EP4688262A1

EP4688262A1 - Device and method for producing a combinatorial microcompartment within a carrier phase

Info

Publication number: EP4688262A1
Application number: EP24715511.2A
Authority: EP
Inventors: Hana SAMET; Christoph Merten
Original assignee: Ecole Polytechnique Federale de Lausanne EPFL
Current assignee: Ecole Polytechnique Federale de Lausanne EPFL
Priority date: 2023-03-24
Filing date: 2024-03-25
Publication date: 2026-02-11
Also published as: CN120897800A; KR20250170073A; EP4434627A1; WO2024200381A1; JP2026511155A

Abstract

The present disclosure provides a method and a microfluidic device for producing one or more combinatorial microcompartment comprising at least two sample species within a carrier phase, the microfluidic device comprising a target channel, a first sample species reservoir comprising a first sample species and at least a second sample species reservoir comprising a second sample species, a first sample injection channel comprising 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 at least a second sample injection channel comprising 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 end of the first sample injection channel and/or the second sample injection channel are valvelessly connected to the target channel and comprise at least one hydrodynamic resistor. The present disclosure also provides a method for co-localizing an entity with a barcode oligonucleotide or set of components thereof in a microfluidic device according to the invention.

Description

Device and Method for Producing a Combinatorial Microcompartment within a Carrier Phase

Embodiments described herein relate to a device, particularly a microfluidic device, and a method for producing one or more combinatorial microcompartment(s) comprising at least two sample species within a carrier phase.

Background of the invention

A problem encountered in microfluidic devices is related to microfluidic backflow. Microfluidic backflow may refer to an unintended flow of liquid, fluid or aqueous samples from one channel or one compartment in the microfluidic device into another channel or another compartment. Backflow therefore results in cross-contamination between, e.g. neighboring channels, microcompartments and/or combinatorial microcompartments which can be a significant problem in microfluidic experiments and assays because liquids, fluids or aqueous samples that should be kept separate can (e.g. unintentionally) mix or disrupt the intended flow of fluids in the microfluidic device.

As mentioned, microfluidic backflow may occur between channels and/or microcompartments. Several factors may contribute to microfluidic backflows in channels, for example pressure differences: if a pressure in one channel is greater than a pressure in another channel, liquid, fluid or aqueous samples can flow from the higher pressure channel into the lower pressure channel resulting in backflow; hydrodynamic flow: when liquid, fluid or aqueous samples are flowing in one channel, they can create a flow that draws liquid, fluid or aqueous samples from other channels into the flowing channel; capillary forces: capillary forces can cause liquid, fluid or aqueous samples to be drawn into adjacent or neighboring channels; and/or incomplete sealing: if seals between channels are not properly designed or are not tightly sealed, liquid, fluid or aqueous samples can leak from one channel into another.

Microfluidic backflow in microcompartments may be caused, for example, by: pressure differences across the micro-channels poor channel design leading to channel blockages; viscous drag forces of the liquid, fluid or the aqueous sample; flow rate mismatches between the inlets and outlet; electrostatic interactions between the liquid, fluid or the aqueous sample and channel wall and/or changes in temperature and viscosity of the liquid, fluid or the aqueous sample.

Typically, the use of flow-controlling and/or switching on-chip valves, such as pneumatic valves (e.g. Quake valves) or Braille valves, can help to prevent or reduce backflow by enabling precise control over the flow of fluids in a microfluidic device.

Valves can be used to generate different aqueous samples. The aqueous samples can be mixed and/or compartmentalized on a microfluidic chip in connection with a co-injection of a carrier phase, such as water or oil, by utilizing the valve's ability to selectively route and control fluid flow in microfluidic channels. To this end, valves can be used to direct the flow of fluids into specific microcompartments within a microfluidic chip, allowing the creation of multiple isolated compartments with, e.g. different chemical or biological environments. These aqueous microcompartments can be used for a variety of purposes, such as conducting chemical reactions, encapsulating cells or other biological materials, or for high throughput screening applications, e.g. allowing for the creation of multiple, isolated environments for screening reactions or assays.

