Device and method for creating a combinatorial microcompartment within a carrier phase

Abstract

The present invention provides a method and microfluidic device for creating one or more combinatorial microcompartments containing at least two sample species in a carrier phase, comprising: a target channel; a first sample species reservoir containing a first sample species and a second sample species reservoir containing a second sample species; a first sample injection channel including a sample injection channel reservoir end connected to the first sample species reservoir and a sample injection channel junction end connected to the target channel; and a second sample injection channel including a sample injection channel reservoir end connected to the second sample species reservoir and a sample injection channel junction end connected to the target channel, wherein the sample injection channel junction ends of the first and / or second sample injection channels are connected to the target channel valveless and include at least one hydrodynamic resistor. The present invention also provides a method for colocalizing entities with a barcode oligonucleotide or a set of its components in a microfluidic device according to the present invention.

Description

[Technical Field]

[0001] Embodiments herein relate to devices, in particular microfluidic devices, and methods for creating one or more combinatorial microcompartments containing at least two sample species within a carrier phase. [Background technology]

[0002] One of the problems encountered in microfluidic devices concerns the backflow of microfluidics. Microfluidic backflow refers to the unintended flow of a liquid, fluid, or aqueous sample from one channel or compartment to another channel or compartment within a microfluidic device. Therefore, backflow can lead to cross-contamination, for example, between adjacent channels, microcompartments, and / or combinatorial microcompartments, which poses a significant problem in microfluidic experiments and analyses because liquids, fluids, or aqueous samples that should be kept separate may (e.g., unintentionally) mix or disrupt the intended fluid flow within the microfluidic device.

[0003] As mentioned above, backflow of microfluidics occurs between channels and / or microcompartments. For example, several factors can cause backflow of microfluidic material within a channel. Pressure difference: If the pressure in one channel is greater than the pressure in another channel, a liquid, fluid, or aqueous sample may flow from the high-pressure channel to the low-pressure channel, potentially causing backflow; Hydrodynamic flow: When a liquid, fluid, or aqueous sample is flowing through one channel, it can create a flow that draws the liquid, fluid, or aqueous sample from another channel into the channel it is flowing through; Capillary force: Capillary force can cause liquid, fluid, or aqueous samples to be drawn into nearby or adjacent channels; and / or incomplete sealing: If the sealing between channels is not properly designed or closed, liquid, fluid, or aqueous samples may leak from one channel to another.

[0004] Backflow of microfluidics within a microcompartment can be caused, for example, by the following: Pressure differences throughout the microchannel, inadequate channel design leading to channel blockage; Viscous drag of liquid, fluid, or aqueous samples; Flow mismatch between inlet and outlet; Electrostatic interactions between the liquid, fluid, or aqueous sample and the channel wall, and / or changes in the temperature and viscosity of the liquid, fluid, or aqueous sample.

[0005] Typically, the use of flow control and / or switching on-chip valves, such as pneumatic valves (e.g., Quake valves) or Braille valves, helps prevent or reduce backflow by enabling precise control over fluid flow within microfluidic devices.

[0006] Using a valve, different aqueous samples can be produced. Aqueous samples are mixed and / or compartmentalized on a microfluidic chip in conjunction with the simultaneous injection of a carrier phase such as water or oil, by utilizing the ability of valves to selectively route and control fluid flow within microfluidic channels. For this purpose, valves can be used to direct fluid flow to specific microcompartments within a microfluidic chip, enabling, for example, the creation of multiple isolated compartments with different chemical or biological environments. These aqueous microcompartments can be used for a variety of purposes, such as carrying out chemical reactions, encapsulating cells or other biological materials, or for high-throughput screening applications, for example, to enable the creation of multiple isolated environments for screening reactions or analyses.

[0007] However, there are some limitations when using valves to produce different aqueous samples or combinatorial mixtures, including the following: i) Complexity; for example, valves are difficult to fabricate and integrate into microfluidic devices due to their small size and complex design; ii) Maintenance; for example, valves are prone to clogging, leaking, or wearing out over time and require regular maintenance and cleaning to ensure proper function; iii) Limitation of the number of flow paths, for example, limiting the number of flow paths that can be controlled using a single valve, thereby limiting the number of screening analyses that can be performed in parallel; iv) Precision requirements, for example, very precise alignment of a valve on an external actuator (e.g., aligning a Braille valve on a Braille display); Furthermore / or v) Scalability limitations, such as the difficulty in scaling up the number of valves for high-throughput screening applications due to complex fabrication processes and the need for precise valve alignment within microfluidic devices. However, the use of valves to generate combinatorial mixtures requires further measures to prevent the backflow of undesirable microfluidics. Furthermore, flexible materials such as PDMS (polydimethylsiloxane), a silicone-based polymer material commonly used in microfluidics due to its transparency, low surface energy, and biocompatibility, are required for the precise function of valves, even though they are difficult to manufacture using standardized and scalable methods.

[0008] U.S. Patent No. 5,726,404, titled “Valveless Liquid Microswitch,” describes a valveless liquid microswitch that can be used to control the flow of fluid in a microfluidic device. The microswitch includes a chamber filled with a liquid that can be actuated by a piezoelectric element or other suitable actuator.

[0009] Therefore, it is beneficial to provide improved microfluidic devices and methods for preventing, reducing, and / or canceling backflow of microfluidic fluids between flow paths and / or microcompartments, while minimizing or eliminating the limitations associated with the use of valves. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent No. 5,726,404 [Overview of the project]

[0011] The subject matter of the present invention is defined in the independent claims, and embodiments of the present invention are defined in the dependent claims.

[0012] The present invention relates to a method and device for combining two or more different sample fluids (or liquids) and target channel fluids in a microfluidic device by transiently injecting at least one of at least two sample fluids from one of at least two sample injection channels into a target channel containing a target channel fluid located at a sample injection junction (which is understood as a channel junction between the target channel and one of the at least two sample injection channels). The target channel includes a continuous flow of target channel fluid supplied via a target channel fluid reservoir. Next, the method and device of the present invention provides at least one sample injection fluid to be injected into the sample injection channel via a pressurized sample injection fluid reservoir, preferably at an injection pressure P i The non-injection pressure P is applied during the injection phase. niThe present invention provides that a fluid is applied during the non-injection phase, and that at least two sample injection channels are each connected valvelessly to a sample injection fluid reservoir via at least one hydrodynamic resistor. Therefore, the present invention provides a sample injection channel supplied by a continuously pressurized sample injection reservoir, both during the injection phase and the non-injection phase, in combination with a hydrodynamic resistor positioned between the sample injection joint and the sample injection reservoir. Such combinations are remarkably effective in avoiding backflow in the injection channel compared to conventional devices and methods. The embodiments of the present invention will be described in detail below with reference to the following.

[0013] In the following detailed description of the present invention, the target channel fluid is also called the carrier fluid or carrier phase, and the injection channel fluid is called the sample species or fluid.

[0014] In light of the above, a microfluidic device and a method for combining at least two fluids within the microfluidic device are provided. Such inventive methods for combining fluids are particularly useful for creating combinatorial microcompartments. Accordingly, in consideration of the above, the present invention further provides a microfluidic device and a method for creating one or more combinatorial microcompartments containing at least two sample species within a carrier phase.

[0015] According to one embodiment, a microfluidic device is provided that includes at least two sample species in the carrier phase. The microfluidic device includes a target channel and a first sample species reservoir containing a first sample species and a second sample species reservoir containing a second sample species. The microfluidic device includes a first sample injection channel, which includes a sample injection channel reservoir end connected to a first sample species reservoir and a sample injection channel junction end connected to a target channel, and a second sample injection channel, which includes a sample injection channel reservoir end connected to a second sample species reservoir and a sample injection channel junction end connected to a target channel. The sample injection channel junction ends of the first sample injection channel and / or the second sample injection channel are connected to the target channel without a valve and are equipped with at least one hydrodynamic resistor. The sample species reservoir included in the microfluidic device of the present invention is pressurized during and after injection of the sample liquid. Furthermore, this microfluidic device is designed to create one or more combinatorial microcompartments.

[0016] According to one embodiment, a method for operating a microfluidic device is provided, which includes the step of injecting a first sample species from a first sample species reservoir into a continuous flow of a carrier phase in a target channel via a first sample injection channel (preferably, the continuous flow of the carrier phase is injected from a carrier phase reservoir into the target channel). The method further comprises the step of injecting at least one second sample species from a second sample species reservoir into a continuous flow of the carrier phase in a target channel via a second sample injection channel, wherein (i) the first and second sample species are injected into the respective first and second seed reservoirs at an injection pressure P i It is injected by applying an injection pressure P i Lower non-injection pressure P ni (ii) a certain amount of water is maintained in each sample type reservoir of each non-injection sample injection channel, and / or (ii) resistance is provided in the first sample injection channel and / or the second sample injection channel using at least one hydrodynamic resistor.

[0017] According to another embodiment, a method is provided for providing one or more combinatorial microcompartments containing at least two sample species. This method includes a step of creating microcompartments by injecting a first sample species from a first sample species reservoir through a first sample injection flow path into a continuous flow of the carrier phase in a target flow path. This method further includes a step of injecting at least one second sample species into the microcompartments created from a second sample species reservoir through a second sample injection flow path to create combinatorial microcompartments, where (i) the first sample species and the second sample species are injected by applying an injection pressure P i to respective first species reservoir and second species reservoir, and a non-injection pressure P i lower than the injection pressure P ni is maintained at each sample species reservoir of each non-injection sample injection flow path, and / or (ii) resistance in the first sample injection flow path and / or the second sample injection flow path is provided using at least one hydrodynamic resistor.

[0018] According to one embodiment, a microfluidic chip including one or more combinatorial microcompartments, wherein a first sample species is injected from a first sample species reservoir through a first sample injection flow path into a continuous flow of the carrier phase in a target flow path to create microcompartments, and at least one second sample species is injected from a second sample species reservoir through a second sample injection flow path into the created microcompartments to create microcompartments, and including at least two sample species generated by this, where (i) the first sample species and the second sample species are injected by applying an injection pressure P i to respective first species reservoir and second species reservoir, while a non-injection pressure P i lower than the injection pressure P ni is maintained at each sample species reservoir of each non-injection sample injection flow path, and / or (ii) resistance in the first injection flow path and / or the second injection flow path is provided using at least one hydrodynamic resistor, is provided.

[0019] According to a further embodiment, a method for colocalizing an entity with a barcode oligonucleotide or a set of its components in a microfluidic device described herein, (i) A step of supplying the substance to the target flow path, (ii) A step of passing the substance through a first sample injection channel and a second sample injection channel, wherein one of the sample injection channels injects an injection pressure (P) into one of the first sample reservoir and the second sample reservoir, respectively. i The process involves applying a solution to supply a barcode oligonucleotide or a set of its components from the sample species reservoir to the target channel via each sample injection channel and each sample injection channel junction end, thereby injecting the barcode oligonucleotide or a set of its components into the target channel. (iii) A method is provided in which step (ii) is optionally repeated, and when step (ii) is repeated, the other of the first sample injection channel and the second sample injection channel supplies a set of barcode oligonucleotides or their components from a sample species reservoir to the target channel.

[0020] According to one embodiment, the method involves applying a non-injection pressure (P) during the non-injection stage. ni The process further includes the step of applying ) to a first sample species reservoir (104) and / or at least one second sample species reservoir (106), P ni is environmental pressure (P 大気 ) is larger than P i >P ni >P 大気 That is the case.

[0021] According to another embodiment, the injection pressure (P i ) and non-injection pressure (P ni This is achieved by pressurizing a first sample species reservoir and at least one second sample species reservoir.

[0022] The supply of barcode oligonucleotides or sets of their components from the sample species reservoir to the target channel is controlled by one or more detection means or sensors.

[0023] The substance is nucleic acid, cells, or drugs.

[0024] The method further includes the step of generating microfluidic droplets containing an entity and a barcode oligonucleotide or its components.

[0025] The method further comprises the steps of fusing a microfluidic droplet containing an entity and a barcoded oligonucleotide or its components with a further microfluidic droplet, and / or injecting a reagent into the microfluidic droplet.

[0026] This method further includes the step of detecting entities and barcode oligonucleotides or their components.

[0027] According to further embodiments, the present invention relates to a method for barcoding a test substance on cells or cell cultures, (i) Using the method of the present invention, a step of co-localizing a substance in a microfluidic droplet with a barcode oligonucleotide or its components and optionally with cells, (ii) If no cells co-localized in step (i), a step of selectively introducing cells into the microfluidic droplets of step (i), (iii) A step of injecting or fusing the reaction mixture containing the reagent into the microfluidic droplets of step (ii), The present invention provides a method comprising (iv) incubating the microfluidic droplets from step (iii) to allow the reaction to proceed in the reaction mixture.

[0028] Further aspects, advantages, and features are evident from the dependent claims, specification, and accompanying drawings.

[0029] To ensure that the above-mentioned features of this disclosure are understood in detail, a more detailed description of this disclosure, which is briefly summarized above, can be obtained by referring to the embodiments. The attached drawings are related to embodiments of the present disclosure and are described below. [Brief explanation of the drawing]

[0030] [Figure 1] A schematic diagram of a microfluidic device for creating a combinatorial microcompartment according to this embodiment is shown. [Figure 2A] A schematic diagram is shown illustrating the injection of the first and second sample species, as well as the occurrence of undesirable backflow due to (i) the maintenance of non-injection pressure on the sample species reservoir in the non-injection sample injection channel and (ii) the lack of a combination of hydrodynamic resistors. [Figure 2B] A schematic diagram is shown illustrating how to avoid undesirable backflow when injecting the first and second sample types while maintaining the non-injection pressure on the sample type reservoir in the non-injection sample injection channel, using a hydrodynamic resistor. [Figure 2C] Schematic diagrams of different microcompartments and combinatorial microcompartments created in the carrier phase of the target channel are shown. [Figure 3] A flowchart of the method according to the embodiments described herein is shown. [Figure 4A] This shows the pressure profile during sample injection when maintaining the non-injection pressure against the sample type reservoir in the non-injection sample injection channel is not combined with the use of a hydrodynamic resistor. [Figure 4B] This figure shows the pressure profile during sample injection while maintaining the non-injection pressure against the sample species reservoir in the non-injection sample injection channel and using a hydrodynamic resistor. [Figure 5A] This shows the flow rate profile during sample injection when maintaining the non-injection pressure against the sample type reservoir in the non-injection sample injection channel is not combined with the use of a hydrodynamic resistor. [Figure 5B]This figure shows the flow rate profile during sample injection while maintaining the non-injection pressure against the sample species reservoir in the non-injection sample injection channel and using a hydrodynamic resistor. [Figure 5C] This shows another flow rate profile during sample injection without using a hydrodynamic resistor while maintaining the non-injection pressure to the sample type reservoir in the non-injection sample injection channel. [Figure 6A] Without maintaining the non-injection pressure on the sample species reservoir in the non-injection sample injection channel, a hydrodynamic resistor is used to measure cross-contamination within a microcompartment created using a microfluidic device. [Figure 6B] The non-injection pressure on the sample species reservoir in the non-injection sample injection channel is maintained, and measurements of cross-contamination within a microcompartment created using a hydrodynamic resistor are shown. [Figure 6C] This shows measured cross-contamination within a microcompartment fabricated on a chip without hydrodynamic resistors, while maintaining the non-injection pressure on the sample type reservoir in the non-injection sample injection channel. [Figure 7A] This shows flow rate data in μL / min (i.e., backflow and / or overshoot) recorded within a microfluidic chip without the use of a hydrodynamic resistor. [Figure 7B] The flow rate data in μL / min (i.e., backflow and / or overshoot) recorded within the microfluidic chip is shown using a hydrodynamic resistor in combination with maintaining the non-injection pressure against the sample species reservoir in the non-injection sample injection channel. [Figure 8A] The present invention illustrates a microfluidic chip configuration including a hydrodynamic resistor, such a chip being used in Figures 4-6B. [Figure 8B] Figures 5C, 6C, and 7A show microfluidic chips that do not have hydrodynamic resistors such as chips used. [Figure 9A] This paper presents experimental setups and results for creating microcompartments using the devices and methods of the present invention. [Figure 9B] This paper presents experimental setups and results for creating microcompartments using the devices and methods of the present invention. [Figure 10A] Further details are provided regarding experimental setups and results for creating microcompartments using the devices and methods of the present invention. [Figure 10B] Further details are provided regarding experimental setups and results for creating microcompartments using the devices and methods of the present invention. [Figure 11] An embodiment of the microfluidic device according to the present invention is shown. [Figure 12] The barcode creation process in this invention is schematically shown. [Figure 13A] This is a schematic visualization of the micro-compartment created in Example 2. [Figure 13B] This is a schematic visualization of the micro-compartment created in Example 2. [Figure 13C] The addition of reagents for cell lysis and reverse transcription into microcompartments is schematically shown. [Modes for carrying out the invention]

[0031] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, and that these may change. It should also be understood that the terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the invention, which is limited solely by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they would be generally understood by those skilled in the art.

