from the group consisting of: FC-3283, FC-40, FC-43, FC-70, 3-ethoxy-1, 1,1,2,3,4,4,5,5,6,6, 6-dodecafluoro-2-trifluoromethyl-hexane, 2,3,3,4,4, 4-heptafluoro-1-butanol, CF3CF2CF2CH2OH, perfluorooctane, perfluorohexane, 1,1, 1-trifluorooctane and 11,1,2, 2-pentafluorodecane.
6. The system of claim 1, wherein at least of the th and second dispersed phases include a hydrophilic agent.
7. The system of claim 1, wherein at least of the th and second dispersed phases include an aqueous agent.
8. The system of claim 1, wherein at least of the th dispersed phase and the second dispersed phase comprise water.
9. The system of claim 1, wherein a surface of the flow path comprises a polymer coating.
10. The system of claim 1, wherein a surface of the flow path comprises a fluorophilic coating.
11. The system of claim 1, wherein a surface of the flow path comprises a fluoropolymer coating.
12. The system of claim 11, wherein said fluoropolymer comprises a reagent selected from the group consisting of: polyvinyl fluoride (PVF), polyvinylidene fluoride (PVDF), Polytetrafluoroethylene (PTFE), Polychlorotrifluoroethylene (PCTFE), perfluoroalkoxy polymers, Fluorinated Ethylene Propylene (FEP), polyethylene tetrafluoroethylene (ETFE), polyethylene chlorotrifluoroethylene (ECTFE), perfluoroelastomers, fluorocarbons, fluoroelastomers, perfluoropolyethers (PFPE), perfluorosulfonic acid (PFSA), and perfluoropolyoxycyclobutane.
13. The system of claim 1, further comprising a surfactant.
14. The system of claim 13, wherein the surfactant comprises an agent selected from the group consisting of fluorocarbons, hydrocarbons, and siloxanes.
15. The system of claim 1, wherein a volume of at least of the th dispersed phase and the second dispersed phase is in a range of about 1 picoliter to about 1 milliliter.
16. The system of claim 1, wherein the flow path comprises an internal hydraulic diameter in a range of about 0.001 "to about 0.5".
17. The system of claim 1, wherein the system comprises multiple copies of a nucleic acid segment.
18. The system of claim 1, wherein the th dispersed phase comprises at least substrates, and wherein the second dispersed phase comprises at least reagents.
19. The system of claim 18, wherein the th dispersed phase comprises a biological sample or th prodrug.
20. The system of claim 18, wherein the second dispersed phase comprises a lysis reagent or a second prodrug.
21. The system of claim 1, wherein the th dispersed phase includes at least nucleic acid molecules, and wherein the second dispersed phase includes nucleic acid amplification reagents.
22. The system of claim 21, wherein the nucleic acid amplification reagents comprise reagents selected from the group consisting of primers, probes, blends, dntps, buffers, and enzymes.
23. The system of claim 1, wherein the detector is configured to measure a fluorescent signal.
24. The system of claim 1, wherein the coalescer comprises an external energy source.
25. The system of claim 24, wherein the external energy source comprises an electric field between at least two electrodes.
26. The system of claim 25, wherein the electrical potential between the at least two electrodes is in the range of about 100V to about 25000V.
27. The system of claim 24, wherein the external energy source comprises at least of a pressure source and a mechanical source.
28. The system of claim 27, wherein the pressure source is a pump.
29. The system of claim 1, wherein the reactor is configured to initiate a chemical reaction.
30. The system of claim 1, wherein the reactor is in communication with an external energy source.
31. The system of claim 1, wherein the reactor comprises at least heat sources.
32. The system of claim 1, wherein the reactor comprises a plurality of heat sources.
33. The system of claim 1, wherein the reactor comprises a th heat source and a second heat source.
34. The system of claim 33, wherein the th heat source is maintained at 85 degrees c
To a temperature in the range of 95 ℃.
35. The system of claim 33, wherein the th heat source is maintained at a temperature of about 90 ℃.
36. The system of claim 33, wherein the second heat source is maintained at 55 degrees c
To a temperature in the range of 70 ℃.
37. The system of claim 33, wherein the flow path is in thermal communication with at least of the heat source and the second heat source.
38. The system of claim 37, wherein the flow path of reagents repeatedly passes through in communication with the th and second heat sources.
39. The system of claim 38, wherein the reagent is subjected to a plurality of iterations.
40. The system of claim 39, wherein the plurality of iterations is at least 30 iterations.
41. The system of claim 39, wherein the plurality of iterations is at least 60 iterations.
42. The system of claim 37, wherein the flow path passes through the th heat source and the second heat source in a figure-8 pattern.
43. The system of claim 1, wherein the reactor is a thermal cycler.
44. The system of claim 1, wherein the controller is configured to specify at least of a length of time and timing for mixing the th dispersed phase with the second dispersed phase.
45. The system of claim 1, wherein the system operates automatically.
Background
Quantification of nucleic acids is an indispensable technique in medical and biological applications. Novel methods for detecting and quantifying nucleic acids, such as emulsion-based digital nucleic acid amplification, including emulsion-based Polymerase Chain Reaction (PCR), provide greater accuracy and convenience compared to conventional nucleic acid amplification, such as conventional Polymerase Chain Reaction (PCR) methods. In particular, emulsion-based digital nucleic acid amplification allows for highly accurate absolute quantification of nucleic acid sequences in a sample. However, performing emulsion-based digital nucleic acid amplification in most systems still requires the preparation of samples in which the nucleic acids are combined with primers or probes prior to analysis by emulsion-based digital nucleic acid amplification.
Disclosure of Invention
An assay system as described herein can be a continuous flow instrument that includes all components for amplification of nucleic acids and detection of products of a desired chemical reaction, including at least of PCR and reverse transcription PCR (RT-PCR).
The systems, devices, and related methods herein facilitate rapid, reliable, and accurate point-of-contact analysis of samples using minimal pre-input processing to analyze samples for analysis under field conditions or under laboratory conditions (non-laboratory conditions) when the sample preparation environment is not viable.A primary sample processing is internal to the device such that a "blood-in-data-out" workflow can be achieved that requires little external sample processing.streamlined sample processing allows data collection to be performed quickly at a point of care (e.g., a clinical environment) or at a sample collection point (e.g., a field or a site of epidemic).
The systems described herein can be configured to include reagents for pre-processing a sample containing a target nucleic acid, such as reagents for nucleic acid purification and/or extraction (e.g., cell lysis), in embodiments, the systems can be configured to include reagents for pre-processing a sample containing a target nucleic acid, such as reagents for nucleic acid purification and/or extraction (e.g., cell lysis), for example, cells, blood, respiratory fluids, and/or urine can be pre-processed by the systems to extract DNA or RNA without having to use different tools for pre-processing.
Is incorporated 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.
Drawings
Fig. 1 depicts a process workflow.
FIG. 2 depicts an exemplary arrangement of system components.
Fig. 3 depicts an exemplary arrangement of a system for conducting an assay, the system including a coalescer (coalescer), a drop generator, a reactor, a detector, and a controller.
FIG. 4 depicts an exemplary arrangement of a system including a coalescer, a reactor, a detector, and a controller.
Fig. 5 depicts a syringe arrangement comprising a stand-alone syringe.
Fig. 6 depicts a syringe arrangement comprising the same syringe with a switch.
Fig. 7 depicts a syringe arrangement including a syringe having a plurality of ports.
Fig. 8 depicts a process workflow for using a syringe.
Fig. 9 depicts the components of the syringe.
Fig. 10 depicts the arrangement of the inlet and outlet of the injector.
Fig. 11 depicts the arrangement of the chambers of the syringe.
Fig. 12 depicts an alternative arrangement of the chambers of the syringe.
Fig. 13 depicts a cross section of a cylindrical cartridge comprising channels.
Fig. 14 depicts a cross section of a cylindrical cartridge comprising two channels.
Fig. 15 depicts a different arrangement of the chambers of the syringe.
Fig. 16 depicts the components of the cartridge.
Fig. 17 depicts the arrangement of the inlet and outlet of the cartridge.
Fig. 18 depicts the arrangement of the chambers of the cartridge.
Fig. 19 depicts an alternative arrangement of the chambers of the cartridge.
Fig. 20 depicts a coalescer arrangement with separate inlets.
FIG. 21 depicts a coalescer arrangement with a common inlet
Fig. 22 depicts a coalescer arrangement with independent inlets of the same height.
Fig. 23 depicts a coalescer arrangement with separate inlets and gravity assisted collection.
Fig. 24 depicts a coalescer arrangement with valves at the continuous phase outlet.
Fig. 25 depicts a cross-section of a gravity trap coalescer.
Fig. 26 depicts a cross section of a gravity trap coalescer with a smaller channel diameter.
Fig. 27 depicts an assembly of gravity trap coalescers with rotary seals.
Fig. 28 depicts an arrangement of inlets and outlets of a gravity trap coalescer.
Fig. 29 depicts an arrangement of chambers of a gravity trap coalescer.
Fig. 30 depicts an alternative arrangement of the chambers of a gravity trap coalescer.
FIG. 31 depicts a process workflow of a probe.
Fig. 32 depicts a configuration of a detector including a photodetector and an excitation source that is not co-located with the photodetector.
Fig. 33 depicts a configuration of a detector including a photodetector, an excitation source, and a dichroic turning mirror.
Fig. 34 depicts a configuration of a detector including a photodetector, an excitation source, a dichroic turning mirror, and a filter.
Fig. 35 depicts a configuration of a detector comprising two photodetectors, an excitation source, and two dichroic mirrors.
Fig. 36 depicts a configuration of a detector comprising two photodetectors, two excitation sources, a filter, and a dichroic mirror.
Fig. 37 depicts a configuration of a detector including a photodetector, two excitation sources, and a controller.
Fig. 38 depicts a configuration of a detector including two excitation sources and a photodetector.
Fig. 39 depicts a configuration of a detector that allows multiplexing.
Detailed Description
In embodiments, or more systems described herein are configured to perform a desired chemical reaction in droplets, including a chemical reaction for analyzing a target analyte (e.g., a nucleic acid) in a sample provided to the system, and/or a chemical reaction for chemical synthesis, including chemical synthesis of a drug compound.
Nucleic acid detection and quantification by nucleic acid amplification is useful in a variety of research and medical applications. There is a need to be able to detect and quantify nucleic acids quickly and accurately. Emulsion-based digital nucleic acid amplification has a higher sensitivity than conventional nucleic acid amplification, such as conventional PCR methods. However, emulsion-based digital nucleic acid amplification typically involves manual preparation of the target nucleic acid sample and/or reagents, as well as stabilization and transport of droplets in the system.
In certain embodiments, the system may be configured to include reagents for performing reactions other than emulsion-based digital nucleic acid amplification, including, for example, reagents for sample nucleic acid purification and/or extraction (e.g., cell lysis). generally, the system described herein is portable.