However, there are several limitations when using Valves to generate different aqueous samples or combinatorial mixtures, including i) complexity, e.g. valves can be difficult to fabricate and to integrate into microfluidic devices due to their small size and intricate design; ii) maintenance, e.g. valves can be prone to clogging, leakage or wear over time, which can require regular maintenance and cleaning to ensure proper function; iii) limited number of channels; e.g. the number of channels that can be controlled using a single valve is limited, which can limit the number of screening assays that can be performed in parallel; iv) accuracy, e.g. the requirement for very accurate alignment of the valve e.g. on an external actuator (e.g. aligning a Braille valve on a Braille display); and/or v) limited scalability, e.g. it can be difficult to scale up the number of valves for high- throughput screening applications due to the complex fabrication process and the need for precise alignment of the valves within the microfluidic device. However, usage of valves for generating combinatorial mixtures additionally requires prevention of undesired microfluidic backflow. Furthermore, flexible materials, such as a PDMS (Polydimethylsiloxane) material which is a silicone- based polymer material that is commonly used in microfluidics due to its transparency, low surface energy, and biocompatibility, are required for correct functioning of the valves, even though the same are difficult to produce in a standardized scalable fashion.

US Patent No. 5,726,404 is titled "Valveless Liquid Microswitch" describes a valveless liquid microswitch that can be used for controlling fluid flow in microfluidic devices. The microswitch includes a chamber filled with a liquid that can be actuated by a piezoelectric element or other suitable actuator.

Accordingly, it is beneficial to provide an improved microfluidic device and methods to prevent, reduce and/or counteract microfluidic backflow between channels and/or microcompartments while minimizing or eliminating the limitations associated with the usage of valves.

Summary of the invention

The subject-matter provided by the present invention is defined in the independent claims, while preferred embodiments of the present invention are defined in the dependent claims.

The invention in its most generic embodiment pertains to a method and device for combining two or more different sample fluids (or liquids) and a target channel fluid within a microfluidic device by temporary injection of at least one of the at least two sample fluids from one of at least two sample injection channels into a target channel comprising the target channel fluid at a sample injection junction (which shall be understood as a channel junction between the target channel and the one of the at least two sample injection channels). The target channel preferably comprises a continuous flow of the target channel fluid which is provided via a target channel fluid reservoir. The method and device of the invention then provides that the at least one sample injection fluid to be injected is provided into the sample injection channel via a pressurized sample injection fluid reservoir, preferably, wherein an injection pressure Pi is applied during injection phase and when non-injection pressure P_m is applied during the non-injecting phase, and wherein the at least two sample injection channels are each valvlessly connected to a sample injection fluid reservoir via at least one hydrodynamic resistor. The invention thus provides sample injection channels that are supplied by continuously pressurized sample injection reservoirs both during injection phases and non-injection phases in combination with hydrodynamic resistors arranged between the sample injection junction and the sample injection reservoir. Such combination is surprisingly effective in avoiding injection channel backflow compared to prior art devices and methods. The invention will be described in the following in detail with reference to specifically preferred embodiments.

In the following detailed description of the invention, the target channel fluid is also referred to as a carrier fluid or carrier phase, wherein the injection channel fluid may be referred to as a sample species or fluid.

In light of the above, a microfluidic device as well as a method for combining at least two fluids in a microfluidic device is provided. Such inventive method for combining fluids is particular useful for producing combinatorial microcompartments. Hence, in view of the above, the invention further provides a microfluidic device as well as a method for producing one or more combinatorial microcompartment(s) comprising at least two sample species within a carrier phase is provided.

According to an embodiment, a microfluidic device comprising at least two sample species within a carrier phase is provided. The microfluidic device comprising a target channel, a first sample species reservoir comprising a first sample species and at least a second sample species reservoir comprising a second sample species. The microfluidic device comprising a first sample injection channel comprising 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 at least a second sample injection channel comprising 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. The sample injection channel junction end of the first sample injection channel and/or the second sample injection channel are valvelessly connected to the target channel and comprise at least one hydrodynamic resistor. In preferred embodiments of the invention, sample species reservoirs comprised in the microfluidic device of the invention are capable of being pressurized during injection and non-injection of sample liquid. In one preferred embodiment the microfluidic device is for producing one or more combinatorial microcompartments. According to an embodiment, a method for operating a microfluidic device is provided, the method comprising: injecting, into a continuous flow of a carrier phase of a target channel (wherein preferably the continuous flow of the carrier phase is injected into the target channel from a carrier phase reservoir), a first sample species from a first sample species reservoir via a first sample injection channel. The method further comprises injecting into the continuous flow of a carrier phase of the target channel at least one second sample species from a second sample species reservoir via a second sample injection channel, wherein (i) the first sample species and the second sample species are injected by applying an injection pressure Pi to the respective first species reservoir and the second species reservoir, while a non-injection pressure P_m being lower than the injection pressure Pi is being maintained on each sample species reservoir of each non-injecting sample injection channel, and/or (ii) a resistance within the first injection channel and/or the second injection channel using at least one hydrodynamic resistor is provided.