[0032] Preferably, the terms used herein are defined as follows: “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, HGW, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0033] Throughout this specification and the following claims, unless otherwise required by context, the word “comprise,” and variations such as “comprises” and “comprising,” mean to include the integer or process or group of integers or processes described, but not to exclude any other integer or process or group of integers or processes. Different aspects of the present invention are defined in more detail in the following sections. Each of the embodiments defined in this way can be combined with any other embodiments unless it is explicitly stated otherwise. Any feature indicated as optional, desirable, or advantageous may be combined with any other feature indicated as optional, desirable, or advantageous.

[0034] Several sources are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing in this specification should be construed as an admission that the present invention does not have any prior rights to such disclosure by prior art. Some of the references cited herein are characterized by being “incorporated by reference.” In the event of any conflict between the definitions or teachings in such incorporated references and the definitions or teachings enumerated herein, the text of this specification shall prevail.

[0035] The elements of the present invention will be described below. These elements are listed with specific embodiments, but they can be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described herein should not be construed as limiting the invention to only those embodiments explicitly described. This description supports and includes embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutations and combinations of all elements described in this application are disclosed in the description of this application unless otherwise indicated in the context.

[0036] Herein, various embodiments of the present disclosure are shown in detail, with one or more examples illustrated in the figures. The drawing is a schematic diagram and not drawn to scale. The drawings may have exaggerated dimensions to emphasize aspects of the present disclosure and / or to clarify the presentation. To facilitate understanding, the same reference numbers are used to indicate identical elements common to the figures, where possible. Elements and features of one embodiment can be beneficially incorporated into other embodiments without further enumeration. Generally, only the differences relating to individual embodiments will be described.

[0037] Each example is provided for illustrative purposes of this disclosure and is not intended to limit this disclosure. Furthermore, features illustrated or described as part of one embodiment can be used with or in conjunction with other embodiments to bring about further embodiments. The explanation is intended to include such modifications and variations.

[0038] Embodiments described herein relate to microfluidic devices for creating one or more combinatorial microcompartments.

[0039] A "microfluidic device" is understood as a miniaturized system for controlling, guiding, and / or manipulating the volume of a fluid, liquid, or aqueous sample within a microliter or nanoliter scale, for example. These devices are fabricated by creating channels and compartments within substrate materials such as glass, plastic, or silicone to create a controlled environment for (micro)fluidic processes. Microfluidic devices can be used to perform a wide range of fluid operations, including mixing, pumping, separation, and reaction, and are used in various applications, including chemical analysis, cell culture, drug discovery, and medical diagnostics. The small size and precise control of microfluidic devices enable efficient and cost-effective experiments, as well as the ability to perform experiments and analyses that would be impossible using conventional large-scale fluid systems.

[0040] A "microcompartment" is understood as a liquid, fluid, or aqueous sample dispersed within a continuous phase, such as an surrounding carrier phase or a carrier fluid such as oil or water. In this respect, microcompartments can be considered miniaturized reaction vessels or analytical vessels. The fluid, liquid, or aqueous sample within the microcompartment is immiscible with the carrier phase fluid or liquid.

[0041] These microcompartments are in the form of surfactant droplets, for example, emulsions having a volume in the picoliter range. A surfactant droplet is, for example, a spherical droplet of a surfactant solution surrounded by a continuous phase, such as oil or water. Surfactants are surface-active compounds that can reduce the surface tension between two immiscible liquids, enabling the formation of stable droplets in a continuous phase. In surfactant droplets, surfactant molecules are adsorbed at the interface between the droplet and the continuous phase, creating a stabilized interface that prevents the droplet and its internal material from merging with the continuous phase. These droplets are also called microfluidic droplets.

[0042] Furthermore, a microcompartment is a (microfluidic) plug having a volume in the nanoliter range, for example, that completely fills the target channel or tube. This plug is then separated by a carrier phase or carrier fluid, such as oil or water. In this case, stabilization with a surfactant is not necessary.

[0043] Furthermore, the microcompartments may also be flow segments, such as microfluidic channels or parts or sections within a pipe, where the fluid flow has a specific and distinct behavior beyond a microliter volume.

[0044] A "combinatorial microcompartment" is understood as a microcompartment containing at least two sample species or substances that are chemically and / or biologically distinct, e.g., sample species (A) and sample species (B). Therefore, combinatorial microcompartments contain a mixture of different species compartmentalized within the carrier phase.

[0045] Possible sample types include molecular drugs or compounds, proteins or enzymes, nucleic acids (DNA, RNA), cells or microorganisms, such as prokaryotic or eukaryotic cells, microscopic tissue samples, particles or nanoparticles and / or biomolecules, such as sugars, lipids or hormones.

[0046] Chemically and / or biologically distinct means substances, entities, or materials that have different chemical / biological properties and composition. This is because they differ in their molecular structure, chemical behavior, and / or biological activity. The distinction may be based on differences in the composition of individual molecules, the types and arrangement of atoms, the presence or absence of specific functional groups, or differences in biological activity. For example, chemically distinct entities include different bioactive substances such as drugs that trigger specific cellular responses. Biologically distinct entities include different types of cells, cells derived from different donors, cell libraries expressing different variants of a given protein, and tissue sections.

[0047] The microfluidic device according to the embodiments discussed herein includes a first sample species reservoir containing a first sample species (A) and a second sample species reservoir containing a second sample species (B).

[0048] A “reservoir” or “sample species reservoir” is understood to be a storage facility or container used to store, contain, or hold a volume of a particular sample species containing an aqueous sample, fluid, or liquid. The pressure applied to the reservoir also regulates the flow or outflow of these sample species. Typical volumes of such sample species reservoirs range from a few picoliters to a few milliliters, particularly a few nanoliters to a few microliters, more specifically a few microliters to a few milliliters, or even higher / lower ranges. A sample species reservoir according to one embodiment of the present invention can be made from a rigid or non-rigid material, but a rigid material is preferred. Preferred materials for the sample species reservoir are plastic materials such as polypropylene or polycarbonate, and glass.

[0049] A “sample species” stored in a sample species reservoir is understood as a fluid, liquid, or aqueous sample containing water, which further includes dissolved or suspended substances of a particular chemical / biological species, e.g., substances or materials having chemically and / or biologically distinct properties, characteristics, compounds, and / or compositions, e.g., (A), (B), (C), etc. The sample species also include carrier phases, such as oil and water. The sample, sample type, and / or carrier phase may be stored in fluid or liquid form, and these terms are used interchangeably herein. The carrier phase fluid or liquid is usually selected from oils, but in certain applications of the present invention, it may also include an aqueous sample liquid.

[0050] In the context of the present invention, “target flow path” is understood as a flow path through which two, three, or more fluids are combined, and the combined fluids are further transported to any analytical area, detection area, storage reservoir, or any other means through which the fluid combination is performed. This term should be understood as a channel or tube in a microfluidic chip, for example, in which the sample according to the present invention is combined. The target channel may be configured to hold, guide, or support the target channel fluid, such as a carrier phase. For example, the "carrier phase" in the target channel is considered to be a fluid or liquid that flows continuously through the tip in order to move or transport the sample through the tip. The carrier phase may be a liquid such as water or oil, and its properties, such as viscosity, surface tension, and pH, can be adjusted to optimize sample transport.

[0051] The term "injection channel" refers to one or more channels through which fluids are injected into a target channel in order to combine one or more fluids according to the present invention. In some cases of the present invention, the injection channel is also called the sample injection channel. In this context, the term "sample" refers to one or more fluids injected into the target channel.

[0052] It is mentioned that the first sample species reservoir contains a first sample species (A) that is different from the second sample species (B) contained in the second sample species reservoir, but it is also possible for both sample species reservoirs to contain the same sample species, for example, the first sample species (A) or the second sample species (B).

[0053] The microfluidic device may include a first sample injection channel and a second sample injection channel. A "sample injection channel" is understood as a (fluid) channel, path, or tube used to transport or deliver a fluid, liquid, or aqueous sample, such as a specific sample type, to, for example, a target channel. The carrier phase is also injected using one or more sample injection channels.

[0054] The sample injection channel is made from glass, silicone, or plastic. The diameter of the sample injection channel ranges from several tens of micrometers to several millimeters. For example, in microfluidic devices used for high-throughput screening, the sample injection channel has a diameter of several micrometers.

[0055] However, a larger diameter is beneficial. For example, when working with cells, a larger channel diameter can be used to avoid channel clogging by allowing cells to pass through without getting trapped or stuck. The first sample injection channel includes a sample injection channel reservoir end connected to the first sample species reservoir and a sample injection channel junction end connected to the target channel, and the second sample injection channel includes a sample injection channel reservoir end connected to the second sample species reservoir and a sample injection channel junction end connected to the target channel.

[0056] At the reservoir end of the sample injection channel, the sample injection channel is connected to the sample type reservoir without a valve. However, the sample reservoir may be pressurized, and the pressure is released by an "external valve," for example, a valve connected to a pressure source (s.below).

[0057] Generally, the combinatorial microcompartment described herein is created using at least two sample injection channels connected to a first sample type reservoir containing a first sample type and a second sample type reservoir containing a second sample type. To create a microcompartment, only one sample injection channel is sufficient.

[0058] The sample injection channel junction ends of the first sample injection channel and / or the second sample injection channel are connected to the target channel without a valve and are equipped with hydrodynamic resistors.

[0059] The sample injection channel junction end defines the end of the sample injection channel that encounters, crosses, merges with, or connects to the target channel. A "valveless" connection is understood as a connection that does not include or use valves to control the flow of fluid. In microfluidic devices with a valveless connection between the sample injection channel and the carrier phase, the sample is introduced into the carrier phase through a small opening or constriction at the end of the sample injection channel junction. The pressure difference between the sample and the carrier phase, along with the shape of the flow path and the fluid resistance of the flow path, determines the flow rate and mixing of the aqueous sample and the carrier phase. Valveless connections offer improved reliability, lower costs, and reduced complexity. They are also easier to manufacture and maintain compared to other devices that use valves.

[0060] Therefore, the terms "valveless" or "without valves" preferably mean that no valves are used to open or close the inlet to the flow path. Preferably, there are no valves associated with the injection of sample species from the sample species reservoir and / or from the sample species reservoir as described herein; for example, each inlet is not opened or closed using a valve. Furthermore, according to a preferred embodiment, to provide resistance to the hydrodynamic resistor and / or the non-injection pressure (P) described herein,ni ) and injection pressure (P i Valves are not used to generate ). Furthermore, this valve does not actively regulate the supply of any entity, such as particles, barcode oligonucleotides, other elements, or fluids, to the injection channel. In other words, all reagents are injected by applying pressure only to the reagent reservoir. Therefore, according to the present invention, the microfluidic device does not have a valve between the sample species reservoir and the target channel. In a more preferred embodiment, there are no valves in the entire microfluidic device. It should be understood that the term "valveless" does not preclude the presence of valves outside the microfluidic device or chip to release pressure after each element or component has been injected or supplied to the target channel or any other part of the microfluidic device. According to one embodiment, it will also be understood that valves that are not involved in the process of supplying elements or components from a sample species reservoir through an injection channel are not excluded by the term "valveless". The opposite of valveless in relation to the supply of entities, barcode oligonucleotides, or other elements or fluids to an injection channel is "valved," which uses valves to open and close each channel.

[0061] A "hydrodynamic resistor" is understood as a device that provides hydrodynamic resistance to the fluid flowing through a sample injection channel in order to control and regulate the fluid flow within a microfluidic system. Pressure drop occurs across resistors that resist the flow of fluid and regulate its flow rate. A hydrodynamic resistor is implemented as a narrow channel, constriction, or constricted region within a microfluidic device, or as a region where fluid resistance is increased, causing a pressure drop or pressure increment, for example, an increase or decrease in pressure over a certain period, thereby generating hydrodynamic / flow resistance.

[0062] The hydrodynamic resistor may be implemented as a narrow channel with a smaller diameter. This is because the resistance provided by the flow path is approximately inversely proportional to the fourth power of the flow path's diameter. In other words, if the diameter of the flow path is reduced by half, the resistance of the flow path increases 16-fold. As a result, the smaller the diameter of the flow path, the higher the resistance, making the flow path more effective in regulating pressure. Therefore, hydrodynamic resistors are implemented as small-diameter channels because they offer higher resistance and better pressure regulation than shorter, wider channels.

[0063] Hydrodynamic resistors contribute to preventing backflow and function as pressure regulators or pressure controllers, ensuring stable pressure within microfluidic devices. For example, when pressure is applied to a reservoir within a microfluidic device, a pressure pulse is generated that causes abrupt flow rate changes, such as abrupt pressure changes due to pressure overshoot and over-regulation in the sample injection channel. This results in undesirable flows and system disruption in one or another direction. By using a hydrodynamic resistor, the pressure pulse is attenuated or dampened, increasing the response time from the application of a specific pressure to the point where it takes effect, e.g., where droplets are formed, thus enabling more stable and precise pressure regulation. This is also important for non-injectable channels, as the system can over-regulate significantly without hydrodynamic resistors, potentially causing pressure pulses and undesirable flow in either direction. Therefore, hydrodynamic resistors maintain the stability and precision of microfluidic devices by regulating or controlling pressure and preventing backflow.

[0064] A hydrodynamic resistor is a specific channel shape, such as a constriction, narrow section, bottleneck, or bend; a porous material; for example, a membrane with a controlled pore size; for example, a hydrogel with controlled swelling properties; and / or a capillary tube with a controlled inner diameter and length.

[0065] Furthermore, the hydrodynamic resistor is implemented as a specific channel shape in a portion of the flow path, or as multiple turns or bends in the cross-section of each channel. In a particularly preferred embodiment, the hydrodynamic resistor is a continuous arrangement of multiple turns or bends in a flow path of about 170–190°, more preferably about 180°, as illustrated in Figure 8A. The multiple turns or bends of the hydrodynamic resistor include 4 to 40 turns / bend, preferably 6 to 30, 8 to 20, 10 to 18, or 12 to 16 turns / bend. In particular, in a continuous arrangement of multiple turns or bends within a flow path, the turns / bends are evenly distributed along each flow path section that is implemented as a hydrodynamic resistor. Each flow path may contain two or more hydrodynamic resistors, for example, two, three, four, five, six, seven, eight, nine, ten, or more hydrodynamic resistors.