The analysis system may include at least zero-dead-volume injectors (zero-dead-volume injectors) configured to automatically and accurately inject sample nucleic acids and/or reagents as will be described in more detail herein at step , the zero-dead-volume injectors may include injection valves, such as injection valves having zero dead volume, such that fluids for the reactions in the system may be automatically and accurately metered without user intervention, nucleic acid samples and reagents, such as primers, probes, polymerases, and/or free nucleotides, may be provided from a cartridge, a reservoir or a sample inlet on the system, and may be combined within the system to form droplets, such as reaction droplets, containing sample nucleic acids and reagents for a desired chemical reaction, hi the case of , the droplets containing sample nucleic acids and the droplets containing reagents are provided to a coalescer where the droplets containing sample nucleic acids and the droplets containing reagents are combined to form droplets containing sample nucleic acids and reagents for an amplification reaction, and the droplets containing reagents may be provided by a loop probe for a PCR reaction, such as a loop probe, a PCR reaction assembly, a PCR reaction kit, a PCR system may be implemented in which may be configured to perform a multiple PCR reaction, such as a PCR reaction, a PCR system, which may be implemented by a reverse transcription reaction system, a PCR system, which may include multiple PCR reaction, a PCR reaction kit, a PCR system, or a PCR system, wherein a PCR system, a.
For example, a droplet containing at least copies of the target molecule will fluoresce and count as the number 1 during detection, while a droplet that does not fluoresce counts as the number 0.
In some embodiments, the assay system may be configured to provide multiplexing, as described herein, that allows for multiple analyses of a single sample, which may be useful for diagnostic purposes, as described herein, the system allowing for multiplexing is configured to be able to detect targets serially and/or in parallel from a single initial sample in some cases the assay system is configured to detect target nucleic acids in parallel such that multiple targets in a sample are analyzed simultaneously in some cases the system may include a detector that includes more than 1 photodetector configured to measure more than emission frequencies from a target nucleic acid and/or emission frequencies from more than target nucleic acids in some cases the parallel multiplexing the serial multiplexing includes analyzing multiple targets sequentially in some cases the serial multiplexing includes analyzing multiple targets sequentially in some cases the serial series of multiple detectors in the flow path.
A system for quantifying nucleic acids is described herein that is more robust and has a wider dynamic range than previous systems.A system as described herein includes or more components capable of automated analysis.in embodiments, the system may include a controller that may be used to control the timing and volume of injections, application of an external source to the coalescer, parameters of the reactor (including reaction time), and processing of signals from the detector or more.
The systems described herein may allow reactions to occur with little or no contamination as compared to traditional emulsion-based digital nucleic acid amplification systems. This is important to provide bioassays that can exhibit the required accuracy and/or reliability, including bioassays for research and/or clinical diagnosis.
For example, prodrug in th volume of dispersed phase is combined with a second prodrug in a second volume of dispersed phase in a coalescer.
The systems and methods described herein enable automated continuous flow reactions without, or substantially without, manual measurement and/or pipetting of samples and/or reagents for chemical reactions. The system may be preloaded with reagents compatible with the chemically reacted sample. The system may be configured to automate the pre-treatment of the sample and the accurate measurement of the chemical reaction reagents. For example, a sample comprising a target nucleic acid can be provided to the system, and the system can perform all of the processes to achieve a desired chemical reaction, and/or provide a desired analysis of the sample. The chemical reaction may be performed without, or substantially without, manual sample quantification and/or manipulation.
Automatic dynamic range of device
Among the reasons, digital PCR is the difficulty in obtaining the proper sample input concentration in order to obtain the proper analyte distribution in the droplets to be analyzed.
Because partitions filled with , two or more DNA molecules are indistinguishable due to the numerical nature of the quantification (i.e., they are all quantified as "" or "present"), only the unfilled partitions can give two degrees of the central parameter in the Poisson distribution and thus the original concentration of a particular DNA sequence in the sample of interest.
If the density of the sample analyte is too high, then at the second aliquot of the sample that is already in the device is diluted and processed, thereby reducing the number of droplets in a single droplet that impede analysis, or if the concentration of the sample analyte in the aliquot is too low (as evidenced by too many droplets not having sample or signal), then in subsequent iterations more concentrated aliquots of the sample in the device are processed in order to achieve optimal or improved analyte concentrations for downstream analysis.
In the example of high dynamic range, th aliquots of the sample are drawn into the instrument, automatically mixed with reagents such as PCR reagents, partitioned, thermocycled, and measured.an automatic controller then determines step below. if the average number of molecules per partition is within an acceptable error range for downstream analysis (such as digital PCR), then the measurement is accepted.in many applications, such as PCR applications, the acceptable range is between 0.0001 and 5 molecules per partition.
In some embodiments, the present invention provides a method of analyzing a sample, including extracting a second aliquot of the sample, and the system will automatically mix with analytical or reaction reagents (such as PCR reagents) from an on-board reservoir, along with dilution water or other suitable diluent the amount of dilution is automatically selected to bring the average number of molecules per partition back within an acceptable or analytically meaningful range, in cases the amount of dilution dilutes the sample to the original or previous concentration of 1/10 to 1/2. then the second aliquot of the sample is partitioned, thermocycled or otherwise subjected to reaction conditions, and measured in the resulting partitions.
Manually obtaining comparable results in other digital PCR instruments can be accomplished by manually mixing th aliquot of sample and PCR reagents at , manually operating the instrument to determine the average number of target molecules per partition, and manually diluting the sample to achieve an acceptable average number of target molecules per partition if too high for accurate quantitation.
For example, where measurements for multiple targets are considered (where the group or multiple targets are at substantially different concentrations, e.g., higher concentrations, than the second group or multiple targets), there is additional benefit.
To achieve this, the sample aliquot is systemically injected and automatically mixed with a detection reagent, such as a PCR reagent, comprising in cases up to four or more primer and probe pairs associated with up to four or more targets from the th group, aliquot partitions, thermocycles and measures, or other reactions and detection methods are employed if the concentration of at least targets in the th group is above the th threshold, a condition associated with that target is diagnosed, or steps can be taken to independently investigate the presence of a condition associated with the detected analyte, if no target is above the concentration threshold associated with that target, the system injects a second aliquot and mixes with a PCR reagent, comprising up to four or more primer and probe pairs associated with four or more targets from the second group, the second aliquot partitions, thermocycles and measures, or other reactions and detection methods are employed if the concentration of at least targets in the second group is above the second target is above the threshold, the four or more targets are associated with the disease detection reagent, thus the cost of the reagent is less likely to be determined by simple automated analysis of the disease or more likely to be less likely to be a rare disease, thus less likely to be able to be detected in parallel with the assay, the third group of a rare disease-related target-related-sample-or-related-to be more likely to be able to be detected in-less likely to be a less-able-to be-able-to-able-to-able-to-detect-to-detect-in-able-detect-in-parallel-to-detect-to-in-parallel-detect-in-parallel-to-detect-parallel-detect-a-less-to-able-to-a-able-to-detect-able-detect-to-in-to-detect-to-in-detect-in.
System and method
FIG. 1 depicts an
In , the system includes a coalescer configured to coalesce at least components of the dispersed phase to provide a coalesced droplet, in , embodiments, the coalesced droplet includes nucleic acid and reagents suitable for a desired chemical reaction (e.g., nucleic acid amplification, including PCR.) for example, droplets from may be coalesced with droplets from the second population in 56110, dispersed phase volume components may be mixed, in , embodiments, mixing within the coalesced droplet may be initiated to facilitate a desired distribution of reagents and/or sample nucleic acid within the droplet, in , embodiments, mixing may be performed to facilitate uniform distribution of reagents and/or nucleic acid within the droplet, in 112, droplets may be generated, in , a plurality of droplets may be generated from the coalesced droplet, for example, to provide a desired volume for processing in the system, in , a reaction product may be detected by a loop detector, in 36116, a loop detector may be configured to detect the concentration of nucleic acid contained within the droplet, such as a loop of nucleic acid flow through the reaction detector.
In cases, the target molecule is a nucleic acid, e.g., the target molecule is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the target nucleic acid treated by systems as described herein may be free-floating or contained within a living or non-living organism, in embodiments systems as described herein may be configured to extract the target nucleic acid contained within a living or non-living organism, e.g., systems may be configured to perform a lysis reaction, in cases, lysis is performed on the system, in systems or more systems may include a reservoir containing a lysis agent.
In cases, the lysis is performed outside the system in cases, the components of the lysis system may be times.
In some cases the target nucleic acid is isolated from a cell, virus, or microorganism in some cases the target nucleic acid is isolated from a bodily fluid (e.g., blood, urine, serum, lymph, saliva, and/or sweat) of the subject in some cases the target nucleic acid is isolated from a tissue in some embodiments the target nucleic acid is isolated from a cell by lysis and subsequent filtration as described herein.
In some embodiments the isolated nucleic acid can be encapsulated in an aqueous fluid then the encapsulated nucleic acid is mixed with an immiscible fluid to form an emulsion in some cases for example, the isolated nucleic acid can be contained in a dispersed phase of an emulsion in some cases the immiscible fluid is an oil exemplary oils are fluorinated, silicone, hydrocarbon and/or mineral oils in some cases the immiscible fluid comprises an oil and or more surfactants in some cases the different densities of the immiscible fluid and the aqueous fluid allow for separation of the two fluids by gravity.
Syringe with a needle
FIG. 2 depicts a schematic of an
The reservoirs for the dispersed
Referring to FIG. 2, the inlet of the zero
The zero
As shown in FIG. 2, the second dispersed phase may include a second component of the chemical reaction, the
As described in further detail herein at , the
In cases, the reaction flow path may comprise a microporous tube composed of a polymer, such as silicone or polyvinyl chloride (PVC), the diameter of the tube may be at least 0.05, 0.10, 0.15, 0.20, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, or greater than 3.0mm in cases, the diameter of the tube may be at most 0.05, 0.10, 0.15, 0.20, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, or greater than 3.0mm in tube diameters may be 0.05, 0.10, 0.15, 0.20, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, or 3.0mm in which case the diameter of the tube may be 0.05, 0.10, 0.15, 0.20, 0.25, 0.50, 0.75, 1.0, 1.5, 2.5, or 3.0mm or in which case the flow path may include a milled or etched flow path through a ceramic material or etched flow path including at least one of the other flow path created in , including a channel, or a channel, a molded material, such as in , including etched or a.
In cases, the reservoir of
FIG. 3 is a schematic diagram of an
The
FIG. 4 is a schematic diagram of another
In embodiments, the system can have separate syringes to form the desired emulsion, for example, the syringes can be arranged separately as in FIG. 5.
In some embodiments, syringes may be used to inject more than dispersed phases, in some embodiments, as shown in fig. 6, the system may include a selector valve configured to control flow to the syringes, in some cases, the system may include a syringe having a plurality of ports, as shown in fig. 7, in some cases, at least 1,2,3,4, 5,6,7, 8, or 8 or more syringes, in some cases, at least 1,2,3,4, 5,6,7, 8, 9, 10, or more than 10 ports, in some cases, at least, about, at most 1,2,3,4, 5,6,7, or 8 syringes, or syringes within a range spanning the above values, in some cases, at least, about, at most 1,2,3,4, 5,6,7, 8, 9, or 10 ports, or ports spanning the above range of values.