According to another embodiment, a method for providing one or more combinatorial microcompartment(s) comprising at least two sample species is provided. The method comprises: injecting, into a continuous flow of a carrier phase of a target channel, a first sample species from a first sample species reservoir via a first sample injection channel to create a microcompartment. The method further comprises injecting, into the created microcompartment, at least one second sample species from a second sample species reservoir via a second sample injection channel to create a combinatorial microcompartment, wherein (i) the first sample species and the second sample species are injected by applying an injection pressure Pi to the respective first species reservoir and the second species reservoir, while a non-injection pressure P_m being lower than the injection pressure Pi is being maintained on each sample species reservoir of each non-injecting sample injection channel, and/or (ii) a resistance within the first injection channel and/or the second injection channel using at least one hydrodynamic resistor is provided.

According to an embodiment, a microfluidic chip is provided, including one or more combinatorial microcompartment(s), comprising at least two sample species produced by injecting, into a continuous flow of a carrier phase of a target channel, a first sample species from a first sample species reservoir via a first sample injection channel to create a microcompartment, and injecting, into the created microcompartment, at least one second sample species from a second sample species reservoir via a second sample injection channel to create a combinatorial microcompartment, wherein (i) the first sample species and the second sample species are injected by applying an injection pressure Pi to the respective first species reservoir and the second species reservoir, while a non- injection pressure P_m being lower than the injection pressure Pi is being maintained on each sample species reservoir of each non-injecting sample injection channel, and/or (ii) a resistance within the first injection channel and/or the second injection channel using at least one hydrodynamic resistor is provided. According to a further embodiment, a method for co-localizing an entity with a barcode oligonucleotide or set of components thereof in a microfluidic device as described herein is provided, said method comprising the steps of

(i) feeding the entity into a target channel;

(ii) passing the entity past a first sample injection channel and at least one second sample injection channel, wherein one of the sample injection channels feeds a barcode oligonucleotide or set of components thereof from the sample species reservoir to the target channel via the respective sample injection channel and a respective sample injection channel junction end by applying an injection pressure (Pi) to the respective one of the first and second sample species reservoirs, thereby injecting the barcode oligonucleotide or set of components thereof into the target channel;

(iii) optionally repeating step (ii), preferably wherein when repeating step (ii) the other of the first sample injection channel and the at least one second sample injection channel feeds a barcode oligonucleotide or set of components thereof from the sample species reservoir to the target channel.

According to one embodiment, the method further comprises applying a non-inj ection pressure (P_m) during a non-injection phase to the first sample species reservoir and/or to the at least one second sample species reservoir, wherein P_m is larger than the pressure of the environment (P atmo sphere), and wherein P i Pni Patmosphere-

According to another embodiment, the injection pressure (Pi) and the non-injection pressure (Pm) are generated by pressurizing the first sample species reservoir and the at least one second sample species reservoir.

According to one embodiment, the feeding of the barcode oligonucleotide or set of components thereof from the sample species reservoir to the target channel is controlled by one or more detection means or sensors.

According to one embodiment, the entity is a nucleic acid or a cell or a drug.

According to one embodiment, the method further comprises generating microfluidic droplets comprising the entity and the barcode oligonucleotide or components thereof.

According to one embodiment, the method further comprises fusing of microfluidic droplets comprising the entity and the barcode oligonucleotide or components thereof with a further microfluidic droplet, and/or the method further comprises injecting reagents into the microfluidic droplet.

According to one embodiment, the method further comprises detecting the entity and the barcode oligonucleotide or components thereof.

According to a further embodiment, the present invention provides a method for barcoding a substance to be tested on a cell or a culture of cells, the method comprises the steps of:

(i) co-localizing the substance with a barcode oligonucleotide or components thereof and optionally a cell in a microfluidic droplet using the method of the present invention; (ii) optionally introducing a cell into the microfluidic droplet of step (i) if no cell is colocalized in step (i);

(iii) injecting or fusing a reaction mixture comprising reagents into the microfluidic droplet of step (ii);

(iv) incubating the microfluidic droplet of step (iii), allowing the reaction mixture to carry out a reaction.