[0066] When used herein, the term "entity" refers to a substance, compound, or particle that can be transported within the flow channel of a microfluidic device according to the present invention. This term encompasses pharmaceuticals, molecular drugs or compounds, chemical or biological substances such as proteins or enzymes, and biomolecules such as sugars, lipids, and hormones. According to a preferred embodiment, the entity is a chemical substance and / or a drug such as a pharmaceutical. The term "substance" also includes particles in its broadest sense. The term “particle” as used herein includes any artificial or natural particle that incorporates, encapsulates, binds to, consists of, or otherwise associates with DNA and / or RNA. In a preferred embodiment, it is a biological particle, preferably a cell, a non-cellular organism, or a DNA and / or RNA carrier, or DNA and / or RNA. Particles are also the transcriptome of a cell or the DNA amplification product from a cell. Most preferably, the particles are cells. And this cell can be any prokaryotic or eukaryotic cell. Preferably, it is a eukaryotic cell, such as a yeast cell, plant cell, or animal cell. Animal cells include insect cells, nematode cells, fish cells, and mammalian cells. More preferably, it is a mammalian cell, such as a mouse, rat, monkey, or human cell. For example, these could be random cells from a heterogeneous cell population (e.g., tissue-derived) or specifically selected cells chosen by FACS. Furthermore, it refers to cells from a cell line or homogeneous culture, for example, primary cells, where "primary" means directly derived from a tissue or organism and not manipulated to have altered characteristics, such as to divide indefinitely. Other examples of cells include developing cells, stem cells, or cancer cells. Examples of noncellular biological forms include viruses, viroids, cosmids, plasmids, and phagemids. Examples of DNA and / or RNA carriers include proteins such as histones or ribosomes. Non-biological particles such as beads are also conceivable. "Beads" (also called "microbeads") are uniform polymer particles having a diameter of up to 1 micrometer, preferably 0.5 to 500 μm, and having a surface to which nucleic acids are bound or linked. The beads referred to herein are typically polyethylene or polystyrene beads, or beads made of a gel matrix.

[0067] The term "barcode oligonucleotide" refers to an oligonucleotide that has at least one so-called variable region, and whose nucleotide sequence is unique to this oligonucleotide compared to other barcode oligonucleotides used. And at least two such barcode oligonucleotides are used. Alternatively, the barcode oligonucleotide may have at least two variable regions, and the combined nucleotide sequence is unique to this oligonucleotide compared to other barcode oligonucleotides used. The term "variable" does not mean that the sequence of a particular oligonucleotide changes, but rather that the structure and sequence are identical except for the sequence of the variable region; in other words, different oligonucleotides exist among oligonucleotides that have the same structure and sequence but a variable region. If a barcode oligonucleotide contains two or more variable regions, each of these variable regions comes from a separate combinatorial component that can be assembled in a combinatorial manner to create different barcode oligonucleotides. The barcode more preferably includes at least one priming region or alternatively one or more displaceable elements.

[0068] Therefore, the "components" of a barcode oligonucleotide are the oligonucleotide itself, which has one variable region. The "set of components" of a barcode oligonucleotide is a complete set of components, consisting of multiple oligonucleotides that make up exactly one barcode oligonucleotide (meaning one identity, not just one molecule). The components of a set are combined by annealing to a linear barcode oligonucleotide, preferably by their annealing regions, and only one linear combination is possible. Therefore, within a set of components, the annealing regions differ among the components, but their variable regions, while identical, are likely to be similarly different.

[0069] The term "colocalization" essentially refers to bringing together two or more entities, such as particles or drugs, and barcode oligonucleotides or their components, preferably into the same microfluidic droplet. This can be achieved, for example, by generating microfluidic droplets from an aqueous fluid containing these entities, by fusing droplets containing each of these entities separately, or by injecting a continuous aqueous phase into pre-formed microfluidic droplets, as described below. The "fusion" of two droplets results in a single microfluidic droplet containing the contents of both originating droplets. Therefore, droplets resulting from such fusion include microfluidic droplet contents containing particles, and microfluidic droplet contents containing barcode oligonucleotides or their components. According to Mazutis et al. (A fast and efficient microfluidic system for highly selective one-to-one droplet fusion. Lab Chip (2009) vol.9(18) pp.2665-2672), such fusion is achieved by one-to-one fusion. Further droplet fusion methods are described in P. Day et al. (eds.), Microdroplet Technology: Principles and Emerging Applications in Biology and Chemistry, Integrated Analytical Systems, DOI 10.1007 / 978-1-4614-3265-4_2,# Springer Science+Business Media, LLC 2012, Chapter 2.

[0070] When used in the context of fluids or liquids, the term "immiscible" refers to a fluid or liquid in which entities such as particles and sample species such as barcode oligonucleotides or their components are immiscible with the fluid or liquid contained in the target flow path. The immiscible liquid is preferably a hydrophobic liquid, preferably an oil. The oil phase should have a viscosity close to that of water and / or be inert to any biological reagents contained within it. Low-viscosity silicone oil or several other oils such as dimethicone, silicone oil, and hydrocarbon oil are used. Preferred oils for use in the context of the present invention are fluorocarbon oils (or fluorinated oils), because even low-viscosity versions of these oils do not cause PDMS to swell. Surfactants are useful in reducing the surface tension of oil-water interfaces and minimizing droplet aggregation, and are therefore present in immiscible fluids or liquids. Surfactants used in droplet-based microfluidics typically consist of a hydrophilic head group and a hydrophobic tail. The amphiphilic properties of these molecules allow them to aggregate at the oil-water interface of a droplet, thereby reducing its interfacial tension and increasing its stability. Surfactants having a nonionic head group are preferred because they minimize the adsorption of macromolecules such as proteins and DNA to the droplet interface, thereby minimizing their impact on the method of the present invention. Suitable fluorosurfactants that can be readily synthesized in the laboratory are well known in the art, for example, described by Clausell-Tormos J et al 2008 Chem. Biol. 15, 427-37 or Sadtler et al. 1996 Angew. Chem. Int. Edn Engl. 35, 1976-8, and many are commercially available, for example, from Sphere Fluidics Limited, UK. Additives to the aqueous phase can also enhance biocompatibility by increasing the retention of small molecules within droplets and minimizing adsorption at the oil-water interface. Different oils can be mixed to optimize the properties of an emulsion for a specific application, and methods for easily characterizing the properties of a selected combination are known in the art (Kaltenbach et al. 2012 Lab Chip 12, 4185).

[0071] As used herein, the term "microfluidic droplet" refers to an aqueous microcompartment of a specific size that encapsulates an aqueous liquid. The size of a microfluidic droplet is expressed as its diameter. The diameter is generally less than 1 mm, for example, about 10 μm to 900 μm, about 20 μm to 800 μm, about 20 μm to 700 μm, about 20 μm to 600 μm, about 20 μm to 500 μm, about 20 μm to 400 μm, preferably 30 μm to 350 μm, 40 μm to 300 μm, 40 μm to 250 μm, 40 μm to 200 μm, or 40 μm to 100 μm (where each narrow range is preferred over the broad ranges mentioned above, and "~" includes the value mentioned). Other forms of microfluidic droplets include elongated droplets, which can be described as having a cylindrical shape, such as a sausage shape; that is, they are longer than they are wide. Such elongated droplets, also referred to herein as “plugs,” have lengths of several millimeters, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9 and up to 10 mm, preferably about 0.2 mm to 6 mm, more preferably about 0.5 mm to 5.5 mm, even more preferably about 1.0 mm to 5 mm, and a diameter of about 300 μm to 900 μm, preferably about 400 μm to 800 μm, more preferably about 500 μm to 700 μm. According to a preferred embodiment, the plug has a diameter of about 300 μm and a length of about 1 mm to 3 mm. According to a preferred embodiment, the plug has dimensions such that it completely fills the target flow path. In such embodiments, individual plugs may be separated by a carrier phase or carrier fluid, such as oil or water. Alternatively, the size of a microfluidic droplet can be defined by its volume. For example, the size of microfluidic droplets is typically less than 1 microliter (μL). Preferably, the size of the microfluidic droplets is less than 900 nanoliters (nl), less than 800 nl, less than 700 nl, less than 600 nl, less than 500 nl, less than 400 nl, less than 300 nl, less than 250 nl, less than 150 nl, less than 100 nl, or less than 50 nl. In preferred embodiments, the size of the microfluidic droplets is 0.05 to 150 nl, preferably 0.05 to 125 nl, 0.05 to 100 nl, 0.05 to 80 nl, or 0.05 to 4 nl (where each narrow range is preferred over the aforementioned broad ranges, with "~" representing the value mentioned). According to a preferred embodiment, the microfluidic droplet is a plug having a volume of about 500 nl.

[0072] The term "generating microfluidic droplets" refers to creating a flow of monodisperse droplets in an immiscible phase. This can be achieved using a droplet generator. Microfluidic droplet generators work by combining two or more flows of immiscible fluids and generating shear forces in the discontinuous phases, causing them to break down into discontinuous droplets. Preferred droplet generators are focused-flow droplet generators and T-shaped droplet generators. A wide variety of such compartmentalization or microencapsulation procedures are available (Benita, S., Ed. (1996). Microencapsulation: methods and industrial applications. Drugs and pharmaceutical sciences. Edited by Swarbrick, J. New York: Marcel Dekker), and are used to create microfluidic droplets used according to the present invention. In fact, the literature identifies more than 200 methods of microencapsulation or compartmentalization (Finch, CA (1993) Encapsulation and controlled release. Spec.Publ.-R.Soc.Chem.138,35). These include membrane-enveloped aqueous vesicles such as lipid vesicles (liposomes) (New, RRC, Ed. (1990). Liposomes: a practical approach. The practical approach series. Edited by Rickwood, D. & Hames, B.O. Oxford: Oxford University Press) and nonionic surfactant vesicles (van Hal, DA, Bouwstra, JA & Junginger, HE (1996). Nonionic surfactant vesicles containing estradiol for topical application. In Microencapsulation: methods and industrial applications (Benita, S., ed.), pp. 329-347. Marcel Dekker, New York.), all of which are incorporated herein by reference. Preferably, the microfluidic droplets or microcompartments of the present invention are formed from an emulsion and are a heterogeneous system of two immiscible liquid phases, in which one phase is dispersed in the other phase as microscopic droplets (Becher, P. (1957) Emulsions: theory and practice. Reinhold, New York; Sherman, P. (1968) Emulsion science. Academic Press, London; Lissant, KJ, ed. Emulsions and emulsion technology. Surfactant Science New York: Marcel Dekker, 1974; Lissant, KJ, ed. Emulsions and emulsion technology. Surfactant Science New York: Marcel Dekker, 1984), all of which are incorporated herein by reference. Emulsions are produced from any suitable combination of immiscible liquids. Preferably, the emulsion of the present invention comprises an aqueous phase (containing particles and other components) as a phase existing in the form of droplets, and a hydrophobic, immiscible liquid (preferably oil) as a surrounding matrix on which these droplets are suspended. Such emulsions are called "water in oil." This has the advantage that the aqueous phase is compartmentalized into separate droplets. The external phase, which is preferably a hydrophobic oil, is generally inert. Emulsions are stabilized by the addition of one or more surfactants. These surfactants act at the water / oil interface to prevent (or at least delay) phase separation. Numerous oils and emulsifiers are used in the production of water-in-oil emulsions, and recent compilations list over 16,000 surfactants, many of which are used as emulsifiers (Ash, M. and Ash, I. (1993) Handbook of industrial surfactants. Gower, Aldershot), which are incorporated herein by reference. The appropriate oils are as described above.

[0073] Figure 1 shows a microfluidic device (100) for creating one or more combinatorial microcompartments according to embodiments described herein.

[0074] The microfluidic device (100) includes a target channel (102) and at least a first sample species reservoir (104) containing a first sample species (A) and a second sample species reservoir (106) containing a second sample species (B). The microfluidic device (100) may, optimally, include further sample species reservoirs, such as a third sample species reservoir (108) containing a third sample species (C) and a fourth sample species reservoir (110) containing a fourth sample species (D). The third sample species (C) and / or the fourth sample species (D) may also be chemically and / or biologically distinct from each other and / or from at least one of the first sample species (A) and the second sample species (B).

[0075] However, combinatorial microcompartments are created within the carrier phase described herein using at least two sample injection channels (114; 116) connected to a first sample type reservoir (104) containing a first sample type (A) and a second sample type reservoir (106) containing a second sample type (B).

[0076] As can be seen from Figure 1, the microfluidic device (100) includes a first sample injection channel (114) which includes a sample injection channel reservoir end (114A) connected to a first sample species reservoir (104) and a sample injection channel junction end (114B) connected to a target channel (102), and a second sample injection channel (116) which includes a sample injection channel reservoir end (116A) connected to a second sample species reservoir (106) and a sample injection channel junction end (116B) connected to a target channel (102). The sample injection channel junction ends (114B; 116B) of the first sample injection channel (114) and / or the second sample injection channel (116) are connected to the target channel (102) without valves and are equipped with at least one hydrodynamic resistor.

[0077] The microfluidic device (100) shown in Figure 1 includes a third sample injection channel (118) which includes a sample injection channel reservoir end (118A) connected to a third sample species reservoir (108) and a sample injection channel junction end (118B) connected to a target channel (102), and a fourth sample injection channel (120) which includes a sample injection channel reservoir end (120A) connected to a fourth sample species reservoir (110) and a sample injection channel junction end (120B) connected to a target channel (102). However, this is optional. The sample injection channel junction ends (118B; 120B) of the third sample injection channel (118) and / or the fourth sample injection channel (120) are connected to the target channel (102) without a valve and are equipped with at least one hydrodynamic resistor.

[0078] As shown in Figure 1, the first sample injection channel (114) and the second sample injection channel (116) are separately connected to the target channel (102) at their respective sample injection channel junction ends (114B; 116B). The same applies to the third sample injection channel (118) and the fourth sample injection channel (120). For this purpose, the first sample injection channel (114) and the second sample injection channel (116) are also considered as individual or separate channels or pathways that enable, for example, the first sample species (A) contained in the first sample species reservoir (104) and the second sample species (B) contained in the second sample species reservoir (106) to be kept separate from each other. Therefore, this prevents mixing of different sample types.

[0079] Although not shown in Figure 1, one or more combinatorial microcompartments flow into either the outlet or readout channel. A "readout channel" is considered a channel used to measure or detect the properties of a fluid, particularly one or more (combinatorial) microcompartments created within it, for example, a channel analyzed to collect information about different samples or sample species (e.g., cells) within a (combinatorial) microcompartment. To do so, fluorescence, luminescence, electrochemistry, and / or mass spectrometry are used.

[0080] The microfluidic device (100) shown in Figure 1 may further comprise one or more sensors (114C; 116C; 118C; 120C). As can be seen in Figure 1, the first sample injection channel (114) is connected to the first sensor (114C), and the second sample injection channel (116) is connected to the second sensor (116C). Correspondingly, the same applies to the third sample injection channel (118) and the fourth sample injection channel (120) shown in Figure 1, as well as the third sensor (118C) and the fourth sensor (120C).

[0081] One or more sensors (114C;116C;118C;120C) are configured to monitor data related to the fluid, liquid, or aqueous sample flowing through each sample injection channel (114,116;118;120), for example, each sample type. For example, the data may include flow data or flow rate indicating the volume of fluid, liquid, or aqueous sample flowing or passing through each sample injection channel (114, 116; 118; 120) per unit time, or pressure data, such as the pressure of the fluid, liquid, or aqueous sample flowing or passing through each sample injection channel (114, 116; 118; 120) per unit time. Exemplary injection flow rates are in the range of 5–40 μL / min, particularly 8–17 μL / min. The preferred pressure is selected from 10 mbar to 10 bar, particularly 100 mbar to 5 bar, and more specifically from 300 mbar to 2000 mbar. One or more sensors (114C; 116C; 118C; 120C) are pressure sensors and / or flow sensors.