Syringe with a needle
A zero dead volume syringe, such as the one described above, can have multiple flow paths therethrough.A flow path can include at least inlets and at least outlets.in some cases , the inlets allow volumes of fluid, such as volumes of fluid including sample nucleic acid and/or reagents, to be introduced into the system from an external source.
In cases, multiple flow paths are used simultaneously, in cases, syringes have at least 1,2,3,4, 5,6,7, or 8 flow paths, in cases, syringes have as many as 1,2,3,4, 5,6,7, or 8 flow paths, in cases, syringes have about 1,2,3,4, 5,6,7, or 8 flow paths, or flow paths that are within a range spanning the above values.
FIG. 8 depicts an
In some cases, the injector may have at least 1, at most, about 1,2,3,4, 5,6,7, 8, or more than 8 ports in some cases the injector has at least, at most, about 1,2,3,4, 5,6,7, or 8 ports, or ports that span the above stated range of values in some cases the surface of the injector may have a greater affinity for the continuous phase than the dispersed phase in some cases the surface of the injector that is attracted to the oil may have an affinity for the side by side water phase in some cases the surface of the injector may be at least of hydrophobic or fluorophilic.
Fig. 9 depicts the components of the syringe. Fig. 10 depicts the arrangement of the inlet and outlet of the injector. Fig. 11 depicts the arrangement of the chambers of the syringe. Fig. 12 depicts an alternative arrangement of the chambers of the syringe.
In some embodiments the sample enters the rotating barrel of the injector.A schematic cross-sectional view of an exemplary cylindrical barrel 1313 is depicted in FIG. 13. cylindrical barrel 1313 includes end caps 1303, 1315 and an internal channel having a fixed internal volume 1311. each cap 1303, 1315 includes a respective channel for fluid flow into or out of the fixed internal volume.cap 1303 includes channels 1305 and 1309 and cap 1315 includes channels 1317 and 1319. in some embodiments , channel 1305 may be configured to align with inlet 1301 of the injector.in some embodiments , channel 1309 may be configured to align with inlet 1307 of the injector.inlet channel 1301 may be connected to a supply of the dispersed phase.inlet 1307 may be connected to a supply of the continuous phase.channel 1317 may be configured to align with outlet 1321 of the injector.channel 1319 may be configured to align with outlet 1323 of the injector.in some embodiments , all surfaces of the barrel include a coating having an affinity for the continuous phase.some embodiments the surfaces of the barrel include a hydrophobic coating.in some embodiments the barrel surfaces include a coating.
A cross-sectional view of a second
Fig. 15 depicts a different arrangement of the chambers of the syringe.
In the cases, the cartridge comprises at least fluid chambers, such as an internal channel with a fixed volume as described herein, in the cases, the cartridge comprises at least 1,2,3,4, 5,6,7, 8, or more than 8 fluid chambers, in the cases, the cartridge comprises at most, about 1,2,3,4, 5,6,7, or 8 fluid chambers, or is included within a range spanning these values, in the 631 cases, the chamber volume may be from about 1 microliter (μ L) to about 50 μ L, in the cases, the chamber volume is at least about 1 μ L, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 50 μ L, 75 μ L, 100 μ L, 250 μ L, 500 μ L, or more than 500 μ L, in the cases, the chamber volume is at most about 1 μ L, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 50 μ L, 75 μ L, 100 μ L, 250 μ L, or 500 μ L, or more than 500 μ L, in the cases, the chamber volume is at most 1 μ L, 10 μ L, 25 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20.
Typically, a sample of nucleic acids and/or reagents is defined in place in a syringe barrel when the chamber of the barrel is fully occupied by the sample of nucleic acids and/or reagents, which has displaced the continuous phase in the barrel by overfilling the barrel chamber with an excess of continuous phase and passing the sample into a waste stream, at the volume of the sample is equal to the size of the chamber.
After the sample is in the syringe barrel, the barrel is rotated in cases, and then the sample passes through a downstream flow path to the coalescer.
Typically, the surface in contact with the sample passing through the downstream path is pre-coated with an immiscible fluid in cases the surface is pre-coated with an oil, such as a fluorinated oil in cases the oil prevents target molecules in the sample from contacting the instrument surface.
In cases, the injector allows selection of primers and probes in cases, the primers and probes are pre-designed and pre-prepared so the user does not need to prepare reagents for the assay
In addition to the sample, sometimes at least reagents, such as enzymes, dntps, primers and probes, are needed to perform nucleic acid amplification, such as pcr, sometimes valves, such as cartridges, provide at least reagents to the systems described herein, in , in 350, the cartridges include at least chambers preloaded with a set volume, in , the cartridges allow the liquid to be stored and subsequently loaded, in , the injection valves have at least 1,2,3,4, 5,6,7, 8 or more than 8 ports, in , the injection valves have about, at most 1,2,3,4, 5,6,7 or 8 ports, or have ports that span the above range of values, in , the cartridges contain 1 flow path, for example, a cartridge with flow paths allows the liquid to be loaded and unloaded in 1 flow path, in , in at least 1,2, 5,6,7, or more than 1, 5, 8, or more than 1,2, 5, 8, or more than 1, 5, 8, or more than 1, 25, 8 at the same time.
At , the cartridge comprises a cartridge having at least fluidic chambers, the chamber volume can be about 1 μ L to about 50 μ L, embodiments have a chamber volume of about 0.05 picoliters (pL) to about 5 milliliters (mL), at , at least about 0.5 μ L, 1 μ L, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 50 μ L, 75 μ L, 100 μ L, 250 μ L, or 500 μ L, at , a chamber volume of at most about 0.5 μ L, 1 μ L, 5 μ L, 10 μ L, 15 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 50 μ L, 75 μ L, 100 μ L, 250 μ L, 500 μ L, at 10 μ L, 355 μ L, 20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 50 μ L, 75 μ L, 100 μ L, 250 μ L, 500 μ L, or 500 μ L, at least about 0.5 μ L, at 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 25 μ L, 10 μ L, 20 μ L, 10 μ L, 20 μ L, 10 μ L.
The surface of the cartridge is typically at least of hydrophobic and fluorophilic, in cases, fluid is introduced through the cartridge using a fluid introduction pump, in cases, when a measured amount of fluid is present, fluid loaded or stored in the cartridge is placed in place within the valve, in cases, the cartridge is placed in place within the valve by monitoring the position of the fluid within the valve, fluid is typically introduced into the cartridge until a specified volume is contained in the cartridge barrel, in cases, a second fluid is introduced into the second channel of the barrel, in cases, the barrel contains at least 1,2,3,4, 5,6,7, 8, or more than 8 fluid channels, in cases, the barrel contains at most, about 1,2,3,4, 5,6,7, or 8 fluid channels, or fluid channels that span the above values.
In some cases , the cartridge includes a reagent reservoir, e.g., the reservoir includes a nucleic acid amplification reagent in some cases the reservoir is refillable in some cases the reagent from the reservoir passes through a zero dead volume valve.
Fig. 16 depicts the components of the cartridge. Fig. 17 depicts the arrangement of the inlet and outlet of the cartridge. Fig. 18 depicts the arrangement of the chambers of the cartridge. Fig. 19 depicts an alternative arrangement of the chambers of the cartridge.
Gravity trap coalescer
In embodiments, a gravity trap coalescer may be configured to coalesce two or more dispersed phases, in embodiments, a gravity trap coalescer may be configured to coalesce a th dispersed phase and a second dispersed phase, for example, an emulsion including a th dispersed phase comprising droplets in a th continuous phase and a second dispersed phase comprising at least droplets in a second continuous phase, a gravity trap coalescer may be configured to combine droplets of a th dispersed phase with droplets of the second dispersed phase to form third droplets, such that the third droplets may be processed as a single entity rather than a plurality of dispersed entities through or more components of a system as described herein, in embodiments, a gravity trap coalescer may be configured to generate third droplets in a third continuous phase.
In embodiments, the droplets of the th dispersed phase containing the reagent are combined with droplets of the second dispersed phase containing the target molecule (e.g., DNA or RNA) to generate third droplets of the third dispersed phase containing both the reagent and the target molecule the reagent may be at least of a primer, a probe, a transcriptase, a buffer, and a dNTP in cases the amount of the reagent may be determined.
As described herein, the dispersed phase can be aqueous, in cases, the dispersed phase comprises greater than about 51% (mass or molar concentration) water, in cases, the continuous phase is immiscible or substantially immiscible with the dispersed phase.
Exemplary fluorinated oils that may be suitable are sold under the trade name FluorinertTM(3M) sold by Fluorinert, including FluorinertTMElectronic Liquid FC-3283, FC-40, FC-43 and FC-70 Another examples of suitable fluorinated oils are under the trade name NovecTM(3M) sale, including NovecTMHFE 7500Engineered Fluid, i.e., 3-ethoxy-1, 1,1,2,3,4,4,5,5,6,6, 6-dodecafluoro-2-trifluoromethyl-hexane, in some cases the fluorochemical is CF3CF2CF2OCH3In NovecTMHFE 7000 is sold under where the fluorochemical is 2,2,3,3,4,4, 4-heptafluoro-1-butanol, CF3CF2CF2CH2In some cases at the fluorinated oil is a perfluorocarbon such as perfluorooctane or perfluorohexane in some cases at the fluorochemical is a partially fluorinated hydrocarbon such as 1,1, 1-trifluorooctane or 1,1,1,2, 2-pentafluorodecane (1,1,1,2, 2-heptafluorodecane).
In some cases , droplets from the th dispersed phase containing the th target molecule are merged with droplets from the second dispersed phase containing the second target molecule.
In cases, the concentration of the nucleic acid is determined, such as mass concentration or molarity.
In the "loaded" state, at least two dispersed phases are located at the joint exit where they are trapped by
As shown in FIG. 21, and the second joint include only inlets, in cases the joint includes at least 1,2,3,4, 5,6,7, 8, or more than 8 inlets, in cases the second joint includes at most about 1,2,3,4, 5,6,7, 8, or more than 8 inlets, in cases the joint includes at most, about 1,2,3,4, 5,6,7, or 8 inlets, or interfaces within a range spanning these values.
As shown in FIG. 22, the joint inlets 2201, 2203 join at a termination point and form channels 2205, 2207 that connect to an outlet joint 2215. in cases, the joint inlets connect at least points with the outlets. in cases, the joint inlets connect with the outlets at multiple points. in cases, a common channel is formed before the joint outlet joint. referring to FIG. 22, the outlets of the joints connect with the joint inlets 2201, 2203 at different points.
Referring to FIG. 23, the outlet has the length of the channel so that the dispersed phase does not flow in the second direction of the vertical channel to the point where the channel deviates from the vertical, as shown in FIG. 23, the portion of the channel that includes inlets 2301, 2303 is oriented so that the central axes of inlets 2301, 2303 deviate from the central axis of channel 2315 by an angle of less than 45.