[0082] According to a preferred embodiment, the supply of sample species, such as a barcode oligonucleotide or a set of its components, from a sample species reservoir (104, 106) to a target channel (102) is controlled by one or more sensors or detection means (114C; 116C; 118C; 120C). In particular, non-injection pressure P ni or injection pressure P i The timing of application can be controlled by one or more sensors or detection means. Time-based control can also be based on the time when entities such as particles are supplied to the target channel, the distance they need to travel to reach and leave the injection channel, and the flow velocity of the particles. For example, when multiple particles are combined with the same sample species in a microfluidic droplet, the time-based control may be arbitrary or based on the frequency or density of particles in the target channel. The detection means can be positioned at the start and / or end of each injection channel and / or above or near the sample species reservoir, and when particles are detected, the non-injection pressure P ni or injection pressure P i This can induce the application of [the substance]. If one or more detection means are positioned at the distance to the start and / or end of each injection channel, a combination of two is also possible. In such cases, control may be based on the time the particle is detected, the distance it needs to travel to reach and leave a series of oligonucleotide inlets, and the particle's flow velocity. Detection means for detecting particles in microfluidic channels are well known in the art and include optical sensors, such as photomultiplier tubes, CMOS or CCD cameras, detection electrodes, or flow sensors such as air flow sensors. Generally, detection means are suitable for detecting particles and / or labels bound to particles, particularly fluorescent labels, and fluorescence or laser spectroscopy, imaging, impedance or magnetic measurements may be used for detection.

[0083] In particular, one or more detection means or sensors are located within 10 mm to 50 mm, preferably 1 mm to 30 mm, and more preferably 200 μm to 500 μm upstream of the first sample injection channel junction end (114B), where upstream means the direction in which the carrier phase drives particles through the target channel. Therefore, a detection means or sensor located upstream of the sample injection channel junction end means that the detection means or sensor is positioned on the target channel in the direction that the carrier phase drives particles through the target channel. Furthermore, the detection means or sensor may be located upstream of only the first sample injection channel junction end (114B), for example, upstream of all second sample injection channel junction ends (116B) or upstream of all sample injection channel junction ends. Another embodiment also includes a detection means located downstream of the generation of microfluidic droplets within the target channel.

[0084] The microfluidic device (100) shown in Figure 1 may further comprise one or more pressure control devices (114D; 116D; 118D; 120D). The first pressure control device (114D) may be connected to the first sample species reservoir (104), and at least the second pressure control device (116D) may be connected to the second sample species reservoir (106). The same applies to the third pressure control device (118D) and the fourth pressure control device (120D), as well as the third sample type reservoir (108) and the fourth sample type reservoir (110).

[0085] The pressure control devices (114D; 116D; 118D; 120D) are connected to a pressure source (122) used to generate pressure within the microfluidic device (100). Examples of pressure sources (122) include syringe pumps, pneumatic pumps, and piezoelectric pumps.

[0086] Pressure control devices (114D; 116D; 118D; 120D) can be used to provide both positive and negative pressure and can be programmed to operate in various ways, such as constant pressure, constant flow rate, or stepped pressure changes. The pressure control devices (114D; 116D; 118D; 120D) control the pressure used for injection, "injection pressure P". i " and the pressure used for non-injection "Non-injection pressure P ni The system is configured to apply different pressures, including '', to the sample type reservoir (104;106;108;110).

[0087] For example, the first pressure control device (114D) controls the first injection pressure P i The first sample type reservoir (104) is used to inject the first sample type (A) through the first sample injection channel (114), and the second pressure control device (116D) controls the second injection pressure P i It is configured to be applied to a second sample type reservoir (106) for injecting a second sample type (B) via a second sample injection channel (116). First injection pressure P i and the second injection pressure P i These may be the same or they may be different.

[0088] "Injection pressure" (P i ) is understood as the (positive) pressure applied for injection, for example, the pressure that drives a fluid, liquid, or aqueous sample from the sample species reservoir (104;106;108;110) through the sample injection channels (114;116;118;120) to the target channel (102). The injection pressure may be applied directly to each sample type reservoir (104;106;108;110) by a pressure control device (114D;116D;118D;120D). Illustrative injection pressure P i This range is 10 mbar to 10 bar, particularly 20 mbar to 7 bar, 100 mbar to 5 bar, 60 mbar to 4 bar, and more specifically, 125 mbar to 2000 mbar, or 300 mbar to 2000 mbar. Such injection pressures result in injection flow rates ranging from 1 to 500 μL / min, particularly 1 to 100 μL / min, and more specifically, 8 to 17 μL / min. However, these flow rates are adjusted or modified depending on the length of the pipe / flow path connected to the outlet.

[0089] Pressure (P i or P ni The pressure is generated by an external pressure source, such as an external mechanical pump, such as a syringe pump, pneumatic pump, and piezoelectric pump, or by an integrated micropump, such as a mechanical micropump, by means known to those skilled in the art. The micropump is selected from the group consisting of syringe micropumps, pneumatic membrane micropumps, piezoelectric micropumps, Braille pin micropumps, electrochemical micropumps, electroosmotic micropumps, acoustic micropumps, magnetohydrodynamic micropumps, electrohydrodynamic micropumps, and gas permeable micropumps, and more preferably, the active micropump is independently selected from the group consisting of syringe micropumps, pneumatic membrane micropumps, and Braille pin micropumps.

[0090] The pressure control device (114D;116D;118D;120D) controls the pressure (e.g., one or more injection pressures P) based on flow data monitored by sensors (114C;116C;118C;120C) in each of the sample injection channels (114;116;118;120). i or one or more non-injection pressures P niTo adjust the settings, each sensor (114C;116C;118C;120C) in the sample injection channel (114;116;118;120) is connected in a communicative manner.

[0091] For example, the first pressure control device (114D) may be communicatively connected to the first sensor (114C) of the first sample injection channel (114), and the second pressure control device (116D) may be communicatively connected to the second sensor (116C) of the second sample injection channel (116), and the first injection pressure P i and the second injection pressure P i These are applied based on monitored flow data of the liquid in the first sample injection channel (114) and the second sample injection channel (116), respectively.

[0092] Needless to say, the third pressure control device (118D) may be communicatively connected to the third sensor (118C) of the third sample injection channel (118), and the fourth pressure control device (120D) may be communicatively connected to the fourth sensor (120C) of the fourth sample injection channel (120), and the third injection pressure P i and the fourth injection pressure P i These are applied based on monitored flow data of the liquid in the third sample injection channel (118) and the second sample injection channel (120), respectively.

[0093] For example, inject pressure P into one or more sample type reservoirs (104;106;108;110) i When applied, one or more sample types are injected into the target channel (102) via one or more sample injection channels (114;116;118;120). With respect to one sample injection channel, for example, the first sample injection channel (114), or other sample injection channels thereafter, for example, the second sample injection channel (116) and / or the third sample injection channel (118), there may be injection or non-injection. In the latter case, "non-injection" should be understood to mean that the flow of the fluid, liquid, or aqueous sample does not substantially flow out of the corresponding sample injection channel at the end of the sample injection channel junction. For example, a sample injection channel in which no sample species has been injected initially is called a "non-injection channel." As a result, the sample injection channel into which the sample species is injected at the first time or at a second time different from the first time is called the "injection channel".

[0094] According to embodiments of the present invention, pressure is also applied to the non-injection channel. Such pressure is called "non-injection pressure" (P ni ) may be called that. For example, non-injection pressures are in the range of 1 mbar to 5600 mbar or 10 mbar to 10 bar, particularly 100 mbar to 5 bar, 50 mbar to 3200 mbar, and more specifically, 300 mbar to 2000 mbar or 100 mbar to 1600 mbar. Such non-injection pressure P ni This effectively reduces the flow rate of fluids, liquids, or aqueous samples at flow rates of less than approximately 30 μL / hour, particularly less than approximately 20 μL / hour, to zero.

[0095] The first pressure control device (114D) and the second pressure control device (116D) control the non-injection pressure P on each sample type reservoir (104;106) of each non-injection sample injection channel (114;116). ni It is configured to maintain this. Non-injection pressure P ni The first injection pressure P i and lower than the second injection pressure Pi, relation P i >P ni >P 大気 It is preferable that it be located in that position. P 大気 The injection pressure P i and non-injection pressure P ni This refers to the environmental or ambient pressure acting on the device, particularly the pressure acting on the sample species reservoir, when no other pressures are applied. Each of the pressure control devices (114D; 116D; 118D; 120D) controls the non-injection pressure P ni It is further configured to increase over time.

[0096] And this non-injection pressure P ni It is increased gradually or continuously with a steady increase; stepwise, for example, in discrete processes with a stable period between each step; for example, pulsed between each pulse and / or randomly, for example, in a series of short, sharp pulses with a randomly changing stable period over time. Therefore, the first pressure control device (114D) and the second pressure control device (116D) control the non-injection pressure P for each sample type reservoir (104;106) of each non-injection sample injection channel (114;116). ni It is configured to increase over time. The same applies to the third pressure control device (118D) and the fourth pressure control device (120D).

[0097] As shown in Figure 1, the target channel (102) is contained within the microfluidic chip (124) and is used to transport or move fluid (including the respective aqueous sample or compartment) from one location to another within the microfluidic chip (124).

[0098] The microfluidic device (100) shown in Figure 1 is used in the field of cancer treatment and / or combinatorial drug screening. In particular, the microfluidic device (100) according to the embodiments described herein enables a high level of automation with different options for primary readout, such as fluorescence spectroscopy, sequencing, and imaging.

[0099] Furthermore, one or more (combinatorial) microcompartments generated using the microfluidic device (100) shown in Figure 1 can be used for high-throughput screening applications. While conventional systems using microtiter plates require approximately 20,000 cells to test a single treatment option against cancer cells, a microfluidic device (100) requires only about 100 cells.

[0100] Furthermore, compared to microfluidic systems with Braille valves where cells are continuously injected into either a droplet generation device or waste, valveless equipment consumes cells only during droplet generation (the cells do not move to the waste). Therefore, the total amount of cells consumed is small, about one-third.

[0101] As will be described in more detail below, the microfluidic device (100) makes it possible to reduce or prevent microfluidic backflow, for example, unintended flow of liquid, fluid, or aqueous sample from one sample injection channel (114;116;118;120) or one combinatorial microcompartment (200A;200B) to one or more other sample injection channels (114;116;118;120) or another combinatorial microcompartment (200A;200B). For this purpose, cross-contamination between adjacent channels, microcompartments, and / or combinatorial microcompartments can be reduced, and liquid, fluid, or aqueous samples can be kept separate. Therefore, unintended mixing of samples is reduced or avoided.

[0102] Furthermore, the fact that the microfluidic device (100) includes at least one hydrodynamic resistor ensures that pressure overshoot and overregulation in the sample injection channel resulting from sudden flow rate and / or pressure changes are balanced, reduced, prevented, or minimized.

[0103] Figure 2A shows a schematic diagram of the first sample injection channel (114) and the second sample injection channel (116) connected to the target channel (102). Figure 2A shows the injection of a first sample type (A) and a second sample type (B) without the use of a hydrodynamic resistor (not shown).

[0104] As can be seen in Figure 2A, the first sample injection channel (114) is connected at its sample injection channel junction end to, for example, the target channel (102) of the microfluidic chip (124). The second sample injection channel (116) is connected to the target channel (102) of the microfluidic chip (124) at its sample injection channel junction end. In Figure 2A, the first sample injection channel (114) is an injection channel, meaning that the first sample type (A) is currently being injected into the target channel (102) for a first time period, while the second sample injection channel (116) is a non-injection channel, meaning that, for example, the second sample type (B) is not currently being injected for a first time period. This can be seen, for example, in the resulting microcompartment (202) in Figure 2A, which contains only sample species (A).

[0105] As can be seen further in Figure 2A, the first sample species (A) not only ends in the target channel (102) to form a microcompartment (202) within it, but also in the second sample injection channel (116) intended for the injection of the second sample species (B). Therefore, this backflow, especially when the second sample injection channel (116) becomes the injection channel, for example, when it is used to inject a second sample type (B) at a second time different from the first time, the remaining trace amount of the first sample type (A) is injected simultaneously, resulting in significant cross-contamination of the created microcompartment (202).

[0106] Although not shown in Figure 2A, such backflow and / or cross-contamination flows not only into the (adjacent) sample injection channel that is currently not injecting, e.g., the second sample injection channel (116), but also into the second sample species reservoir (106). Correspondingly, the same applies to the other non-injectable sample injection channels present in the microfluidic device (100) shown in Figure 1.

[0107] As can be seen further from Figure 2A, the created microcompartments (202) are not of uniform size and have non-uniform shapes. For example, the microcompartments (202) are bulky, deformed, or strained due to pressure differences associated with backflow and / or pressure / flow differences associated with overshoot or over-regulation due to sudden flow rate changes, as seen in Figure 2A. With respect to combinatorial microcompartments (see, for example, combinatorial microcompartments (200A;200B) in Figure 2C), this also results in cross-contamination combinatorial microcompartments, i.e., places where two species are mixed.

[0108] Figure 2B shows a schematic diagram of the first sample injection channel (114) and the second sample injection channel (116), which are connected to the target channel (102) without valves, similar to Figure 2A. Figure 2B shows the injection of a first sample type (A) and a second sample type (B) using a hydrodynamic resistor (not shown).

[0109] In Figure 2B, the first sample injection channel (114) is connected to the target channel (102) of the microfluidic chip (124) via a valveless connection at its sample injection channel junction end. Similarly, the second sample injection channel (116) is connected valvelessly at its sample injection channel junction end to, for example, the target channel (102) of the microfluidic chip (124). The sample injection channel junction ends (114B; 116B) of the first sample injection channel (114) and / or the second sample injection channel (116) are provided with at least one hydrodynamic resistor (not shown).

[0110] In Figure 2B, the first sample injection channel (114) is an injection channel, meaning that the first sample type (A) is currently being injected into the target channel (102) for a first time period, while the second sample injection channel (116) is a non-injection channel, meaning that the second sample type (B) is not currently being injected for a first time period. This can be seen in the resulting microcompartment (202) in Figure 2B, which contains only sample species (A).

[0111] As can be seen from Figure 2B, the first sample type (A) does not reach the second sample injection channel (116) intended for the injection of the second sample type (B). This is the non-injection pressure P to the second sample reservoir (106) of the second sample injection channel (116). ni This is maintained, and this is due to the fact that it is currently not injected, for example, during the initial period and during the use of hydrodynamic resistors associated with valveless connections. As a result, "backflow" of sample type (A) in the second sample injection channel (116) is avoided. In particular, when the second sample injection channel (116) becomes the injection channel, and is used to inject a second sample type (B) at a second time different from the first time, the remaining trace amount of the first sample type (A) is not injected at the same time, thereby reducing or preventing cross-contamination. Furthermore, pressure and / or flow rate differences associated with overshoot or over-regulation due to rapid flow rate changes can be offset, reduced, prevented, or minimized.

[0112] Furthermore, compared to the microcompartment (202) created in Figure 2A, the microcompartment (202) created in Figure 2B is of a more uniform size and shape, for example, continuous, which results in reduced or avoided backflow, i.e., non-injection pressure P against the sample species reservoir (106) of the non-injection second sample injection channel (116) in combination with the use of a hydrodynamic resistor. ni This is to maintain it.

[0113] Figures 2A and 2B illustrate the creation of a microcompartment (202), and these embodiments are not limited thereto; that is, the first sample injection channel (114) and the second sample injection channel (116) are similarly used to create the combinatorial microcompartment (200A;200B) shown in Figure 2C, which contains a mixture of the first sample species (A) and the second sample species (B).

[0114] Figure 2C shows the non-injection pressure P applied to the sample reservoir (106) of the non-injection second sample injection channel (116). ni A schematic diagram is shown of different microcompartments created within the carrier phase of the target channel (102), which are generated using at least one hydrodynamic resistor while maintaining the same characteristics.