Referring to FIG. 23, the inlet of the joint is oriented vertically such that the axis of the cross-section perpendicular to the inlet is oriented degrees from an axis parallel to the direction of the gravitational field, which is at most about 45 degrees, in cases, the angle is at most about 20 degrees, the vertical orientation is directed relative to the direction of decreasing field, which may increase the tendency of the dispersed phase to move toward the outlet of the joint.
Referring to FIG. 24, a
FIG. 25 depicts a schematic cross-sectional view of a gravity trap coalescer according to embodiments FIG. 25 shows an exemplary workflow for detecting and quantifying at least target molecules using a gravity trap coalescer as described herein FIG. 25 shows an exemplary workflow where a 0 portion of the dispersed phase is introduced into the device at point 2503 where it flows through a flow channel 2507. a second portion of the dispersed phase is introduced into the device at point 2519 where it flows through a second flow channel 2515. the flow channel and second flow channel meet at junction 2505 and are referred to as a inlet and a second inlet of the junction has a outlet and a second outlet through which the continuous and dispersed phases may exit the junction through a outlet and a second outlet the outlet and second outlet share a central axis perpendicular to the cross-section of the channel including the outlet and the second outlet the flow path of the fluid has a vertical or near vertical section (referred to as a "vertical channel") for the outlet, the flow is hindered by the removable end of the vertical channel ("2509") to allow unimpeded flow.
Referring to fig. 25, the orientation of the outlets allows at least part of the dispersed phase to travel towards the dead end 2511 sometimes the density of the dispersed phase is less than the continuous phase such that the unit vector aligned with the central axis of the th outlet directed towards the dead end deviates from the vector aligned with the local gravity field and directed in the direction of decreasing gravity field strength by an angle of less than 45 deg. alternatively or in combination the density of the dispersed phase is greater than the density of the continuous phase such that the unit vector aligned with the central axis of the th outlet directed towards the dead end deviates from the vector aligned with the local gravity field and directed in the direction of increasing gravity field strength by an angle of less than about 45 deg. the amount of deviation may vary, in cases the amount of deviation is at most about 10 deg., 15 deg., 20 deg., 25 deg., 30 deg., 35 deg., 40 deg. or 45 deg. in 56 cases the amount of deviation is at least 10 deg., 15 deg., 20 deg., 25 deg., 30 deg., 35 deg., 40 deg., or more than 45 deg. in deg., the amount of deviation is about 10 deg., 15 deg., 25 deg., 30 deg., or more than 5910 deg. in the above cases.
In FIG. 25, at least two portions of the dispersed phase are trapped by gravity at by the dead end in the th outlet of junction 2505 the continuous phase flows freely through the second outlet of junction 2517 at least portions of the dispersed phase may have at least surface energies.
In some cases , at least of the dispersed phases are surrounded by surfactant, the surfactant can be electrically polarized, in some cases with an external source, at least of the dispersed phases are distorted due to the surfactant being polarized by the external source.
FIG. 26 shows a fitting formed from a monolith, as shown, the monolith takes an approximately cylindrical shape, there is a axis of the
Fig. 27 depicts components of an example of a gravity trap coalescer. Fig. 28 depicts an example inlet and outlet arrangement for a gravity trap coalescer. Fig. 29 depicts an example cavity arrangement of a gravity trap coalescer. Fig. 30 depicts an alternative arrangement of the chambers of an example of a gravity trap coalescer.
In the case of cases, the dispersed phase has a greater mass density than the continuous phase, and the orientation of the joint outlet is reversed.the joint outlet passes through at least lower planes of the monolith, through an upper plane of the monolith, or a cylindrical side plane of the monolith.the inlet of the joint comprises at least points on the axis of the joint, and the inlet continues through the cylindrical side plane of the monolith.in the case of , the distance between the lower plane of the monolith and the point where the joint inlet intersects the cylindrical side plane of the monolith is not equal to the distance from the lower plane of the monolith to the point where the inlet of the joint intersects the cylindrical side plane of the monolith.
In some cases the continuous phase is in contact with the surface of the monolith, in some cases the continuous phase is a fluorinated oil, in some cases the dispersed phase comprises a fluorophilic material an exemplary fluorophilic material is a fluoropolymer in some cases the fluoropolymer is selected from the series of materials including, but not limited to, polyvinyl fluoride (PVF), polyvinylidene fluoride (PVDF), Polytetrafluoroethylene (PTFE), Polychlorotrifluoroethylene (PCTFE), perfluoroalkoxy polymers, Fluorinated Ethylene Propylene (FEP), polyethylene tetrafluoroethylene (ETFE), polyethylene chlorotrifluoroethylene (ECTFE), perfluoroelastomers, fluorocarbons, fluoroelastomers, perfluoropolyethers (PFPE), perfluorosulfonic acid (PFSA), and perfluoropolyoxycyclobutane, in some cases the entire monolith is made of fluoropolymer so as to not require lining or coating the fluid channels with the fluorophilic material.
At cases, the monolith surface intended to be in fluid contact with the continuous phase is lined or coated with a fluorophilic material at cases, the lining is achieved by inserting an annular section of fluoropolymer into a cylindrical open region of the monolith, the cylindrical open region including an inlet and an outlet of the joint, wherein the outer diameter of the fluoropolymer section and the inner diameter of the cylindrical open region of the monolith are within such a range that the outer diameter of the fluoropolymer section is slightly larger than the inner diameter of the cylindrical open region of the monolith so as to compact and apply an elastic force perpendicular to the inner diameter of the cylindrical open region of the monolith and form a liquid tight seal with the monolith.
In some cases , the coating is achieved by means not limited to Chemical Vapor Deposition (CVD), Physical Vapor Deposition (PVD), induced chemical vapor deposition (i-CVD), Atomic Layer Deposition (ALD), Molecular Layer Deposition (MLD), Atmospheric Pressure CVD (APCVD), Low Pressure CVD (LPCVD), ultra-high vacuum CVD (UHVCVD), Aerosol Assisted CVD (AACVD), Direct Liquid Injection CVD (DLICVD), microwave plasma assisted CVD (MPCVD), Plasma Enhanced CVD (PECVD), and Combustion Chemical Vapor Deposition (CCVD).
In cases, the coalescer includes a removable restrictor the removable restrictor may be at least of a gate valve, needle valve, ball valve, rotating pin, or sliding pin, according to an exemplary removable restrictor on the junction outlet of the monolith material, a cylindrical port extends from a point coaxial with the axis of the junction outlet, the axis being perpendicular to the junction and a cross-section of the point, inserting a cylindrical plug material compatible with the monolith into the port to substantially cover the cross-section of the th outlet of the junction, portions of the cylindrical plug extend into the junction outlet comprised of a fluorine-philic material, openings in the cylindrical plug allow series of rotational positions so that the outlet is closed so that no portion of the dispersed phase can pass through, openings in the cylindrical plug also allow series of rotational positions so that the outlet is open and the dispersed phase can exit, in cases, the outer diameter of the cylindrical plug is greater than the inner diameter of the cylindrical port, in some cases, a tight seal is provided between the cylindrical plug and the monolith.
According to a second example of a removable restriction at the outlet of the junction, the outlet of the junction includes a th cavity in the monolith and fluid channels connected to the monolith by leak-tight seals.
In cases, the dispersed phase coalesces using physical or electronic forces (such as electrostatic forces, magnetic forces, pressure, or shear forces) forces may be provided by an external source, for example, increased pressure is applied to the continuous phase such that the separated phase is separated, in cases, the coalescer uses gravity to lift the water phase without oil rising, the shape of the chamber, such as a conical shape, may improve coalescence in cases, part of the dispersed phase and the second part of the dispersed phase spontaneously combine, in cases, at least 2,3,4, 5,6,7, 8, 9, 10, or more than 10 dispersed phases are combined, in cases, at most about 2,3,4, 5,6,7, 9, 10, or more than 10 dispersed phases are combined, in cases, at most about 2,3,4, 5,6,7, 8, 9, or 10 dispersed phases are combined, or in cases, turbulent coalescence of the dispersed phases is enhanced by turbulent fluid mixing of turbulent flow after swirling, e.g., turbulent fluid droplets are mixed by swirling .
In some embodiments, the connector comprises a first connector inlet and a second connector outlet, the first connector inlet and the second connector outlet are connected to a first connector outlet, the first connector inlet and the first connector outlet are connected to a second connector outlet, the first connector inlet and the first connector outlet are connected to a first connector outlet, the first connector inlet and the first connector outlet are connected to a second connector outlet, the first connector inlet and the first connector outlet, the first connector outlet.
According to an embodiment, the power supply provided at the outlet of the junction may comprise a electrode and a second electrode located on opposite sides of the outlet of the junction, generating a potential difference across the outlet of the junction between the electrodes, polarizing the surfactant around the dispersed phase in the outlet of the junction by an electric field, and providing energy to combine at least two of the dispersed phases in the case of the electrodes are shaped as sharp points so as to increase the electric field strength across the outlet of the junction in the case of , a th electrode is inserted into the th port of the monolith in the case of , and a second electrode is inserted into the second port of the monolith in the case of the potential difference between the electrodes is at least 100V in the case of , the potential difference between the electrodes is at least 100V 200, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 1000, 200, 1000, 200, 1000, 200, 1000, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200, 500, 200.
At the conductivity of the dispersed phase is greater than the conductivity of the continuous phase the difference in conductivity can cause movement of charges or ions to partially or completely cancel the electric field the surface of the dispersed phase has a non-zero surface charge where different volumes of the dispersed phase have opposite charges and attract each other then the continuous phase can be replaced .
According to a further example of adding an external source to the th outlet of the joint, the removable restriction may prevent passage of the continuous and dispersed phases, the inlet port is located between the joint outlet and the removable restriction the inlet port may be filled with the continuous phase and connected to a means to increase the pressure of the continuous phase, during a loaded state, the th removable restriction is open while the second removable restriction is closed so that a portion of the dispersed phase is collected by gravity trapping, once all portions of the dispersed phase are collected, the th removable restriction is closed coalescence may be initiated where the pressure of the continuous phase is increased at the inlet port to combine portions of the dispersed phase.
Droplet generator
In some embodiments, a merged droplet formed by coalescing a th droplet comprising the nucleic acid sample and a second droplet comprising the agent can be provided to a droplet generator.
In some cases , the droplet generator includes a T-junction partitioner (fractionator) configured to segment a fluid, such as a coalesced droplet, alternatively or in combination, the droplet is generated by fluid agitation, microfluidic flow junctions, and/or spontaneously.
In some cases , the droplet generator further steps divide the droplet into a plurality of droplets in some embodiments , the droplet generator may be configured to generate at least about 1000, 2000, 4000, 6000, 8000, 10000, 12000, 14000, 16000, 18000, 20000, 25000, 30000, 40000, 50000, 100000, 200000, 500000, 1 million, 2 million, 3 million, 4 million, 5 million, 1 million, in some embodiments , the droplet generator may be configured to generate at most about 1000, 2000, 4000, 6000, 8000, 10000, 12000, 14000, 16000, 18000, 20000, 25000, 30000, 40000, 50000, 100000, 200000, 500000, 1 million, 2 million, 3 million, 4 million, 5 million, 1 million droplets in some embodiments , the droplet generator may be configured to generate at least about 1000, 14000, 10000, 6000, 18000, 50000, 200000, 50000, 200001 million, 50000, 200001 million, 100000, 200001 million, in some embodiments , the droplet generator may be configured to generate at least about 1000, 14000, 4000, 200000, 50000, 200001 million, 50000, or a range of droplets spanning a range of the aforementioned range.
In some cases , the droplet generated by the droplet generator has a diameter of at least about 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, or more than 1000 micrometers (μm) in some cases the droplet has a diameter of at most about 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 900, 925, 950, 975, or 1000 μm in some cases , 575, 600, 650, 675, 600, 650, 600, 750, 1000, 775, 800, 825, 850, 900, 600, 1000, 600, 1000, 150, 500, 150, 500, or 300, 150, 600.
Reactor with a reactor shell
Systems for detecting and quantifying at least target molecules are described herein under cases, a chemical reaction is initiated by a reactor, the reactor can include at least of a temperature, an electric field, a magnetic field, acoustic energy, and a source of electromagnetic radiation configured to initiate the chemical reaction.
A thermal cycler, as described herein, may include at least two temperature zones at respective temperatures that are fixed.A temperature may be selected based on the chemical reaction that the reactor is configured to initiate. a droplet containing a sample nucleic acid in quantitative amounts and reagents for a desired chemical reaction, or the reaction droplet is typically brought into thermal contact with at least two temperature zones.
In some embodiments, the reaction droplet may be flowed through the reactor or thermal cycler to effect nucleic acid amplification in the reaction droplet, for example, the reaction droplet may be maintained at the temperature for a duration specified at , and at the second temperature for a second specified duration, in some embodiments, the thermal contact between the reaction droplet and at least two temperature zones may be changed back and forth to effect a desired thermal distribution, in some embodiments, the reaction droplet may be maintained at the temperature for a duration specified at , and at the second temperature for a second specified duration, in some embodiments, the thermal contact between the reaction droplet and at least two temperature zones may be changed to effect a desired thermal distribution, in some embodiments, the reactor thermal cycler may have at least 1,2,3,4, 5,6,7, 8, or more than 8 zones or sources, in some embodiments, the reactor or thermal cycler may have at least 1,2,3,4, 5,6,7, or 8 zones or more than the temperature zone or source or the temperature range of the activation temperature of the reaction or the source may be at least 50, in some embodiments, the temperature range of the aforementioned temperature range of the activation temperature range of the reaction or the activation of the reaction droplet may be equal to 200 ℃ or the temperature range of the temperature of the 3695, 5, 7, 5, 7, 95, or more than 60, or 50, or more than 60, or more than the temperature range of the temperature range of the activation temperature of the activation of the second temperature of the amplification of the nucleic acid in some embodiments, or the nucleic acid in some embodiments, 10 temperature range of the nucleic acid in some embodiments, 10 ℃ of the activation temperature range of the amplification of the nucleic acid, 10, or the amplification of the nucleic acid, 10, or the.
The thermal cycler typically includes a tube wrapped around or more components configured to be maintained at different temperatures, the reaction droplets may flow through the tubing such that the reaction droplets reach a desired thermal profile of the reaction droplets. in the case, the tube is wrapped around a heating block. in the case, the thermal cycler includes a tube wrapped around two heating blocks, each heating block configured to be maintained at a particular temperature. in the case, the tube is wrapped in a serpentine fashion. the internal hydraulic diameter of the tube may be in the range of about 0.000001 inches (") and about 0.25". the internal hydraulic diameter of the tube may be in the range of about or less than 0.000001 inches (") to about or greater than 5.0". in the case, the internal diameter is at least about 0.000001 ", 0.000005", 0.00001 ", 0.00005", 0.0005 ", 0.000001", 0.00005 ", 0.00000.00005", 0.00000 ", 0.00005", a microfluidic channel spanning at least about 0.05 ", 0.00005", a microfluidic channel spanning the range of the aforementioned range of 0.05 ", 0.00000.05", 0.00005 ", 0.05", 0.00000.00000.00005 ", 0.05", 0.1 ", 0.00005", 0.00000.05 ", 0.1", 3 ", 3.1", 3.05 ", 3.1", 3.1.1.1 ", 3.1", 3.1.1 ", 3.1", 1.1.1.1.1 ", 3.1.1.1.1", 1.1.0.1 ", 1.1.1.1", 1.1.1.
In some cases , the reaction time is determined by the flow rate within the tube and/or channel in some cases , the reaction time is determined by the length of the tube and/or channel in some cases, typically, the reaction time is varied by varying at least of the flow rate and length in some cases , the reaction droplet is passed through the tube and/or channel in contact with at least two heat blocks more than times.
In conventional thermocyclers, to accommodate multiple wraps around the heating block, the axis of the channel is oriented such that buoyancy problems exist. In particular, droplets of the dispersed phase cannot completely fill the channel diameter, and due to buoyancy, droplets of different sizes may move at different speeds. This can lead to axial droplet breakup, droplet collisions, and potential coalescence, damage, and contamination.
A thermal cycler as described herein may address buoyancy issues by constraining flow to a planar or substantially planar arrangement.in embodiments , the thermal cycler includes or more heating blocks including a slab configuration.
For example, a thermal cycler includes or more blocks, including channels within every of or more blocks.
In embodiments, the thermal cycler includes tubes and channels in thermal contact with or more heat blocks in embodiments, the thermal cycler includes a free standing flexible tube adhered to the heat blocks in cases, the flexible tube is adjusted to match the desired surface pattern on the heat blocks in cases, the flexible tube is mounted in the heat blocks within pre-cut channels on the block surface.
In cases, the thermal cycler is configured to improve at least of conductive heat transfer and temperature uniformity this is accomplished, for example, by using a highly thermally conductive slurry to eliminate air gaps within the thermal cycler (such as air gaps between the conduit and or more surfaces of the heating block).
In some cases , the thermal cycler initiates chemical reactions of reverse transcription prior to amplification, in some cases , this is achieved by providing a separate heating block at a temperature configured to initiate reverse transcription, the heating block may include a rectangular shape, in some cases , the system includes sets of selector valves to allow a user to select whether to flow reaction droplets through the heating block configured to initiate reverse transcription, in some cases , the separate heating block is provided at a temperature configured for nucleic acid amplification, the nucleic acid amplification including a "hot start" step for enzyme activation (e.g., polymerase activation), in some embodiments , the "hot start" step may be performed at a temperature of about 95 ℃ to activate the polymerase.
Alternatively to or in combination with temperature, initiating a reaction by applying electromagnetic radiation, in cases the electromagnetic radiation is provided outside the reaction flow path, wherein the reaction flow path is partially transparent to electromagnetic radiation of at least frequencies, in cases the reaction flow path comprises a tube of at least of glass, ceramic or polymeric material, in cases the reaction flow path is a channel in a material that is transparent to at least frequencies of electromagnetic radiation, in cases the wavelength of the electromagnetic radiation is at least about 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 900, 925, 950, 975, 1000 or in excess of 1000nm, in cases, the wavelength of the electromagnetic radiation is at most about 10, 240, 1000, 600, 75, 100, 125, 150, 240, 600.
Droplet detection
After passing the reaction through a reactor, such as a thermal cycler, the droplets are typically analyzed individually as described herein, in embodiments the system includes a detector downstream of the reactor, in cases the system includes a means for introducing fluid (e.g., through a fluid junction) into the stream of droplets to separate the droplets from one another in steps, separates the droplets, often setting the detector so that each passing droplet obtains at least 1 data point, in cases each droplet collects at least 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 15, 500, or more than 500 data points, in cases each droplet collects at most about 1, 5, 10, 15, 20, 25, 30, 40, 50, 375, 150, 425, 450, 500, or more than 500 data points, in cases each droplet collects at most about 1, 5, 10, 15, 20, 25, 30, 40, 375, 50, 375, 150, 500, or more than 500 data points, 150, 500, or more than about 100, 150, 500, 100, 600, 200, 150, 600, 500, 100, 500, 600, 200, 500, 100, 500, 600.
In cases, the droplets then flow through a droplet detector and a signal is detected. typically, the droplets are measured in at least of optical, electrical, mechanical, and magnetic ways the signal can be, but is not limited to, at least of the intensity of the electromagnetic radiation, the frequency of the electromagnetic radiation, the intensity of the electric or magnetic field, or the orientation of the electric or magnetic field in cases the droplets are compared to the background.
Generally, the mathematical operating principles of or more systems described herein apply to target molecules that follow a poisson distribution model when an event occurs under certain conditions, poisson distribution models the number of times an event occurs within a time interval.
K is the number of target molecules that may be found in a single droplet. k may be 0, 1,2, etc.
The occurrence of a drop with a certain value k does not affect the probability that other drops occur with any other value k. That is, droplet occupancy of the target molecule occurs independently in each droplet. The rate at which drops are generated at a certain value k is constant. The ratio is not higher in some time intervals and lower in other time intervals.
The probability that a droplet with concentration k will occur in a certain interval is proportional to the length of the interval.
When these conditions are satisfied, k is called a poisson random variable, and the distribution of k is a poisson distribution. Thus, the distribution of molecular targets in the droplets is presented as,
where P [ k ] is a poisson distribution, which may be interpreted as the probability that a randomly selected droplet will contain k copies of a target molecule when μ is the average concentration of molecules per droplet in an interval, k is an integer, μ is 0 to some real number between the maximum of k in the interval for a determination of the concentration of the target component, the units or molecules of the target component will be distributed between droplets according to the poisson distribution, and the probability that a given droplet will contain k units or molecules of the target component is given by P [ k ], the detector in the system cannot distinguish 1 component from more than 1 component, but it can distinguish 0 copies from 1 copy for a determination of the concentration of the target component of the droplet, the probability of obtaining zero copies can be estimated by P [0] = N [0]/(N [0] + N [1+ ]), where N [0] is the number of droplets with zero copies and N [1+ ] is the number of droplets with or more copies, for the system, the emission intensity can be determined by calculating the number of droplets below the emission intensity threshold value, and the total number of N [0]/(N + N) is the number of droplets in which is the total number of droplets in a direct measurement of the target concentration of the target component of the droplet in the droplet population, where the emission interval is given by the concentration of the target interval of the target component of the droplet N + N
C＝μ∑Vd，
Wherein C is the concentration of the target molecule in the sample, VdIs the drop volume, ∑ VdThis is a direct method of target concentration quantification by the highly accurate and repeatable nature of the sample injector, coalescer and droplet generator, specifically by controlling the total volume injected, the droplet volume is known in advance]And N1]The instrument is able to quantify target molecule concentrations at lower theoretical detection limits, within an arbitrary dynamic range, and with greater accuracy than other currently used commercial methods, while eliminating the need for abnormal droplets in the measurement.