[0115] Figure 2C shows two combinatorial microcompartments (200A; 200B) containing at least two sample species with different compositions, and a microcompartment (202) containing only one sample species. The first combinatorial microcompartment (200A) contains a first sample type (A) and a second sample type (B). The second combinatorial microcompartment (200B) contains multiple sample types (A-D), including the first sample type (A), the second sample type (B), the third second sample type (C), and the fourth second sample type (D). Furthermore, these sample species (A-D) may be chemically and / or biologically distinct. The number of sample species in a combinatorial microcompartment (200A) is not limited as long as it contains at least two sample species.

[0116] The two combinatorial microcompartments (200A; 200B) and microcompartment (202) in Figure 2C are fabricated by the microfluidic device (100) shown in Figure 1, and therefore have a uniform shape and / or equal size. As can be seen with respect to the combinatorial microcompartments (200A;200B), there is no cross-contamination between different sample types (A-D) within the created combinatorial microcompartments (200A;200B). The sample species (A-D) are clearly separated from each other, divided, or of different species.

[0117] Figure 3 shows a flowchart of the method (300) according to the embodiment described herein. As shown in Figure 3, the method (300) is for providing one or more combinatorial microcompartments (200A;200B) each containing at least two sample species (A;B). This method includes the steps of creating a microcompartment (202) by injecting a first sample species (A) from a first sample species reservoir (104) through a first sample injection channel (114) into a continuous flow of the carrier phase in a target channel (102), and creating a combinatorial microcompartment (200) by injecting at least one second sample species (B) from a second sample species reservoir (106) through a second sample injection channel (116) into the created microcompartment (202), and creating a combinatorial microcompartment (200). The method further includes the step (306) of providing resistance in the first injection channel (114) and / or in the second injection channel (116) using at least one hydrodynamic resistor. The first sample species and at least one second sample species are barcode oligonucleotides or sets of their components. Preferably, the set of barcode oligonucleotides or their components for the first sample species is different from the set of barcode oligonucleotides or their components for at least one second sample species.

[0118] The resistance is hydrodynamic resistance. This resistance is provided between i) the injection of the first sample type (A) (302) and / or the injection of the second sample type (B) (304) and ii) the non-injection of the first sample type (A) and / or the non-injection of the second sample type (B).

[0119] The first sample type (A) and the second sample type (B) are injected into their respective first sample type reservoirs (104) and second sample type reservoirs (106) at an injection pressure P i It is injected by applying an injection pressure P i Lower non-injection pressure P ni This is maintained in each sample type reservoir (104;106) of each non-injection sample injection channel (114;116).

[0120] This method may optionally include a step (306) of repeating the step (302; 304) for creating another combinatorial microcompartment in order to create a series of combinatorial microcompartments within the carrier phase of the target channel (102).

[0121] In the method shown in Figure 3, the injection of the second sample type (B) is performed either consecutively or simultaneously with the injection of the first sample type (A). That is, both the first sample type (A) and the second sample type (B) are injected during the first time period. Alternatively, the first sample type (A) may be injected at a first time and the second sample type (B) at a second time different from the first time, or vice versa.

[0122] In the method shown in Figure 3, the first sample type (A) does not reach the second sample injection channel (116) intended for the injection of the second sample type (B). This is the non-injection pressure P ni This is due to the fact that a second sample species reservoir is maintained, and resistance is given in the first injection channel (114) and / or the second injection channel (116) using at least one hydrodynamic resistor. As a result, "backflow" of sample type (A) from the first sample injection channel (114) to the second sample injection channel (116) is reduced or avoided. Furthermore, pressure and / or flow rate differences associated with overshoot or over-regulation due to rapid flow rate changes can be offset, reduced, prevented, or minimized. Furthermore, this avoids cross-contamination, for example, when a second sample injection channel (116) is used to inject a second sample type (B), because the remaining trace amount of the first sample type (A) is not co-injected.

[0123] Figure 4A shows the non-injection pressure P for the non-injection sample reservoir. ni This shows the pressure profile during sample injection when the condition is not maintained. In Figure 4A, the x-axis represents time in seconds, and the y-axis represents pressure in mbars.

[0124] In Figure 4A, the non-injection pressure P is shown. ni Without maintaining this condition, it is clear that an irregular pressure profile or pressure peak will persist or occur, indicating a high probability of undesirable backflow and therefore cross-contamination. The resulting (combinatorial) microcompartments have low uniformity and are of unequal sizes (for example, the microcompartments created in Figure 2A). This can also cause overshoot or over-regulation due to rapid changes in flow rate.

[0125] On the other hand, Figure 4B shows the non-injection pressure P. ni Maintaining this, the pressure profile during sample injection using a hydrodynamic resistor is shown. In Figure 4B, the x-axis represents time in seconds, and the y-axis represents pressure in mbars.

[0126] As can be seen from the figure, the pressure profile of the pressure peak is quite uniform when compared to the measurements shown in Figure 4A, resulting in the formation of uncontaminated (combinatorial) microcompartments of equal size (see, for example, Figure 2B or Figure 2C). For the microcompartments (see, for example, Figure 2C), this results in a reduction or prevention of cross - contamination and a reduction of overshoot or over - regulation due to rapid flow rate changes.

[0127] Note in Figure 4B that the linear increase in pressure is due to the increase in backpressure within the target flow channel (102) (see Figure 1) with an increase in the number of samples.

[0128] Figure 5A shows the flow rate profile during sample injection without maintaining the non - injection pressure P ni The x - axis of Figure 5A shows time in seconds, and the y - axis of Figure 5A shows the flow rate in μL / min. The flow rate profiles shown in Figure 5A are preferably as similar to each other as possible because doing so results in (combinatorial) microcompartments being created with uniform and / or equal sizing.

[0129] This also reduces cross - contamination. In the figure of Figure 5A, the flow rate on the y - axis includes both positive and negative flow rate values.

[0130] As can be seen from the figure, for the second sample type B (which corresponds to sample 2 in the figure of Figure 5A), a negative (less than 0) flow rate is observed. Such a negative flow rate indicates an undesirable backflow and cross - contamination between adjacent sample injection channels as described above (see, for example, Figure 2A).

[0131] ​As can be seen in Figure 5A, a "backflow" is observed, indicated by the negative flow rate between adjacent sample injection channels, causing significant cross-contamination between sample type (A) (which corresponds to sample 1 in the diagram in Figure 5A) and sample type (B) (which corresponds to sample 2 in the diagram in Figure 5A).

[0132] On the other hand, Figure 5B shows the non-injection pressure P according to an embodiment of the present disclosure. ni Maintain the current flow rate profile using a hydrodynamic resistor. In Figure 5A, the x-axis represents time in seconds, and the y-axis represents flow rate in μL / min.

[0133] As can be seen in Figure 5B, the negative flow rate associated with backflow between adjacent channels is significantly reduced (to virtually zero), thereby avoiding cross-contamination between adjacent sample injection channels. To this end, a more uniform flow rate is achieved, and equally sized, uncontaminated (combinatorial) microcompartments are formed (see, for example, Figures 2B and 2C). With respect to combinatorial microcompartments, this also results in a reduction or prevention of cross-contamination (see, for example, Figure 2C).

[0134] Figure 5C shows the non-injection pressure P without using a hydrodynamic resistor. ni This shows another flow profile for a microfluidic chip that maintains [the same behavior]. In Figure 5C, the x-axis represents time in seconds, and the y-axis represents flow rate in μL / min.

[0135] Figure 5C further illustrates how the flow rate changes over time as the three samples (Samples 1-3) are injected. At the start of injection, a sudden change in flow rate occurs, such as an overshoot, where the flow rate briefly exceeds the desired or target flow rate before returning to normal or the target flow rate. In other words, in Figure 5C, the overshoot peak represents the case where the flow rate exceeds the desired flow rate. These overshoots or peaks can negatively impact the accuracy and consistency of flow rates in microfluidic systems, potentially leading to backflow and cross-contamination.

[0136] To measure cross-contamination, microcompartments were created using samples of different injection phosphors, such as green, orange, and blue fluorescent dyes, as in Figures 6A, 6B, and 6C. Fluorescent dyes are a type of molecule that absorbs light of one wavelength and re-emits it at longer wavelengths, producing visible fluorescence of different colors depending on the phosphor used, for example. These dyes are used to visualize specific structures within microcompartments or to track the movement of molecules within cells. In this case, a fluorescent dye can be used to detect cross-contamination within the (combinatorial) microcompartment. By exciting fluorescent dyes with a specific light source, a bright and easily detectable fluorescent signal can be generated, which indicates cross-contamination.

[0137] Figure 6A shows the non-injection pressure P. ni The measured cross-contamination in a combinatorial microcompartment containing at least two sample types (A;B) prepared without maintaining the original state is shown.

[0138] In the example shown in Figure 6A, cross-contamination is monitored while injecting green, orange, and blue fluorescent dyes into the microcompartments to create combinatorial microcompartments (200A; 200B), i.e., those containing at least two sample types. In the embodiment shown in Figure 6A, the combinatorial microcompartment is located at a non-injection pressure P ni It is created without maintaining it. The x-axis in Figure 6A represents time in seconds, and the y-axis in Figure 6A represents fluorescence intensity in arbitrary units, such as values ​​in the range of 0 to 6 A.U., selected by the experimenter rather than based on internationally recognized standards.

[0139] The Z coefficient is used in fluorescence analysis. The Z-coefficient is a statistical measure used to assess the quality of the analysis, or in this case, the species of samples within a combinatorial microcompartment. Therefore, cross-contamination alters fluorescence readings, for example, decreasing the Z-coefficient, so the Z-coefficient corresponds to a measure of cross-contamination between at least two species within a combinatorial microcompartment. Generally, a Z coefficient between 0.5 and 1.0 is considered good (e.g., no or reduced cross-contamination), while a Z coefficient value less than 0.5 or even negative values ​​indicate cross-contamination between species within a combinatorial microcompartment.

[0140] As can be seen from the measurements shown in Figure 6A, the fluorescence response is rather heterogeneous, indicating cross-contamination between fluorescent dyes. The Z-coefficient measured for the green fluorescent dye was -4.03, the Z-coefficient measured for the orange fluorescent dye was -0.726, and the Z-coefficient measured for the blue fluorescent dye was -1.462.

[0141] In the measurement shown in Figure 6A, the non-injection pressure P ni The Z-coefficient measured in combinatorial microcompartments created without maintaining the original structure was negative, indicating significant cross-contamination within the combinatorial microcompartments.

[0142] In the embodiment of FIG. 6B, while injecting green, orange, and blue fluorescent dyes into the microcompartments, cross - contamination was monitored to create combinatorial microcompartments (200A; 200B), that is, those containing at least two sample types. In the embodiment of FIG. 6B, the non - injection pressure P according to the embodiment of the present disclosure ni was maintained, and a hydrodynamic resistor was used to create combinatorial microcompartments. The x - axis of FIG. 6B represents time in seconds, and the y - axis of FIG. 6B represents fluorescence intensity in measurement units of an arbitrary scale including values from 0 to 1 AU, which is not based on an internationally recognized standard but is selected by the experimenter.

[0143] As can be seen from the measurements shown in FIG. 6B, the fluorescence response is fairly uniform, indicating that cross - contamination between the fluorescent dyes is significantly reduced when compared with the measurements shown in FIG. 6A. The Z - factor measured for the green fluorescent dye was 0.612, the Z - factor measured for the orange fluorescent dye was 0.74, and the Z - factor measured for the blue fluorescent dye was 0.714.

[0144] In the measurements shown in FIG. 6B, the non - injection pressure P ni was maintained, and the Z - factor measured within the combinatorial microcompartments created by using a hydrodynamic resistor was not only a positive value but also within the range of 0.5 to 1.0, indicating that cross - contamination within the combinatorial microcompartments is significantly reduced or avoided when compared with the measurements shown in FIG. 6A.

[0145] The combinatorial microcompartments measured in FIG. 6B were created using the microfluidic device (100) shown in FIG. 1 and / or the method shown in FIG. 3.

[0146] Figure 7A shows flow rate data (i.e., backflow and overshoot) recorded with a microfluidic chip without using a hydrodynamic resistor, and Figure 7B shows the non-injection pressure P ni The flow rate data (i.e., backflow and overshoot) recorded on the microfluidic chip using a hydrodynamic resistor while maintaining the current flow rate.

[0147] Figure 8A shows a microfluidic chip according to the present invention, which includes hydrodynamic resistors implemented as bends or turns of approximately 180° in each channel. Figure 8B shows a microfluidic device without hydrodynamic resistors.

[0148] Comparing both recorded backflow and / or recorded overshoot, we find that microfluidic chips incorporating hydrodynamic resistors exhibit significantly lower backflow and / or overshoot compared to microfluidic chips without hydrodynamic resistors.

[0149] In a further embodiment, the present invention provides a method for colocalizing an entity with a barcode oligonucleotide or a set of its components in a microfluidic device. Barcoding of cellular mRNA with unique identifiers, such as the incorporation of specific nucleotide sequences during cDNA synthesis, is widely used in genomic applications (AESaliba, AJ Westermann, SAGorski, J. Vogel, Single-cell RNA-seq: advances and future challenges. Nucleic Acids Research 42, 8845 (2014)), and each method is described, for example, in International Publication No. 2016 / 207441, which is incorporated herein by reference in its entirety.

[0150] Microfluidic devices used in a method for colocalizing an entity with a barcode oligonucleotide or a set of its components are preferably devices disclosed herein. With respect to Figure 1, the device preferably includes a target channel (102) containing a target channel fluid such as a carrier phase, a first sample species reservoir (104) containing a first sample species (A) in the form of a first barcode oligonucleotide or a set of its components, and a second sample species reservoir (106) containing a second sample species (B) in the form of a second barcode oligonucleotide or a set of its components. The carrier phase is an immiscible phase, more preferably an immiscible fluid. The first barcode oligonucleotide and the second barcode oligonucleotide or their component sets may be the same or different. Furthermore, this first barcode oligonucleotide and the second barcode oligonucleotide, or the set of its components, are different from each other. The device may include not only a first and second sample species reservoir having a first barcode oligonucleotide and a second barcode oligonucleotide or a set of its components, but also a plurality of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, for example, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, or 200 sample species reservoirs, each with a set of its respective number of barcode oligonucleotides or its components. Figure 1 shows the third sample type reservoir (108) and the fourth sample type reservoir (110). The barcode oligonucleotides or their component sets in each of the multiple sample species reservoirs are different from one another.

[0151] The device further includes a first sample injection channel (114) which includes a sample injection channel reservoir end (114A) connected to a first sample species reservoir (104) and a sample injection channel junction end (114B) connected to a target channel (102). The device also includes a second sample injection channel (116) which includes a sample injection channel reservoir end (116A) connected to a second sample species reservoir (106) and a sample injection channel junction end (116B) connected to a target channel (102). The device includes not only a first sample injection channel (114) and a second sample injection channel (116) including a sample injection channel reservoir end and a sample injection channel junction end, but also, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 sample injection channels, for example, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, or more sample injection channels, each including a sample injection channel reservoir end and a sample injection channel junction end. Figure 1 shows the third sample injection channel (118) and the fourth sample injection channel (120), each having its own injection channel (118, 120). The sample injection channel junction ends (114B; 116B) of the first sample injection channel (114) and / or the second sample injection channel (116) are connected to the target channel (102) without valves. The term "valveless" is defined herein as described above. This term means that no valves are provided at the junctions of the flow channels, preferably, no valves are provided anywhere between the sample injection channel and the target channel and their junctions to the respective sample type reservoirs.

[0152] In one embodiment, the injection channel is an integrated channel. According to an alternative embodiment, the injection channel consists of two or more different sections. In one such embodiment, the injection channel has a first section connected to a sample species reservoir, for example, a tube, and a second section connected to a target channel, for example, a microchannel. The first section and the second section are interconnected by an adapted adapter. If the first and second sections have different diameters, the adapter has a tapered shape that allows connection of the two flow path sections having different diameters and / or being made of different materials.