The drop detector includes at least of an optical excitation source, collimating optics, dichroic filters, objective optics, emission filters, detectors, excitation filters, and pinhole filters.an exemplary optical excitation source is a broadband source of lasers, light emitting diodes, photodiodes, photomultiplier tubes, and filtering light.in cases, the excitation source is power controlled or continuously powered.in cases, a pinhole filter is located in front of the detector to block adjacent drops and fluid from the center drop being analyzed.alternatively or in combination, when positioned at the back focal plane of the objective, the pinhole acts as a confocal aperture.in cases, the aperture excludes auto-emission and scattering from outside the focal volume.in cases, the drop detector contains a fluid channel.
In some cases , the detector measures the emitted electromagnetic radiation after exposure to an excitation source such as electromagnetic radiation in some cases , the detector includes at least 1,2,3,4, 5,6,7, 8, 9, 10 or more than 10 excitation sources in some cases , in some cases the detector includes 3 excitation sources in some cases , the detector includes at least, up to about 1,2,3,4, 5,6,7, 8, 9 or 10 excitation sources or excitation sources in a range spanning the above values in some cases , in some cases excitation is in the range of about 300nm to about 900nm in some cases, excitation may be at least about 300, 350, 450, 400, 500, 550, 600, 650, 700, 800, 850 or 900nm, in some cases up to about 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 900nm in some cases, 600, 650, 700, 600, 850, 900 or 900nm, in some cases 56, at least about 300, 350, 600.
FIG. 31 shows an example of a process workflow including a detector that modulates excitation to perform multiplexing, in cases the detector workflow includes an excitation source and detection As shown in FIG. 31, a continuous phase may be added to the flow path to increase the distance between adjacent droplets prior to introducing the droplets into the detector.
Referring to FIG. 32, the detector includes a continuous phase source and an inlet for the continuous phase, which is located upstream of where the disperse phase is irradiated by the electromagnetic radiation. The continuous phase was injected to spatially separate the dispersed phase volumes (fig. 32).
Referring to fig. 33 and 34, the detector includes an excitation source, a photodetector, at least dichroic mirrors 3303, 3403, and at least filters 3405, 3407 in cases, the detector includes at least 1,2,3,4, 5,6,7, 8, 9, 10, or more than 10 dichroic mirrors, in cases, the detector includes at most, about 1,2,3,4, 5,6,7, 8, 9, or 10 dichroic mirrors, or a number in a range spanning the above values, in cases, the detector includes at least 1,2,3,4, 5,6,7, 8, 9, 10, or more than 10 filters, in cases, the detector includes at most, about 1, 3502, 3,4, 5,6,7, 8, 9, or 10 filters, or a number in a range spanning the above values, in cases, the detector includes 2 dichroic mirrors 3503, 3505, 4,5, 6,7, 8, 9, or 10 filters, or a number in a range spanning the above values, in cases, the detector includes 2 dichroic mirrors (e.g., 3503, 3505, 4, 7, 9, or 10 filters), or more than 2, and at least 3605, at least 36035, including at least 36035, a reaction path between the photodetector, and at least 3535, and at least 3512, 35, at least 36035, 35, and at least 3512, 35, 2.
In some cases , the detector includes a controller the controller can be used to alternate between on and off states of the excitation source (e.g., FIG. 37). for example, the controller alternates between an off state of the excitation source and an on state of the second excitation source. in some cases , the controller applies signal filtering to increase the signal-to-noise ratio.exemplary signal filtering is a lock-in amplifier. in some cases , portion of the reaction flow path is transparent.
In cases, the reaction flow path is transparent in portions, the reaction flow path may be transparent to the excitation source such that the dispersed phase may be excited by electromagnetic radiation and the radiation intensity may be associated with the reaction progress in cases, the detector includes a dispersion grating to spatially distinguish the electromagnetic radiation emitted by sources from the electromagnetic radiation emitted by a second source (e.g., FIG. 38). FIG. 39 depicts a configuration of the detector that allows multiplexing.
Examples of dyes include SYBR Green I, SYBR Green II, SYBR gold, ethidium bromide, methylene Blue, pyronine Y, DAPI, acridine orange, Blue View or phycoerythrin under conditions signals are generated by the activity of optically responsive substances such as dyes or fluorescent probes exemplary reactive fluorescent probes may also be used fluorophore may be aromatic or heteroaromatic compounds fluorescent may be pyrene, anthracene, naphthalene, acridine, stilbene, benzoxazole, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate, anthranilate, xanthene dyes, coumarin exemplary xanthene dyes include, for example, fluorescein and rhodamine dyes including, but not limited to, 6-carboxyfluorescein (FAM), 2'7' -dimethoxy-4 '5' -dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyDIP [ R6 ] 25 ], N, N, N, 5' -dichloro-6-carboxyfluorescein (JOE), 2-carboxyquinoline-5-bis-aminoquinoline-2-5-oxocoumarin, such as benzamidine-2-bis (3-oxo-2-5-phenylethyl) cyanine, 2-oxoindole-6-7-carboxylate-2-oxoindole-6-carboxylate-2-oxoindole-2-6-carboxylate-2-carboxylate-2-6-carboxylate-2-carboxylate-2-carboxylate-2-carboxylate-2-6-carboxylate-2-carboxylate-2-carboxylate-2-carboxylate-2-carboxylate-2-carboxylate-2-6-carboxylate-2-carboxylate-2-carboxylate-2-carboxylate.
In some cases, at most about 1,2,3,4, 5,6,7, 12,13, 18, 13, 14, 19, 20, or more than 20 multi-sequence specific probes are used in some cases at , at most about 1,2,3,4, 5,6,7, 12,13, 14, 15, 17, 18, 19, 20, at , at most, about 1,2,3,4, 5,6,7, 8, 9, 10, 11, 13, 10, 11, 12,13, 14, 16, 14, 15, 16,17, 18, 20, or more than 20 multi-sequence specific probes are used in some cases, at most, about 1,2,3,4, 5,6,7, 8, 9, 10, 11, 12,13, 14, 15, 16,17, 18, 19, or 20, or multiple sequences across the above ranges of values, multiple sequences can be used in multiple cycles to perform multiple cycles between excitation and multiple cycles, at , multiple levels of excitation and multiple cycles of signal detection can be performed in some cases, at no time, multiple cycles, at , multiple cycles of excitation probes can be performed at the same time, multiple cycles, and multiple cycles, using multiple excitation probes can be performed at the detection of.
In some cases , the systems and methods disclosed herein include at least computer programs or uses thereof computer programs include a series of instructions executable in the CPU of a digital processing device that are written to perform specified tasks computer readable instructions may be implemented as program modules such as functions, objects, Application Programming Interfaces (APIs), data structures, etc. that perform specific tasks or implement specific data types.
In examples, the computer program includes a stand-alone application that is a program that runs as a stand-alone computer process rather than an add-on component to an existing process, e.g., not a plug-in.
In cases, the computer includes an external device, in cases, communication between the computer and the external device occurs through at least of physical cables, storage devices, memory devices, and wireless connections, in cases, the system interfaces with a software system on a personal computer, tablet computer, or mobile device.
For example, the system includes storage and/or memory devices.A storage and/or memory device is or more physical devices for temporarily or permanently storing data or programs.in some cases, the device is volatile memory and requires power to retain stored information.alternatively or in combination, the device is non-volatile memory and retains stored information when the digital processing device is not powered.for example, non-volatile memory includes at least of flash memory, Dynamic Random Access Memory (DRAM), Ferroelectric Random Access Memory (FRAM), phase change random access memory (PRAM). in some cases, the device is a storage device including, as non-limiting examples, CD-ROM, DVD, flash memory devices, disk drives, tape drives, optical disk drives, and cloud computing based storage.in some cases, the storage and/or memory device is a combination of those disclosed herein.alternatively or in combination, data may be stored in a database that may be accessed or analyzed by a third party application.
Definition of
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term "comprises," "comprising," and grammatical equivalents thereof specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of or more other features, integers, steps, operations, elements, components, and/or groups thereof.
As used herein, the term "about" with respect to a number or a numerical range should be understood to mean the number and the number +/-10% thereof or, for the value of the range listed, from 10% below the lower limit listed to 10% above the upper limit listed, unless expressly stated or apparent from the context.
As used herein, the singular forms "," "," and "the" include plural referents unless the context clearly dictates otherwise.
As used herein, the terms "amplifying" and "amplification" are used interchangeably and generally refer to the production of or more copies of a nucleic acid.
As used herein, the terms "nucleic acid" and "nucleic acid molecule" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs) or analogs thereof, nucleic acids may have any three-dimensional structure and may perform any function, known or unknown non-limiting examples of nucleic acids include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), Peptide Nucleic Acids (PNAs), coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), (miRNA), microRNA (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, nucleic acids may comprise or more modified nucleotides, such as methylated nucleotides and nucleotide analogs, e.g., Locked Nucleic Acids (LNA), Fluorinated Nucleic Acids (FNA), nucleic acid bridges and thioated nucleotides, modified nucleotides may be used in a nucleic acid sequence assembly process, if other nucleic acid molecules are of the type that may be incorporated into a polymeric form, such as a linker 3632, or other nucleic acid molecule may be further processed by other means such as a linker 3632.
As used herein, the term "primer" generally refers to a nucleic acid molecule that is capable of hybridizing to a template nucleic acid molecule and is capable of being extended in a template-directed manner by the template nucleic acid molecule.
As used herein, the terms "target nucleic acid" and "target nucleic acid molecule" are used interchangeably and generally refer to nucleic acid molecules having a target sequence in an initial population of nucleic acid molecules for which the presence, amount, and/or nucleotide sequence, or changes in or more of these, are desired to be determined.in cases, the target nucleic acid molecule can be double-stranded.in cases, the target nucleic acid molecule can be single-stranded.
As used herein, the term "cylindrical" or grammatical equivalents thereof refers to a three-dimensional shape that includes a surface formed by projecting a closed two-dimensional curve along an axis that intersects a plane of the curve.