[0153] According to a particular preferred embodiment, each sample injection channel includes at least one hydrodynamic resistor. A hydrodynamic resistor is as defined herein and is implemented as a specific flow path shape in a portion of the flow path, preferably as a number of turns or bends in each section of the flow path. In particular, the hydrodynamic resistor is a continuous arrangement of multiple turns or bends in a flow path of about 170 to 190°, preferably about 180°, as shown in Figure 8A, for example. The multiple turns or bends of the hydrodynamic resistor include 4 to 40 turns / bend, more preferably 6 to 30, 8 to 20, 10 to 18, or 12 to 16 turns / bend. In particular, in a series of turns or bends within a flow path, the turns / bends are evenly distributed along the flow path sections that function as hydrodynamic resistors. Furthermore, the distance from one turn or bend in a hydrodynamic resistor to the next turn or bend in the hydrodynamic resistor is essentially the same. It is understood that each individual flow path may contain two or more hydrodynamic resistors, for example, two, three, four, five, six, seven, eight, nine, ten, or more hydrodynamic resistors.

[0154] A method for colocalizing an entity with a barcode oligonucleotide or a set of its components in a microfluidic device includes, as a first step, supplying the entity to a target channel. This target channel is one of the target channels (102) of the devices disclosed herein. The entity is a substance such as a drug or particles containing nucleic acids, preferably DNA and / or RNA. According to a preferred embodiment, the particles are cells. In a more preferred embodiment, the particles are drugs.

[0155] In the second step, the method includes passing an entity through a first sample injection channel and a second sample injection channel, one of which supplies a set of barcode oligonucleotides or their components from the first sample species reservoir or the second sample species reservoir to the target channel via the respective sample injection channel and the respective sample injection channel junction. This involves injecting pressure (P) into one of the first and second sample type reservoirs. i This is done by applying a solution, thereby injecting a barcode oligonucleotide or a set of its components into the target channel. The first sample injection channel and the second sample injection channel, the first sample species reservoir and the second sample species reservoir, and the respective injection channel junction ends are preferably as defined herein. Therefore, the second step is to pass the substance through the first sample injection channel (114) and the second sample injection channel (116), wherein one of the sample injection channels injects the first sample reservoir (104) and the second sample reservoir (106) to each of them at an injection pressure (P i The process includes applying a solution to supply a barcode oligonucleotide or a set of its components from the sample species reservoir (104, 106) to the target channel (102) via the respective sample injection channels (114, 116) and the respective sample injection channel junction ends (114B, 116B), thereby injecting the barcode oligonucleotide or a set of its components into the target channel (102).

[0156] In an optional third step, the method includes repeating the second step. This iteration is not limited to a single iteration, but includes multiple iterations selected according to individual needs. Then, when the second step is repeated, the other of the first sample injection channel and the second sample injection channel supplies a set of barcode oligonucleotides or their components from the other of the first sample species reservoir and the second sample species reservoir to the target channel. Therefore, if in the second step a set of barcode oligonucleotides or their components is supplied from the first sample species reservoir to the target channel via the first sample injection channel, then in the third step a set of barcode oligonucleotides or their components is supplied from the second sample species reservoir to the target channel via the second sample injection channel. The third step can be repeated multiple times, depending on the circumstances and individual needs, with or without alternating supply of barcode oligonucleotides or sets of their components from the first and second sample species reservoirs. A specific iteration sequence of step (ii) of the method, involving the supply of a set of barcoded oligonucleotides or their components from the same or different sample species reservoirs, is set to meet individual needs. Therefore, the present invention is not limited to any particular order of supply from any specific sample species reservoir, but is adapted accordingly. This also applies to the number of sample type reservoirs and the number of injection channels for each.

[0157] The method of the present invention involves a non-injection pressure (P) during the non-injection step. ni The process further includes the step of applying ) to a first sample species reservoir (104) and / or a second sample species reservoir (106). Non-injection pressure P ni is the environment (P 大気 It is preferable that the pressure is greater than the pressure of ). Injection pressure P i and non-injection pressure P ni This refers to the environmental or ambient pressure acting on the device, particularly the pressure acting on the sample species reservoir, when no other pressures are applied. Therefore, it is the same as the pressure surrounding the device. However, when the pressure within the device acting on the components of the device, particularly the sample species reservoir, is different from the pressure surrounding the device, the environmental pressure refers to the pressure acting on the sample species reservoir. P i is greater than P ni and P ni is greater than P 大気 as well. In particular, the ratio between P i and P ni is from about 1.25 to about 2.5, preferably from about 1.5 to about 2.25, from about 1.75 to about 2.0, and most preferably about 2.0.

[0158] And P ni is from about 5 to 5600 mbar, P i is from about 10 to 7000 mbar, more preferably P ni is from about 50 to 3200 mbar, P i is from about 60 to 4000 mbar, and most preferably P ni is from about 100 to 1600 mbar, P i is from about 125 to 2000 mbar. According to the invention, all the pressures shown are above the ambient pressure or P 大気 plus (i.e., in addition thereto), in other words, the pressure values shown are gauge pressure values.

[0159] According to the invention, when an injection pressure P i is applied to one sample species reservoir, simultaneously a non-injection pressure P ni is applied to the other sample species reservoir to prevent any sample species from entering the non-injection flow path from the injection sample species reservoir and the injection flow path. Depending on the actual use of each of the device and the method, to enable a controlled mixing of a set of barcode oligonucleotides or components thereof from different sample species reservoirs, it will be understood that the injection pressure P i is applied simultaneously to two or more sample species reservoirs. However, in most cases, the injection pressure P i This is applied to only a single sample type reservoir, but simultaneously, i.e., the injection pressure P i As long as it is applied to one sample type reservoir, the non-injection pressure P ni This is applied to all other sample reservoirs.

[0160] According to the present invention, the non-injection pressure P for each sample type reservoir in each non-injection sample injection channel is ni It increases over time. Non-injection pressure P ni If the non-injection pressure P increases over time, ni Nevertheless, the injection pressure P i Not exceeding P i and P ni The ratio between these two values ​​is preferably maintained at approximately 0.25 to approximately 2.5.

[0161] And the injection pressure (P i ) and non-injection pressure (P ni This is produced by pressurizing the first sample species reservoir (104) and / or the second sample species reservoir (106). This is carried out under the control of pressure control devices such as the pressure control devices (114D, 116D, 118D, 120D) shown exemplified in Figure 1. This pressure control device is a microfluidic flow controller, such as a Microfluidic Flow Control System (MFCS).

[0162] According to one embodiment of the present invention, the supply of a set of barcode oligonucleotides or their components from a sample species reservoir to a target channel is controlled by one or more detection means or sensors. The detection means or sensors are as defined above and are shown in Figure 1 by reference numerals 114C, 116C, 118C, and 120C, respectively. The arrangement shown in Figure 1 is merely an example and therefore does not limit the present invention. Therefore, the detection means or sensors may be placed alternatively or additionally at other locations within the device, for example, at the start and / or end of each injection channel, on the target channel, or in the vicinity of the sample species reservoir, for example, between the sample species reservoir and the connection to the outside of the microfluidic device (for example, in the filling line for filling the sample species reservoir with sample species). Detection means for detecting particles (also called particle detection means) and other components within microfluidic channels are well known in the art and include optical sensors, such as photomultiplier tubes, CMOS or CCD cameras, or detection electrodes. Generally, detection means are suitable for detecting particles and / or labels bound to particles, particularly fluorescent labels, and fluorescence or laser spectroscopy, imaging, impedance or magnetic measurements may be used for detection. The detection means is, for example, a sensor such as a highly sensitive microfluidic flow sensor based on microelectromechanical systems (MEMS) technology, preferably a flow sensor. A more preferred detection means is, for example, a high-precision thermal flow sensor, which is particularly useful for monitoring the flow rate of liquids and cells. Further sensors useful in the present invention include, for example, Coriolis mass flow sensors. According to a preferred embodiment, the sensor is a flow sensor that detects the fluid supplied to the sample species reservoir. For example, such a flow sensor detects the amount of gas, such as air, that corresponds to the amount of sample type supplied from the sample type reservoir to each injection channel. In other words, for each volume of sample species supplied from the sample species reservoir to the injection channel and then to the target channel, the corresponding volume of fluid, such as air, enters the sample species reservoir. The flow sensor is positioned to detect this volume of fluid entering the sample reservoir. Additionally or alternatively, the device may include detection means or sensors located at one or more of the ends of each injection channel, i.e., the injection channel junction to the sample species reservoir and / or the injection channel junction to the target channel and / or the injection channel itself. The arrangement of a detection means or sensor at or near the sample species reservoir, injection channel, and / or injection channel junction is also called upstream arrangement or upstream detection. A preferred means for upstream detection is a flow sensor, such as those described herein.

[0163] In addition to or as an alternative to the upstream arrangement described above, the device may include detection means or sensors positioned in or on the target flow path. Such an arrangement of detection means or sensors is referred to herein as a downstream arrangement. Particularly preferred detection means or sensors for downstream placement or downstream detection are particle detection means, in particular, particle detection means that enable the detection of solid particles, such as cells. Particularly preferred detection means for downstream detection are those capable of imaging or detecting fluorescence signals.

[0164] According to a preferred embodiment, the set of barcode oligonucleotides or their components supplied to the targeting channel while the entity passes through the sample injection channel junction end is predetermined or recorded.

[0165] To control the supply of a set of barcode oligonucleotides or their components from a sample species reservoir to a target channel, one or more detection means or sensors, upon detecting an entity, apply a non-injection pressure P to each of the sample species reservoirs. ni or injection pressure P i It is preferable to induce the application of [a certain substance]. This entity is then detected in the target channel (102).

[0166] Non-injection pressure P into sample type reservoir ni or injection pressure P i Applying a certain force can also be triggered, or alternatively, by time. Time-based control can be based on the point in time when the substance is supplied to the target channel, the distance it needs to travel to reach and exit the sample injection channel junction, and the flow velocity of the substance.

[0167] According to embodiments of the present invention, a method for colocalizing entities with a barcode oligonucleotide or a set of its components in a microfluidic device includes a first entity being supplied to a targeting channel before a further entity, and then repeating all steps one or more times with the further entity. For each entity passing through the joint end of each sample injection channel, a different sample reservoir is used, with an injection pressure P i The other sample reservoir is pressurized at the non-injection pressure P. ni It is pressurized. The set of barcode oligonucleotides or their components that are supplied to the targeting channel while this entity passes through the sample injection channel junction end is predetermined or recorded.

[0168] In embodiments in which multiple entities colocalize with barcode oligonucleotides or their components, a preferred embodiment includes repeating all steps one or more times while the entities are continuously supplied to the target channel, and different sample species reservoirs are preferably injected at a time-dependent injection pressure P i Then it is pressurized. Preferably, the order of the barcode oligonucleotides or sets of their components supplied to the target channel is recorded.

[0169] This method generates microfluidic droplets containing entities and barcode oligonucleotides or their components. The method further includes fusing a microfluidic droplet containing an entity and a barcode oligonucleotide or its components with a further microfluidic droplet containing a further entity, such as a cell. Alternatively, further entities can be injected into a microfluidic droplet containing the entity and barcode oligonucleotides or their components.

[0170] Microfluidic droplets are generated, handled, and / or controlled within the microfluidic devices described herein. The generation of microfluidic droplets is based on the manipulation of a continuous liquid flow through a microfabricated channel. Furthermore, this liquid flow operation is performed by injecting the sample type reservoir at non-injection pressure P. ni or injection pressure P i This is carried out by an (external) pressure source, an (external) mechanical pump, or an (integrated) mechanical micropump.

[0171] Microfluidic droplets are produced, for example, by generating independent compartments using two-phase microfluidics, where aqueous droplets surrounded by an immiscible oil phase function as closed containers, as described in International Publication No. 2016 / 207441.

[0172] According to one embodiment, the method further includes the step of injecting additional components or elements into a microfluidic droplet containing an entity and a barcode oligonucleotide or a set of its components. Additional components or elements include reagents such as reaction mixtures, particularly one or more of the following: ligation mix, primer extension mix, reverse transcription mix (RT), PCR mix, RT-PCR mix, rearrangement mix, and / or lysis buffer. Such additional components or elements are particularly suitable when the microfluidic droplets contain entities such as drugs, barcode oligonucleotides or their components, and at least one cell. According to one embodiment, the injection is performed by injecting the aqueous phase containing each additional component or element into the microfluidic droplet.

[0173] When colocalizing multiple entities with a barcode oligonucleotide or a set of its components in the same microfluidic droplet, time control may be arbitrary or based on the frequency or density of particles in the colocalization channel.

[0174] This method further includes the step of detecting entities and barcode oligonucleotides or their components. The entities are detected by the respective barcode oligonucleotides or their associated components using any suitable means known to those skilled in the art. For example, entities and barcode oligonucleotides or their components can be detected by sequencing.

[0175] According to one embodiment, if the entity is a cell, the cell is phenotyped after colocalizing with a barcode oligonucleotide or a set of its components, either in the target channel or after the cell has left the target channel. Phenotyping is performed while cells co-localized with barcode oligonucleotides or a set of their components are within the target channel. Alternatively, phenotyping can be performed when the cells colocalize with a barcode oligonucleotide or set of components and leave the target channel. If the microfluidic droplets containing entities and barcoded oligonucleotides or their components further contain the cells described above in this specification, and optionally further further elements or components of the form of the (reaction) reagent, phenotyping is performed after incubation, which allows the reagent to perform the desired reaction, e.g., lysis and / or PCR. Those skilled in the art can easily select the respective incubation parameters for the desired reaction to occur. Phenotyping may involve detecting any biophysical or biochemical characteristics of a cell, such as size, shape, morphology, staining, or, for example, immunostaining or ligand binding. This can be achieved by applying imaging techniques, such as bright-field or fluorescence imaging, and spectroscopic methods including fluorescence spectroscopy. Phenotypic determination makes it possible to correlate specific cellular phenotypes, particularly single-cell phenotypes, with their transcriptome or genetic abnormalities such as mutations, or with the effects of specific drugs on specific cells or cell populations.

[0176] In a further embodiment, the microfluidic device may further comprise one or more additional injection channels connected to the target channel, which supply additional components to the target channel.

[0177] Furthermore, the present invention provides a method for determining the effect of a drug on a cell or a cellular transcriptome or a DNA amplification product from a cell, the method comprising the step of barcoding the drug or the cellular transcriptome or a DNA amplification product from a cell using the method of the present invention described herein. The barcode sequence in the drug or barcoded transcriptome or barcoded DNA amplification product preferably indicates the drug to which the cells are exposed or the cells exposed to the drug.

[0178] Methods for co-localizing microfluidic devices and entities with barcode oligonucleotides or sets of their components can be used to barcode substances such as drugs being tested on cells or cell cultures. In such applications, the present invention is (i) A step of co-localizing a substance, such as a drug, in a microfluidic droplet with a barcoded oligonucleotide or its components and optionally with cells using a method described herein, (ii) If there are no colocalized cells in step (i), the step of introducing cells into the microfluidic droplets in step (i) is performed, (iii) a step of injecting or fusing a reaction mixture containing, for example, reagents for reverse transcription and PCR, and / or for selectively lysing cells, into a microfluidic droplet of step (ii), (iv) The steps include incubating the microfluidic droplets from step (iii), and optionally annealing the barcode oligonucleotides to the RNA or DNA of the lysed cells in the reaction mixture within the microfluidic droplets, optionally lysing the cells and / or carrying out a reaction, such as reverse transcription and PCR.

[0179] According to one embodiment, this method is (v) a step of inactivating any enzyme in a microfluidic droplet in any order and / or a step of destroying the microfluidic droplet, (vi) further comprising the step of analyzing the contents of microfluidic droplets by arbitrarily selecting arrangement determination.

[0180] If step (ii) is present, cells are introduced into a microfluidic droplet by fusing a microfluidic droplet containing the substance and barcode oligonucleotides or their components with a further microfluidic droplet containing cells. Alternatively, cells can be injected into microfluidic droplets containing a substance and barcoded oligonucleotides or their components.

[0181] In step (v), the enzyme is inactivated to prevent enzymatic activity such as polymerase activity or reverse transcriptase activity. Enzymes can be inactivated, for example, by changing the temperature (e.g., thermal inactivation), pH, or buffer composition, or by adding enzyme-specific inhibitors to achieve conditions under which the enzyme does not function.

[0182] Any or all of the steps of the above method can be performed using the microfluidic devices described herein, i.e., the microfluidic devices according to the present invention. Furthermore, at least step (i) and optionally step (ii) are carried out on a microfluidic device according to the present invention.

[0183] The reaction mixture may include a buffer, nucleotides, culture medium and / or expression medium, and / or enzymes such as reverse transcriptase, polymerase, ligase, RNAse and / or DNAse inhibitors. The reaction mixture contains an RT-PCR mix for performing RT-PCR, and optionally a lysis buffer for lysing cells and an RNAse inhibitor to prevent RNA degradation. According to one embodiment, the reaction mixture is selected from the group consisting of RT mix, RT-PCR mix, PCR mix, and rearrangement mix. The individual components in the reaction mixture and their concentrations can be easily selected by those skilled in the art depending on the intended purpose.

[0184] The methods for barcoding substances such as drugs described herein are not limited to barcoding a single substance or drug, but two or more substances or drugs, such as combinations of different substances and / or drugs, are also used herein in the context of the methods described herein. In other words, this method can be used, for example, to barcode combinations of substances or drugs, such as combinatorial therapy with different drugs.

[0185] The device used in the method for barcoding substances such as drugs as described herein is preferably a microfluidic device as described herein. In the current setup for barcoding substances such as drugs, it is preferable that the sample species reservoir not only contains barcode oligonucleotides and / or their components, but also that at least one of the sample species reservoirs contains the substance drug to be barcoded. When two or more substances or drugs are barcoded, the different substances or drugs may be contained in a single species reservoir (e.g., as a premix), or they may be contained individually or in other pre-mixed combinations in two or more sample species reservoirs. The methods and devices of the present invention enable the combination of different substances and drugs in different combinations. It will be understood that cells or other entities such as DNA and / or RNA can also be supplied from their respective sample species reservoirs to the target channel via their respective injection channels.

[0186] In particular, according to the method for barcoding substances such as drugs to be tested with respect to cells or cell cultures described herein, the sample species reservoir of a microfluidic device includes a set of barcode oligonucleotides or their components, the substance and / or drug to be barcoded, and optionally, the cells on which the substance and / or drug is to be tested.

[0187] Furthermore, this method may include a subsequent step of phenotyping the cells described herein. Phenotyping is performed after the cells have exited the target channel, more preferably after incubation in step (iv).

[0188] The reaction mixture is injected into the T-junction channel as shown in Figure 13C.

[0189] Microfluidic devices and methods for co-localizing entities with barcode oligonucleotides or sets of their components can also be used, for example, to barcode the transcriptome of a cell, to barcode DNA amplified from a cell, or to barcode the genome of a cell. For such applications, this method is (i) a step of colocalizing cells with barcode oligonucleotides in a microfluidic droplet using the method described herein, (ii) A step of dissolving cells in microfluidic droplets, (iii) A step of annealing a barcode oligonucleotide to RNA or DNA of lysed cells in a microfluidic droplet, (iv) A step of performing a reverse transcription (RT), RT-PCR, PCR, or rearrangement reaction in a microfluidic droplet or in an aqueous phase in which a microfluidic droplet has been disrupted, using the annealed barcode oligonucleotide as a primer or rearrangement element, respectively, thereby generating a barcoded transcriptome, a DNA amplification product, or a genome. The RT mix, RT-PCR mix, PCR mix, or rearrangement mix is ​​preferably contained in the target channel, in the microfluidic droplets fused to the microfluidic droplets generated in the method of the present invention, or in the aqueous phase from which the microfluidic droplets have been destroyed. According to one embodiment, this method is (v) A step of inactivating any enzyme in a microfluidic droplet containing a barcode oligonucleotide and destroying the microfluidic droplet in any order, (vi) further comprising the step of analyzing a barcoded transcriptome, a barcoded DNA amplification product, or a barcoded genome.

[0190] In step (v), the enzyme is inactivated to prevent enzymatic activity, such as polymerase activity or reverse transcriptase activity, using components of the microfluidic droplet (primers, DNA, or RNA) that were not present in the same microfluidic droplet (for example, in different microfluidic droplets if the droplets are pooled). Enzymes can be inactivated, for example, by changing the temperature (e.g., thermal inactivation), pH, or buffer composition, or by adding enzyme-specific inhibitors to achieve conditions under which the enzyme does not function.

[0191] Analyzing barcoded transcriptomes, barcoded DNA amplification products, or barcoded genomes includes sequencing by next-generation sequencing. Preferably, the barcoded transcriptome, barcoded DNA amplification product, and / or barcoded genome are sequenced together. This allows for performing only one analytical step, such as a single sequencing reaction, on all cells being analyzed, instead of performing separate analyses on each cell.

[0192] Furthermore, the present invention provides a method for correlating the phenotype of a single cell with its transcriptome, a DNA amplification product derived from the cell, or its genome, comprising barcoding the transcriptome of a single cell, barcoding a DNA amplification product from a single cell, or barcoding the genome of a single cell, using the methods of the present invention described herein, wherein the cell is further phenotypic, and the sequence of barcodes in the barcoded transcriptome, amplification product, or genome indicates the phenotype of the cell from which the transcriptome, DNA amplification product, or genome is derived. Phenotypic determination can be performed as described above in this specification.

[0193] As shown herein, the method and device of the present invention simplify the system architecture by using a single sample species reservoir to generate diverse concentration gradients for each sample species stored in the reservoir. Previously, a separate reservoir was required for each reagent concentration, which reduced the risks associated with contamination and device malfunction. Furthermore, the present invention allows for continuous adjustment of concentrations over a wide range, rather than having only reagents of different concentrations pre-filled in a reservoir. This also reduces overall reagent consumption and accelerates the preparation process.

[0194] The present invention further relates to the following items.

[0195] Item 1: Microfluidic device (100), A target channel (102) containing a target channel fluid such as a carrier phase, A first sample species reservoir (104) containing a first sample species (A) and a second sample species reservoir (106) containing a second sample species (B), A first sample injection channel (114) includes a sample injection channel reservoir end (114A) connected to a first sample species reservoir (104) and a sample injection channel junction end (114B) connected to a target channel (102), A second sample injection channel (116) includes a sample injection channel reservoir end (116A) connected to a second sample species reservoir (106) and a sample injection channel junction end (116B) connected to a target channel (102), and Includes, A microfluidic device (100) having sample injection channel junction ends (114B; 116B) of a first sample injection channel (114) and / or a second sample injection channel (116) connected to a target channel (102) without valves and comprising at least one hydrodynamic resistor.

[0196] Item 2: The microfluidic device (100) according to Item 1, wherein a first sample species reservoir (104) and a second sample species reservoir (106) are preferably configured to be pressurized during both the injection and non-injection phases.

[0197] Item 3: A microfluidic device (100) as described in Item 1 or Item 2, wherein a first sample injection channel (114) and a second sample injection channel (116) are separately connected to a target channel (102) at their respective sample injection channel junction ends (114B; 116B).

[0198] Item 4: The microfluidic device (100) according to any one of items 1 to 3, wherein the microfluidic device generates one or more combinatorial microcompartments (200A;200B) containing at least two sample species (A;B) in the carrier phase, the first sample species (A) and the second sample species (B) being chemically distinct, and the combinatorial microcompartments (200A;200B) being combinatorial droplets or combinatorial plugs.

[0199] Item 5: A microfluidic device (100) according to any one of items 1 to 4, wherein the carrier phase is an immiscible phase and one or more combinatorial microcompartments (200A; 200B) flow into the outlet channel or readout channel.

[0200] Item 6: A microfluidic device (100) according to any one of Items 1 to 5, wherein each of the first sample injection channel (114) and the second sample injection channel (116) is connected to a sensor (114C; 116C) configured to monitor the flow data of the liquid in the respective first sample injection channel (114) and the second sample injection channel (116).

[0201] Item 7: A microfluidic (100) device according to any one of items 1 to 6, comprising a first pressure control device (114D) connected to a first sample species reservoir (104) and a second pressure control device (116D) connected to a second sample species reservoir (106), wherein the first pressure control device (114D) and the second pressure control device (116D) are connected to a pressure source (122).

[0202] Item 8: The first pressure control device (114D) injects the first sample type (A) into the first sample type reservoir (104) via the first sample injection channel (114) at a first injection pressure P i The second pressure control device (116D) is configured to apply a second injection pressure P to the second sample type reservoir (106) which injects the second sample type (B) through the second sample injection channel (116).i A microfluidic device (100) as described in item 7, configured to apply a [value].

[0203] Item 9: A first pressure control device (114D) is communicatively connected to a first sensor (114C) of a first sample injection channel (114), and a second pressure control device (116D) is communicatively connected to a second sensor (116C) of a second sample injection channel (116), and the first injection pressure P i and the second injection pressure P i However, the microfluidic device (100) described in item 8 is applied based on the monitored flow data of the liquid in each sample injection channel (114;116).

[0204] Item 10: The first pressure control device (114D) and the second pressure control device (116D) apply the non-injection pressure P to each sample type reservoir (104;106) of each non-injection sample injection channel (114;116). ni It is configured to maintain, preferably, a non-injection pressure P ni However, the first injection pressure P i and the second injection pressure P i Lower than, relationship:P i >P ni >P 大気 Accordingly, optionally, the first pressure control device (114D) and the second pressure control device (116D) control the non-injection pressure P on each sample type reservoir (104;106) of each non-injection sample injection channel (114;116). ni A microfluidic device (100) described in any one of items 7 to 9, configured to increase over time.

[0205] Item 11: A microfluidic (100) device as described in Item 10, wherein the non-injection pressure applied to each sample type reservoir (104;106) of each non-injection sample injection channel (114;116) causes a liquid flow within each sample injection channel (114;116).

[0206] Item 12: A method (300) for providing one or more combinatorial microcompartments (200A;200B) containing at least two sample species (A;B), The process (302) involves injecting a first sample species (A) from a first sample species reservoir (104) into the continuous flow of the carrier phase in a target channel (102) via a first sample injection channel (114) to create a microcompartment (202), Step (304) to create a combinatorial microcompartment (200) by injecting at least one second sample species (B) from a second sample species reservoir (106) into a prepared microcompartment (202) via a second sample injection channel (116), The steps include: providing resistance in the first injection channel (114) and / or the second injection channel (116) using at least one hydrodynamic resistor; Methods that include...

[0207] Item 13: The first sample type (A) and the second sample type (B) are injected into their respective first sample type reservoirs (104) and second sample type reservoirs (106) at pressure P i It is injected by applying an injection pressure P i Lower non-injection pressure P ni The method according to item 12, wherein each non-injectable sample injection channel (114;116) is maintained in each sample species reservoir (104;106).

[0208] Item 14: Non-injection pressure P for each sample type reservoir (104;106) in each non-injection sample injection channel (114;116). ni The process includes increasing the injection pressure P over time. i and non-injection pressure P ni However, relationship: P i >P ni >P 大気 The method described in item 13, in accordance with the instructions.

[0209] Item 15: The method according to any one of items 10 to 14, wherein the injection of the second sample species (B) occurs sequentially or incidentally to the injection of the first sample species (A), the carrier phase is an immiscible phase, the first sample species (A) and the second sample species (B) are chemically distinct, and the combinatorial microcompartment (200) is a combinatorial droplet or combinatorial plug containing at least one prokaryotic or eukaryotic cell.

[0210] Item 16: A microfluidic chip (122) comprising one or more combinatorial microcompartments (200) containing at least two sample species (A;B) produced by the method described in any one of Items 10 to 15.

[0211] Item 17: Use of a microfluidic device to combine a first sample type (A) and a second sample type (B) to create a target flow channel fluid, During the non-injection stage, the first sample type reservoir (104) containing the first sample type (A) is subjected to a non-injection pressure P. ni Apply and P to the second sample reservoir (106) containing the second sample (B) during the non-injection stage. ni The process of applying, During the injection phase, an injection pressure P is applied to at least one of the first sample reservoir (104) and the second sample reservoir (106). i The step includes applying a solution, thereby injecting sample type A and / or sample type B into the target flow path fluid, P i >P ni >P 大気 And, The device being used is A target channel (102) containing the target channel fluid, A first sample injection channel (114) includes a sample injection channel reservoir end (114A) connected to a first sample species reservoir (104) and a sample injection channel junction end (114B) connected to a target channel (102), The second sample injection channel (116) includes a sample injection channel reservoir end (116A) connected to a second sample species reservoir (106) and a sample injection channel junction end (116B) connected to a target channel (102), The sample injection channel junction ends (114B; 116B) of the first sample injection channel (114) and / or the second sample injection channel (116) are connected to the target channel (102) without valves and are equipped with at least one hydrodynamic resistor.

[0212] Item 18: A method for colocalizing an entity with a barcode oligonucleotide or a set of its components in a microfluidic device as described in any one of Items 1 to 11, (i) A step of supplying the substance to the target flow path (102), (ii) A step of passing the substance through a first sample injection channel (114) and a second sample injection channel (116), wherein one of the sample injection channels (114, 116) injects an injection pressure (P) into one of the first sample reservoir (104) and the second sample reservoir (106). i The process involves applying a solution to supply a barcode oligonucleotide or a set of its components from the sample species reservoir (104, 106) to the target channel (102) via each sample injection channel (114, 116) and each sample injection channel junction end (114B, 116B), thereby injecting the barcode oligonucleotide or a set of its components into the target channel (102), (iii) Step (ii) is optionally repeated, preferably, when step (ii) is repeated, the other of the first sample injection channel (114) and the second sample injection channel (116) supplies a barcode oligonucleotide or a set of its components from the sample species reservoir (104, 106) to the target channel (102) and A method that includes this.

[0213] Item 19: Non-injection pressure (P) during the non-injection phase niThe process further includes the step of applying ) to a first sample species reservoir (104) and / or a second sample species reservoir (106), P ni environmental pressure (P 大気 ) is larger than P i >P ni >P 大気 The method described in item 18.

[0214] Item 20: Injection pressure (P i ) and non-injection pressure (P ni The method described in item 18 or item 19, which is produced by pressurizing the first sample species reservoir (104) and the second sample species reservoir (106).

[0215] Item 21: The method according to any one of items 18 to 20, wherein the supply of a barcode oligonucleotide or a set of its components from a sample species reservoir (104, 106) to a target channel (102) is controlled by one or more detection means or sensors.

[0216] Item 22: A method according to any one of items 18-21, wherein the entity is a nucleic acid, a cell, or a drug.

[0217] Item 23: The method according to any one of Items 18 to 22, further comprising the steps of generating microfluidic droplets containing entities and barcoded oligonucleotides or their components, or further comprising fusing microfluidic droplets containing entities and barcoded oligonucleotides or their components with further microfluidic droplets, and / or further comprising injecting a reagent into the microfluidic droplets.

[0218] Item 24: The method according to any one of items 18 to 23, further comprising the step of detecting an entity and a barcode oligonucleotide or its components.