Numbering implementation
Numbered embodiment 1 includes a system for performing an assay automatically in continuous flow, the system comprising (a) a flow path comprising a inlet and a outlet for a continuous phase, (b) a -zero dead volume syringe configured to provide a th dispersed phase, (c) a second zero dead volume syringe configured to provide a second dispersed phase, (d) a coalescer, (e) a reactor, (f) a detector, and (g) a controller, numbered embodiment 2 includes the system of numbered embodiment 1, wherein the reactor is configured to initiate a chemical reaction, numbered embodiment 3 includes the system of numbered embodiments 1-2, wherein the reactor comprises an external energy source, numbered embodiment 4 includes the system of numbered embodiments 1-3, wherein the reactor is a thermal cycler, numbered embodiment 5 includes the system of numbered embodiments 1-4, wherein the continuous phase comprises a hydrophobic oil, embodiment 6 includes the system of numbered embodiments 1-5, wherein the second dispersed phase and the dispersed phase of FC 3-3, wherein the dispersed phase comprises at least one of fluorine fluoride, FC 3-wherein the reactor comprises the external energy source, wherein the reactor comprises the system of numbered embodiment 4-comprises the system of numbered embodiment 1-3, wherein the reactor comprises the thermal cycler comprises the hydrophobic oil, wherein the system of numbered embodiment 6-comprises the system of numbered embodiment 1-5, wherein the fluorine fluoride, wherein the dispersed phase comprises the fluorine fluoride, wherein the dispersed phase, FC 3-comprises the fluorine-3-comprises the system, wherein the fluorine-3-comprises the fluorine-fluoride, wherein the fluorine-3-fluoride, wherein the system,CF3CF2CF2CH2Number 9 includes the system of number 1-8, wherein the fluoropolymer is at least of polyvinyl fluoride (PVF), polyvinylidene fluoride (PVDF), Polytetrafluoroethylene (PTFE), Polychlorotrifluoroethylene (PCTFE), perfluoroalkoxy polymer, Fluorinated Ethylene Propylene (FEP), polyethylene tetrafluoroethylene (ETFE), polyethylene chlorotrifluoroethylene (ECTFE), perfluoroelastomer, fluorocarbon, fluoroelastomer, perfluoropolyether (PFPE), perfluorosulfonic acid (PFSA), and perfluoropolyoxetane number 10 includes the system of number 1-9, further includes a surfactant number 11 includes the system of number 1-10, wherein the surfactant is at least 2 of number 1-10, wherein the system of number 12 includes the embodiment 1-11, wherein the system of number 12 includes the embodiment 1-10, wherein the surfactant is a fluorocarbon, hydrocarbon, and silicone, and wherein the system of number 12 includes the number 1-13, wherein the number 14 includes a probe number 15, wherein the system of amplification of nucleic acids number 1-13 includes a loop amplification of a loop-10, wherein the loop amplification of the nucleic acid comprises a loop-10, wherein the loop amplification of the loop system of the loop-10, wherein the loop of loop-1, the loop of loop-loop probe includes the loop probe number 1, wherein the loop of loop-13, the loop of loop probe includes the loop of the loop probe, the loop system of loop-loop, wherein the loop-loop system of loop-loop system of loop-loop system includes the loop-Numbering embodiment 20 includes the system of numbering embodiments 1-19, wherein the external energy source is provided by an electric field between at least two electrodes, numbering embodiment 21 includes the system of numbering embodiments 1-20, wherein the electric potential between the at least two electrodes is in the range of about 100V to about 25000V numbering embodiment 22 includes the system of numbering embodiments 1-21, wherein the external energy source is at least of a pressure source and a mechanical source, numbering embodiment 23 includes the system of numbering embodiments 1-22, wherein the pressure source is provided by a pump, numbering embodiment 24 includes the system of numbering embodiments 1-23, wherein the reactor includes a fixed temperature zone, numbering embodiment 25 includes the system of numbering embodiments 1-24, wherein the reactor includes at least two fixed temperature zones, wherein numbering embodiment 26 includes the system of numbering embodiments 1-25, wherein a flow path circulates between the at least two fixed temperature zones, numbering embodiment 27 includes the system of numbering embodiments 1-26, wherein numbering embodiment 26 includes the system of numbering embodiments 1-25, wherein a dead phase flow path control system 3527 includes a second syringe 3627, wherein the second syringe 35 d, No. 20 includes a syringe , a syringe 367, a syringe , a syringe, and a syringe, wherein the syringe, a syringe, wherein the syringe, a syringe, wherein the syringe, a syringeNumbering embodiment 32 comprising the system of numbering embodiments 1-31, wherein the droplet generator generates droplets in a number in the range of about 2000 to about 1000000, numbering embodiment 33 comprising the system of numbering embodiments 1-32, wherein the droplet generator generates droplets in a number in the range of about 3000 to 30000 numbering embodiment 34 comprising the system of numbering embodiments 1-33, wherein the system performs emulsion-based digital nucleic acid amplification, numbering embodiment 35 comprising the system of numbering embodiments 1-34, wherein the th disperse phase comprises at least target nucleic acid molecules, and wherein the second disperse phase comprises nucleic acid amplification reagents, numbering embodiment 36 comprising the system of numbering embodiments 1-35, wherein the nucleic acid amplification reagents are at least one of primers, probes, blends, dnps, buffers and enzymes, numbering embodiment 37 comprises the system of numbering embodiments 1-36, wherein the embodiment measures the aggregation of droplets using a poisson distribution, numbering embodiment 32 comprises the system of numbering embodiments 1-31, wherein the numbering embodiment 1-34 comprises a multiplex automatic clustering system for the number-34, wherein the number of the multiplex number-probes comprises a multiplex number of fluorescent probes based on a number of a primer, a probe, a number of a multiplex number of a number of 1-35, a number of an embodiment 1-35, wherein the number of a multiplex signal detector comprises a multiplex automatic clustering system of a multiplex of a primer, a multiplex of a fluorescent probe, a multiplex system, a fluorescent probe, a fluorescent signal for a fluorescent probe, a fluorescent signal detector, a fluorescent probe, a fluorescent signal detector, a fluorescent probe, a fluorescentFormula 45 includes the system of numbered embodiments 1-44, wherein the surface of the flow path includes fluoropolymer, and wherein at least of the th and second dispersed phases do not contaminate the flow path numbered embodiment 46 includes the system of numbered embodiments 1-45, wherein the fluorinated oil is FC-3283, FC-40, FC-43, FC-70, 3-ethoxy-1, 1,1,2,3,4,4,5,5,6,6, 6-dodecafluoro-2-trifluoromethyl-hexane, 2,3,3,4,4, 4-heptafluoro-1-butanol, CF3CF2CF2CH2Numbering embodiment 47 includes the system of numbering embodiments 1-46, wherein the fluoropolymer is at least of polyvinyl fluoride (PVF), polyvinylidene fluoride (PVDF), Polytetrafluoroethylene (PTFE), Polychlorotrifluoroethylene (PCTFE), perfluoroalkoxy polymer, Fluorinated Ethylene Propylene (FEP), polyethylene tetrafluoroethylene (ETFE), polyethylene chlorotrifluoroethylene (ECTFE), perfluoroelastomer, fluorocarbon, fluoroelastomer, perfluoropolyether (PFPE), perfluorosulfonic acid (PFSA), and perfluoropolyoxetane numbering embodiment 48 includes the system of numbering embodiments 1-47, wherein the surfactant is at least of fluorocarbon, hydrocarbon, and silicone, numbering embodiment 49 includes the system of numbering embodiments 1-48, wherein the reactor cycles from a third temperature region to a second temperature region to perform nucleic acid amplification in embodiment 1,1, 1-trifluorooctane, and 1,1,1,2, 2-pentafluorodecane, wherein the annealing occurs at a temperature in the second embodiment 5-55 temperature range from about ℃ to about 50 ℃ in embodiment 51. the system numbering embodiment 51-55, wherein the annealing occurs at least 50 ℃ in the second embodiment 5-55 temperature range from about ℃ to about 50 ℃ in the second embodiment 5-55. the system numbering embodiment, wherein the annealing occurs at least the temperature range of numbering embodiment 1-55 ℃ in the system numbering embodiment 1-55, wherein the annealing occurs at least the temperature range of numbering embodiment No. 5-55, the system of numbering embodiment No. 55, the system of numbering embodiment 1-55, the numbering embodiment, the numbering system of No. 5The method includes the steps of providing a system according to embodiments 1-54, further including a third temperature zone, embodiment 56 including embodiments 1-55, wherein reverse transcription occurs in the third temperature zone, embodiment 57 including embodiments 1-56, wherein a "hot start" step of nucleic acid amplification occurs in the third temperature zone, embodiment 58 including embodiments 1-57, wherein the second dispersed phase includes at least of RNA, DNA and protein, embodiment 59 including embodiments 1-58, wherein the second dispersed phase includes at least of antigen, reporter, primer, probe, blend, buffer and enzyme, embodiment 60 includes a method for automated determination in continuous flow, comprising (a) preparing a sample including at least 2 target molecules in the dispersed phase of , (b) automatically injecting into a flow path containing the continuous phase the first dispersion phase the first amount of the first target molecules, and/or the second dispersed phase a buffer, and/or a buffer, and (c) comparing the number of the first dispersed phase with the number of the first target molecules detected by the first dispersion mode 120-7, the first dispersion mode, and/or the second dispersion phase includes at least one of RNA, DNA and/or protein, and/or a final dispersion phase detection by the third embodiment 2. the first dispersion method comprises (c) comparing the number of the first dispersion phase detected target molecules detected by the first dispersion reaction with a threshold number of the second dispersion reaction, 2,3, 2,3, 7, 3, 7, 3, 7, 3, 7, 3, 7, 3, 7, and 7, and/6, 7Numbering embodiment 66 comprises any of numbering embodiments 1-65, wherein a defined volume of water is automatically injected after the result of the th assay.no. 67 comprises any of numbering embodiments 1-66, wherein the sample is at least of a cellular, viral, microbial fluid, numbering embodiment 68 comprises any of numbering embodiments 1-67, wherein the sample is from a subject, numbering embodiment 69 comprises a system for metering the volume of the dispersed phase of an emulsion, the system comprising (a) a drum comprising a monolith having a fixed internal open volume, (b) a second cover of the drum comprising a channel for inlet, (c) a second cap for the drum comprising a channel for any outlet, (d) a means for initiating the flow of dispersed phase through an inlet to a flow path 6867, (e) a second continuous phase flow path through an inlet to a second flow path 355, further comprising a channel for any outlet, wherein the inlet of a continuous phase flow path comprises a number 1-99, a constant volume of a continuous phase flow path for the emulsion, and wherein the number of the continuous phase flow path comprises a number 1-75, wherein the number of the sample comprises a number of a constant volume of the sample, and a number of the sample comprises a number of the number ofAnd wherein the axis of the third flow channel forms an angle of less than 45 with a second axis aligned with the direction of the increasing gravitational field, (d) a removable restriction, (e) a fourth flow channel comprising a second outlet connected to an outlet for the continuous phase, and wherein for at least portion of the length of the fourth flow channel, a third axis forms an angle of less than 45 with a fourth axis, (f) the third flow channel, the second flow channel, the third flow channel, and the fourth flow channel interface, (g) an external source, and (h) a controller for automatically coalescing a numbered embodiment 77 comprising any of the numbered embodiments 1-76 wherein the external source is applied to the third flow channel, numbered embodiment 78 comprising any of the numbered embodiments 1-77, wherein the numbered embodiments flow channel, the second flow channel, the third flow channel, and the fourth flow channel comprise at least one of the numbered embodiments 1-7, wherein the numbered embodiments 23-7 include at least one of the numbered embodiments 358, the numbered embodiments 23-7, and the number of the fourth flow channel 99-80, wherein the numbered embodiments 23-7 flow channel, the number of the third flow channel, and the fourth flow channel, include at least one of the numberNumbering embodiment 88 comprises any 0 of numbering embodiments 1-87, wherein the external source is at least 1 of pressure source and mechanical source, numbering embodiment 89 comprises any 2 of numbering embodiments 1-88, wherein the pressure source is provided by a pump, numbering embodiment 90 comprises any 3 of numbering embodiments 1-89, wherein the controller controls the timing of coalescence, numbering embodiment 91 comprises any 394 of numbering embodiments 1-90, wherein any volume of dispersed phase comprises a target molecule, numbering embodiment 92 comprises any of numbering embodiments 1-91, wherein the at least 467 volume of dispersed phase comprises any 465 volume of target molecule, numbering embodiment 587 of embodiments 1-90, wherein the at least a fifth 99 th inlet of a third continuous flow channel is connected to a third continuous flow channel 94, wherein the third inlet of the third continuous flow channel is connected to a third continuous flow channel 94, wherein the third continuous flow channel 99 comprises a fifth 99 th inlet of a third continuous flow channel 94, a fifth 99 th continuous flow channel 99 th inlet of a buffer, a third continuous flow channel 99 th continuous flow channel 99, a third continuous flow channel 99 th continuous flow channel, a third continuous flow channel 99 th, a third continuous flow channel 99, a third continuous flow channel 99 th continuous flow channel, a third continuous flow channel 99, a third continuous channel, a third continuous flow channel, a third continuous channel 99, a third continuous channel, a third continuous flow channel, a third continuous flow channel, a third continuous channel, a fourth continuous channel, a third continuous flow channel, a third continuous channel, a fourth continuous channel, a third continuous channelThe system of the invention comprises a syringe 100, a syringe 102, a syringe 100, a syringe 100, a syringe 102, a syringe 100, a syringe, a syringe 100, a syringe,numbering embodiment 104 comprises any item of numbering embodiments 1-103, wherein said external source is an electric field between at least two electrodes, numbering embodiment 105 comprises any item of numbering embodiments 1-104, wherein the potential between said at least two electrodes is in the range of about 100V to about 10000V, numbering embodiment 106 comprises any item of numbering embodiments 1-105, wherein the potential between said at least two electrodes is in the range of about 500V to about 2000V, numbering embodiment 107 comprises any item of numbering embodiments 1-106, wherein said electric field has a current in the range of about 10kHz to about 100kHz, numbering embodiment 108 comprises any item of numbering embodiments 1-107, wherein said external source is at least items of pressure source or mechanical source, numbering embodiment 109 comprises at least one item of embodiments 1-108, wherein said dosing embodiment provides at least one of a sample volume after said sample volume is introduced into said sample volume metering system 112, wherein said dosing system comprises at least one of sample volume after said sample volume is measured by said sample volume measuring system 113-7371, 113, 112, 113, or similar to a sample volume, 113, or similar to a sample volume, 113, or similar to a sample volume, or similar, 113, or similar to a volume, or similar, wherein said system for a sample volume of said system is introduced into said system, wherein said system for a sample volume of a predetermined volumeNumber embodiment 117 includes any of number embodiments 1-116, wherein the system compares a measurement of the presence of an analyte in an individual droplet of an emulsion to a threshold for the frequency of the presence of an analyte in an individual droplet of an emulsion number embodiment 118 includes any of number embodiments 1-117, wherein the system dilutes a remaining aliquot of the sample when the measurement of the presence of an analyte in an individual droplet of an emulsion is above the threshold, number embodiment 119 includes any of number embodiments 1-118, wherein the system measures the presence of an analyte in an individual droplet of an emulsion generated from the diluted remaining aliquot of the sample number embodiment 120 includes any of number embodiments 1-119, wherein the system concentrates the remaining aliquot of the sample number embodiment 120 includes any 632 of number embodiments 1-119, wherein the system does not require the presence of an analyte in a number of number embodiments 120 when the measurement of the presence of an analyte in an individual droplet of an emulsion is below the threshold, wherein the number embodiment 120 includes no more than one of the number of analyte metering reagent metering systems 123, or more than three times equal or three times, wherein the number of the number embodiments includes no more than one of the number embodiments 1-125, 2, wherein the number of the number embodiments 1-125 includes no more than one of the number embodiments of the number embodiments 1,2 of the number embodiments 1, 120 includes no more than one of the number embodiments 1-125 of the number embodiments of the method ofThe system measures a second sample no more than 5 minutes after the th sample is quantitated, embodiment 128 includes any of embodiments 1-127, wherein the system measures a second sample no more than 5 minutes after the th sample is quantitated, embodiment 129 includes any of embodiments 1-128, wherein the sample quantitates at least 20 samples, embodiment 130 includes any of embodiments 1-129, wherein the sample quantitates at least 50 samples, embodiment 131 includes any of embodiments 1-130, wherein the premeasured kit includes sufficient reagent to analyze at least 10 samples, embodiment 132 includes any of embodiments 1-131, wherein the premeasured kit includes sufficient reagent to analyze at least 20 samples, embodiment 133 includes sufficient reagent to analyze any of the 20 samples, embodiment of embodiments 1-137, wherein the premeasured kit includes sufficient reagent to analyze at least 50 samples, embodiment 5819 includes sufficient reagent for a serial loop assay of embodiments 1-137, embodiment 6326 includes at least one reagent for a loop prediction of loop-loop prediction of loopNumber embodiment 141 includes any of number embodiments 1-140, wherein the continuous phase includes a hydrophobic reagent number embodiment 142 includes any of number embodiments 1-141, wherein the continuous phase includes an oil number embodiment 143 includes any of number embodiments 1-142, wherein the continuous phase includes a hydrophobic fluorinated oil number embodiment 144 includes any of number embodiments 1-143, wherein the hydrophobic fluorinated oil includes a reagent selected from the group consisting of FC-3283, FC-40, FC-43, FC-70, 3-ethoxy-1, 1,1,2,3,4,4,5,5,6,6, 6-dodecafluoro-2-trifluoromethyl-hexane, 2,3,3,4, 4-heptafluoro-1-butanol, CF, and CF-r3CF2CF2CH2Number embodiment 145 includes any of number embodiments 1-144, wherein at least of the dispersed phase and the second dispersed phase include a hydrophilic agent number embodiment 146 includes any 1 of number embodiments 1-145, wherein at least 8295 of the dispersed phase and the second dispersed phase include an aqueous agent number embodiment 147 includes any of number embodiments 1-146, wherein at least of the dispersed phase and the second dispersed phase include water number embodiment 148 includes any of number embodiments 1-147, wherein the surface of the flow path includes a polymeric coating layer number embodiment 149 includes any 464 of number embodiments 1-148, wherein the surface of the flow path includes a hydrophilic coating layer number embodiment 150 includes a polymeric coating layer number 1-149, wherein the surface of the flow path includes a fluorinated fluoropolymer of the fluorinated fluoropolymer of number embodiment 1-149, wherein the fluorinated fluoropolymer of number embodiment 1-150 includes a fluorinated ethylene-tetrafluoroethylene (pfe) and a fluorinated ethylene-tetrafluoroethylene (pfe) wherein the fluorinated ethylene-tetrafluoroethylene (pfe) includes a fluorinated ethylene-tetrafluoroethylene) copolymer (pffe), a fluorinated ethylene-tetrafluoroethylene (pfe) includes a fluorinated ethylene-tetrafluoroethylene copolymer (pffe) and a fluorinated ethylene-tetrafluoroethylene copolymer (pffe) wherein the fluorinated ethylene-tetrafluoroethylene copolymer includes a fluorinated ethylene-tetrafluoroethylene copolymer number 1-150, a fluorinated ethylene-tetrafluoroethylene copolymer (pffe) and a fluorinated ethylene-tetrafluoroethylene copolymer, wherein the fluorinated ethylene-tetrafluoroethylene copolymer includes a fluorinated ethylene-tetrafluoroethylene copolymer (pffe) includes a fluorinated ethylene copolymer number 1-tetrafluoroethylene copolymer (pffe) and a fluorinated ethylene copolymer, a fluorinated ethylene copolymer (pffe) and a fluorinated ethylene copolymer, a fluorinated ethylene copolymer (pffe) wherein the fluorinated ethylene copolymer number 150 includes a fluorinated ethylene copolymer, aNumbering embodiment 152 comprises any of numbering embodiments 1-151 including a surfactant embodiment 153 includes any of numbering embodiments 1-152 wherein the surfactant includes a reagent selected from the group consisting of fluorocarbons, hydrocarbons and siloxanes, numbering embodiment 154 includes any of numbering embodiments 1-153 wherein the volume of at least of the dispersed phase and the second dispersed phase is in the range of about 1 picoliter to about 1 milliliter numbering embodiment 155 includes any 3 of numbering embodiments 1-154 wherein the flow path includes an internal hydraulic diameter in the range of about 0.001 "to about 0.5", numbering embodiment 156 includes any 8 of numbering embodiments 1-155, wherein the multiple copies of any nucleic acid segment of the numbering embodiment 157 include any 8 of the dispersed phase of numbering embodiments 1-156 wherein the second dispersed phase probe embodiment includes at least one of a primer 26, a primer 163, a probe 161, a probe 162, a probe 161, a probe 162, a probe 161, a probe 162, a probe 161, a probe 162, a probe 161, a probe 162, a probe 161, a probe including a probe 161, a probe for an energy detector for detecting a probe for aNumber embodiment 166 includes any item of number embodiments 1-165, wherein the external energy source includes at least of a pressure source and a mechanical source, number embodiment 167 includes any item of number embodiments 1-166, wherein the pressure source is a pump, number embodiment 168 includes any item of number embodiment 1-167, wherein the reactor is configured to initiate a chemical reaction, number embodiment 169 includes any item of number embodiments 1-168, wherein the reactor is in communication with the external energy source, number embodiment 170 includes any item of number embodiments 1-169, wherein the reactor includes at least 4, number embodiment 171 includes any item of number embodiments 1-170, wherein the reactor includes a plurality of heat sources, number embodiment 172 includes any item of number embodiments 1-171, wherein the reactor includes a number embodiment 1-170, wherein the reactor includes a number of any item , wherein the number embodiment 172 includes a number of items 3755, number embodiment 99, wherein the number embodiment includes a number of heat source 85, a number of items 150, a number embodiment 99-150 includes a number of items 3755, a number of items 150, a number embodiment 1-150, a number embodiment 99, a number embodiment includes a number of items 595, a number of items 3755, a number of items 150, a number embodiment 1-150, a number embodiment includes a number of items 150, a number embodiment 1-55, a number of items 150, a number of heat source, a number of items 150, a heat source, a number of a heat sourceNumber heat source and the second heat source number embodiment 182 includes any of the items of number embodiments 1-181, wherein the reactor is a thermal cycler number embodiment 183 includes any of the items of number embodiments 1-182, wherein the controller is configured to specify at least of a time period and a timing for mixing the dispersed phase with the second dispersed phase number embodiment 184 includes any of the items of number embodiments 1-183, wherein the system operates automatically.
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