[0219] The foregoing applies to embodiments of the present disclosure, but other and further embodiments of the present disclosure may be devised without departing from its basic scope, the scope of which is determined by the following claims. [Examples]

[0220] Example 1: Creation of microcompartments to hold reagents of different concentrations This embodiment demonstrates how the pressure-controlled microfluidic device according to the present invention can create a microfluidic microcompartment containing reagents of various concentrations stored in a seed sample reservoir. The experimental setup includes a pressurized reservoir filled with carrier oil, the fluorescent dye Resazurin (7-hydroxy-10-oxidephenoxazine-10-ium-3-one, sodium), and FreeStyle® 293 expression medium. As shown in Figure 9A, microfluidic microcompartments were created by injecting different reagents into the flow channel network. By adjusting the pressure applied to the reservoir, the relative flow rate of the aqueous sample (fluorescent dye and buffer solution) was dynamically changed, and different final concentrations of the dye in the microfluidic microcompartment were obtained while injecting oil at a constant flow rate.

[0221] In this example, microcompartments were created at 1 / 4 and 3 / 4 concentrations of the fluorescent dye loaded into the reservoir. Under condition A, a microcompartment was created by injecting 8 μL / min of red fluorescent dye and 24 μL / min of buffer. For condition B: Microcompartments were created by injecting 24 μL / min of red fluorescent sample and 8 μL / min of buffer. For both conditions A and B, P ni It is 100-600 mbar, P i The pressure ranged from 125 to 2000 mbar.

[0222] Using the equipment described in Panwar, J., Autour, A. & Merten, CA (Design and construction of a microfluidics workstation for high-throughput multi-wavelength fluorescence and transmittance activated droplet analysis and sorting. Nat Protoc 18, 1090-1136 (2023). https: / / doi.org / 10.1038 / s41596-022-00796-2, incorporated herein by reference), the fluorescence signals of samples were determined after the creation of microfluidic microcompartments by laser spectroscopy. The results are shown in Figure 9B. The measured fluorescence signal of the microcompartment created under condition A was 0.068 [au], and the measured fluorescence signal of the microcompartment created under condition B was 0.209 [au]. The ratio of the fluorescence signals in the microcompartment under condition B to condition A is 3.077 [au], which is consistent with the concentration of the obtained dye ([au] = arbitrary unit, used to describe quantitative signals whose amplitude depends on amplification).

[0223] In the alternative setup shown in Figure 10A, the same method was used to create microcompartments with concentrations equivalent to 0.0909 times and 0.909 times the fluorescent dye in the sample reservoir (stock concentration of 11 μM).

[0224] Under condition C, a microcompartment was created by injecting 24 μL / min of red fluorescent solution and 2.4 μL / min of buffer solution. Under condition D, a microcompartment was created by injecting 2.4 μL / min of red fluorescent solution and 24 μL / min of buffer. The results are shown in Figure 10B. The measured fluorescence signal of the microcompartment created under condition C was 8.924 [au], and the measured fluorescence signal of the microcompartment created under condition D was 0.895 [au]. The ratio of the fluorescence signal of the microcompartment under condition D to condition C is 9.969 [au], which is consistent with the 10-fold difference expected from the flow rate.

[0225] These results demonstrate that this method enables the creation of microfluidic microcompartments containing a wide range of stock solution concentrations present within the sample species reservoir.

[0226] Example 2: Creation of microcompartments to accommodate different combinations of barcode oligonucleotides This example demonstrates how the device can be used to create microfluidic microcompartments for co-localizing cells with different combinations of barcode oligonucleotides. The experimental setup includes a pressurized sample species reservoir filled with carrier oil, a cell suspension, and a drug mixed with a barcode of a known oligonucleotide sequence (Combi-seq barcode as incorporated herein by reference, as described in Mathur L, Szalai B, Du NH et al. Combi-seq for multiplexed transcriptome-based profiling of drug combinations using deterministic barcoding in single-cell droplets. Nat Commun 13, 4450 (2022). https: / / doi.org / 10.1038 / s41467-022-32197-0). As shown in Figure 11, microfluidic microcompartments were created by injecting different reagents into the flow channel network. By varying the combination of injected reagents, microcompartments containing different combinations of drugs and barcoded oligonucleotides were created. Specifically, 18 different combinatorial barcodes were created according to Table 1 below. The ligated barcode was double-stranded and had a ligation region (gcggc) between the 5'-barcode and the 3' poly-T barcode. The barcode ligation scheme is shown in Figure 12. Table 1 shows the barcode elements used in this embodiment. [Table 1] JPEG2026511155000002.jpg250168

[0227] Figures 13A and 13B schematically illustrate the microcompartments created for condition number 6 (Figure 13A) and condition number 11 (Figure 13B), which are detailed in Table 1 above.

[0228] After generation, the microfluidic microcompartments were incubated for 16 hours in an incubator maintained at 37°C and 5% CO2. After incubation, as shown in Figure 13, the two barcode fragments were ligated into a single combinatorial oligonucleotide, and the microcompartment was injected into the T-junction channel for the injection of additional reagents for reverse transcription. The microcompartment was injected at a flow rate of 30 μL / min, and the reagent was injected at 10 μL / min. The composition of each reagent is shown in Table 2. [Table 2]

[0229] The microcompartments were then incubated at room temperature for 40 minutes, followed by incubation at 50°C for 30 minutes, and finally at 85°C for 5 minutes. Next, the microcompartment was combined with a single aqueous solution for library preparation for sequencing, as described by reference in Mathur, L., Szalai, B., Du, NH et al., Nat Commun 13, 4450 (2022), https: / / doi.org / 10.1038 / s41467-022-32197-0, without performing cDNA purification using C1 dynabeads. The library was sequenced using a NextSeq 500 instrument (Illumina) with an average sequencing depth of 16.9 million reads per sample. Table 3 lists the number of reads obtained for each ligated barcode. [Table 3] JPEG2026511155000005.jpg250168

[0230] Numerous sequencing reads were obtained for all barcodes generated as combinatorials, and the desired on-demand sample composition was successfully generated using the method and device of the present invention. It was demonstrated that the desired barcodes were functional and could be used to amplify cellular RNA and generate barcoded cDNA for all samples.

Claims

1. A microfluidic device (100), A target channel (102) containing a target channel fluid such as a carrier phase, A first sample species reservoir (104) containing a first sample species (A) and a second sample species reservoir (106) containing a second sample species (B), A first sample injection channel (114) includes a sample injection channel reservoir end (114A) connected to the first sample species reservoir (104) and a sample injection channel junction end (114B) connected to the target channel (102), The second sample injection channel (116) includes a sample injection channel reservoir end (116A) connected to the second sample type reservoir (106) and a sample injection channel junction end (116B) connected to the target channel (102), and Includes, A microfluidic device (100) wherein the sample injection channel junction ends (114B; 116B) of the first sample injection channel (114) and / or the second sample injection channel (116) are connected to the target channel (102) without a valve and are equipped with at least one hydrodynamic resistor.

2. The microfluidic device (100) according to claim 1, wherein the first sample species reservoir (104) and the second sample species reservoir (106) are configured to be pressurized between both the injection and non-injection phases.

3. The microfluidic device (100) according to claim 1 or 2, wherein the first sample injection channel (114) and the second sample injection channel (116) are separately connected to the target channel (102) at their respective sample injection channel joint ends (114B; 116B).

4. The microfluidic device generates one or more combinatorial microcompartments (200A; 200B) containing at least two sample species (A; B) within the carrier phase, The first sample species (A) and the second sample species (B) are chemically distinct species. The microfluidic device (100) according to any one of claims 1 to 3, wherein the combinatorial microcompartment (200A; 200B) is a combinatorial droplet or a combinatorial plug.

5. The carrier phase is an immiscible phase, A microfluidic device (100) according to any one of claims 1 to 4, wherein one or more combinatorial microcompartments (200A; 200B) flow into an outlet channel or a read channel.

6. The microfluidic device (100) according to any one of claims 1 to 5, wherein each of the first sample injection channel (114) and the second sample injection channel (116) is connected to a sensor (114C; 116C) configured to monitor the flow data of the respective liquids in the first sample injection channel (114) and the second sample injection channels (114; 116).

7. The system comprises a first pressure control device (114D) connected to the first sample species reservoir (104) and at least a second pressure control device (116D) connected to the second sample species reservoir (106), The microfluidic (100) device according to any one of claims 1 to 6, wherein the first pressure control device (114D) and the second pressure control device (116D) are connected to a pressure source (122).

8. The first pressure control device (114D) injects the first sample type (A) into the first sample type reservoir (104) via the first sample injection channel (114) at a first injection pressure P ｉ It is configured to apply, The second pressure control device (116D) injects the second sample type (B) into the second sample type reservoir (106) via the second sample injection channel (116) using a second injection pressure P ｉ A microfluidic device (100) according to claim 7, configured to apply a certain substance.

9. The first pressure control device (114D) is communicated to the first sensor (114C) of the first sample injection channel (114), The second pressure control device (116D) is communicated to the second sensor (116C) of the second sample injection channel (116), The first injection pressure P ｉ and the second injection pressure P ｉ The microfluidic device (100) according to claim 8, wherein the fluid is applied based on the monitored flow data of the liquid in each of the sample injection channels (114; 116).

10. The first pressure control device (114D) and the second pressure control device (116D) control the non-injection pressure P to each sample type reservoir (104; 106) of each non-injection sample injection channel (114; 116). ｎｉ It is configured to maintain the non-injection pressure P ｎｉ is lower than the first injection pressure P ｉ and the second injection pressure P ｉ and satisfies the relationship: P ｉ > P ｎｉ > P 大気 in accordance with The first pressure control device (114D) and the second pressure control device (116D) control the non-injection pressure P to each sample type reservoir (104; 106) of each non-injection sample injection channel (114; 116). ｎｉ A microfluidic device (100) according to any one of claims 7 to 9, configured to increase over time.

11. The microfluidic (100) device according to claim 10, wherein the non-injection pressure applied to each sample type reservoir (104;106) of each non-injection sample injection channel (114;116) causes a liquid flow within each of the sample injection channels (114;116).

12. A method (300) for providing one or more combinatorial microcompartments (200A; 200B) containing at least two sample species (A; B), The process (302) involves injecting a first sample species (A) from a first sample species reservoir (104) into the continuous flow of the carrier phase in a target channel (102) via a first sample injection channel (114) to create a microcompartment (202), Step (304) of creating a combinatorial microcompartment (200) by injecting at least one second sample species (B) from a second sample species reservoir (106) into the created microcompartment (202) via a second sample injection channel (116), The steps include: applying resistance to the first injection channel (114) and / or the second injection channel (116) using at least one hydrodynamic resistor; Methods that include...

13. The first sample type (A) and the second sample type (B) are injected into the respective first sample type reservoir (104) and second sample type reservoir (106) at an injection pressure P ｉ It is injected by applying, The injection pressure P ｉ Lower non-injection pressure P ｎｉ The method according to claim 12, wherein each non-injected sample injection channel (114; 116) is maintained in each sample type reservoir (104; 106).

14. The non-injection pressure P is applied to each sample type reservoir (104; 106) of each non-injection sample injection channel (114; 116). ｎｉ This includes a process of increasing over time, The injection pressure P ｉ and the non-injection pressure P ｎｉ However, relationship: P ｉ >P ｎｉ >P 大気 The method according to claim 13.

15. The injection of the second sample type (B) occurs continuously with or in conjunction with the injection of the first sample type (A). The carrier phase is an immiscible phase, The first sample species (A) and the second sample species (B) are chemically different species. The method according to any one of claims 10 to 14, wherein the combinatorial microcompartment (200) is a combinatorial droplet or combinatorial plug containing at least one prokaryotic or eukaryotic cell.

16. A microfluidic chip (122) comprising one or more combinatorial microcompartments (200) containing at least two sample species (A; B) produced by the method according to any one of claims 10 to 15.

17. The use of a microfluidic device to combine a first sample type (A) and a second sample type (B) to create a target flow channel fluid, During the non-injection stage, the non-injection pressure P is applied to the first sample type reservoir (104) containing the first sample type (A). ｎｉ Apply and inject P into at least the second sample type reservoir (106) containing the second sample type (B) during the non-injection stage. ｎｉ The process of applying, During the injection stage, an injection pressure P is injected into at least one of the first sample type reservoir (104) and the second sample type reservoir (106). ｉ The process includes the step of applying a solution, thereby injecting the first sample type A and / or the second sample type B into the target flow channel fluid, P ｉ >P ｎｉ >P 大気 And, The microfluidic device used is The target channel (102) containing the target channel fluid, A first sample injection channel (114) includes a sample injection channel reservoir end (114A) connected to the first sample type reservoir (104) and a sample injection channel junction end (114B) connected to the target channel (102), The second sample injection channel (116) includes a sample injection channel reservoir end (116A) connected to the second sample type reservoir (106) and a sample injection channel junction end (116B) connected to the target channel (102), The sample injection channel junction ends (114B; 116B) of the first sample injection channel (114) and / or the second sample injection channel (116) are connected to the target channel (102) without a valve and are equipped with at least one hydrodynamic resistor.

18. A method for colocalizing an entity with a barcode oligonucleotide or a set of its components in a microfluidic device according to any one of claims 1 to 11, (i) A step of supplying the substance to the target flow path (102), (ii) A step of passing the substance through the first sample injection channel (114) and the second sample injection channel (116), wherein one of the sample injection channels (114, 116) injects the first sample type reservoir (104) and the second sample type reservoir (106) with an injection pressure (P ｉ The process involves applying a solution to supply a barcode oligonucleotide or a set of its components from the sample species reservoir (104, 106) to the target channel (102) via the respective sample injection channels (114, 116) and the respective sample injection channel junction ends (114B, 116B), thereby injecting the barcode oligonucleotide or a set of its components into the target channel (102), (iii) Step (ii) is optionally repeated, preferably, when step (ii) is repeated, the other of the first sample injection channel (114) and the second sample injection channel (116) supplies a barcode oligonucleotide or a set of its components from the sample species reservoir (104, 106) to the target channel (102) and A method that includes this.

19. Non-injection pressure (P) during the non-injection phase ｎｉ The process further includes the step of applying the first sample species reservoir (104) and / or the second sample species reservoir (106), P ｎｉ However, environmental pressure (P 大気 Larger than P ｉ >P ｎｉ >P 大気 The method according to claim 18.

20. The injection pressure (P ｉ ) and the non-injection pressure (P ｎｉ The method according to claim 18 or 19, wherein the result is produced by pressurizing the first sample species reservoir (104) and the second sample species reservoir (106).

21. The method according to any one of claims 18 to 20, wherein the supply of the barcode oligonucleotide or set of its components from the sample species reservoir (104, 106) to the target channel (102) is controlled by one or more detection means or sensors.

22. The method according to any one of claims 18 to 21, wherein the entity is a nucleic acid, a cell, or a drug.

23. The process further includes generating microfluidic droplets containing the aforementioned entity and the barcode oligonucleotide or its components, or further comprising the step of fusing a microfluidic droplet containing the entity and the barcode oligonucleotide or its components with a further microfluidic droplet, The method according to any one of claims 18 to 22, further comprising the step of injecting a reagent into the microfluidic droplets.

24. The method according to any one of claims 18 to 23, further comprising the step of detecting the entity and the barcode oligonucleotide or its components.

25. A method for barcoding a test substance on cells or cell cultures, (i) A step of co-localizing the substance to be tested in a microfluidic droplet with a barcode oligonucleotide or its components and optionally with cells using the method according to any one of claims 18 to 24, (ii) If there are no colocalized cells in step (i), the step of selectively introducing cells into the microfluidic droplets of step (i), (iii) A step of injecting or fusing a reaction mixture containing a reagent into the microfluidic droplets of step (ii), (iv) A step of incubating the microfluidic droplets of step (iii) and allowing the reaction to occur in the reaction mixture. Methods that include...

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