Further aspects, advantages, and features are apparent from the dependent claims, the description, and the accompanying drawings.

Description of the drawings

So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments. The accompanying drawings relate to embodiments of the disclosure and are described in the following:

FIG. 1 shows a schematic view of a microfluidic device for producing a combinatorial microcompartment according to embodiments described herein;

FIG. 2A shows a schematic view of injecting a first sample species and a second sample species and the occurrence of undesired back-flow due to a lack of combining (i) maintenance of a non-injection pressure on sample species reservoirs of non-injecting sample injection channels with (ii) hydrodynamic resistors;

FIG. 2B shows a schematic view of avoiding undesired backflow when injecting a first sample species and a second sample species while maintaining a non-injection pressure on sample species reservoirs of non-injecting sample injections channels while using a hydrodynamic resistor according to embodiments of the present disclosure;

FIG. 2C shows a schematic view of different microcompartments and combinatorial microcompartments created in a carrier phase of a target channel according to embodiments of the present disclosure;

FIG. 3 shows a flow diagram of a method according to embodiments described herein;

FIG. 4A shows a pressure profile during sample injection without combining maintaining a non-injection pressure on sample species reservoirs of non-injecting sample injections channels with using a hydrodynamic resistor;

FIG. 4B shows a pressure profile during sample injection while maintaining a non- injection pressure on sample species reservoirs of non-injecting sample injections channels and using a hydrodynamic resistor according to embodiments of the present disclosure; FIG. 5 A shows a flow rate profile during sample injection without combining maintaining a non-injection pressure on sample species reservoirs of non-injecting sample injections channels with using a hydrodynamic resistor;

FIG. 5B shows a flow rate profile during sample injection while maintaining a noninjection pressure on sample species reservoirs of non-injecting sample injections channels and using a hydrodynamic resistor according to embodiments of the present disclosure;

FIG. 5C shows another flow rate profile during sample injection while maintaining a noninjection pressure on sample species reservoirs of non-injecting sample injections channels without using hydrodynamic resistors;

FIG. 6A shows a measurement of cross contamination in a microcompartment created with a microfluidic device without maintaining a non-injection pressure on sample species reservoirs of non-injecting sample injections channels but using a hydrodynamic resistor;

FIG. 6B shows a measurement of cross contamination in a microcompartment created maintaining a non-injection pressure on sample species reservoirs of non-injecting sample injections channels but using a hydrodynamic resistor according to embodiments of the present disclosure;

FIG. 6C shows a measurement of cross contamination in a microcompartment created while maintaining a non-injection pressure on sample species reservoirs of non-injecting sample injections channels but on a chip without a hydrodynamic resistor;

FIG. 7A shows flow rate data in ul/min (i.e. backflow and/or overshoot) recorded within a microfluidic chip without using a hydrodynamic resistor;

FIG. 7B shows flow rate data in ul/min (i.e. backflow and/or overshoot) recorded within a microfluidic chip using a hydrodynamic resistor in combination with maintaining a non-injection pressure on sample species reservoirs of non-injecting sample injections channels in accordance with the preferred embodiments of the invention.

FIG. 8A shows a microfluidic chip setup including hydrodynamic resistors in accordance with the invention, such a chip used for Figs. 4 to 6B;

FIG. 8B shows a microfluidic chip setup without hydrodynamic resistors such as a chip used for Fig. 5C, 6C, and 7A.

FIG. 9 shows an experimental set up and results for generating microcompartments using the device and method of the present invention.

FIG. 10 shows a further experimental set up and results for generating microcompartments using the device and method of the present invention.

FIG. 11 shows a preferred embodiment of a microfluidic device according to the present invention.

FIG. 12 schematically shows the generation of barcodes in accordance with an embodiment of the present invention. FIG. 13 A and B are schematic visualizations of two exemplary microcompartments generated in Example 2. FIG. 13 C schematically shows the addition of reagents for cell lysis and reverse transcription to the microcompartments.

Detailed description of the invention

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. Any feature indicated as being optional, preferred or advantageous may be combined with any other feature or features indicated as being optional, preferred or advantageous.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being “incorporated by reference”. In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Reference will now be made in detail to the various embodiments of the disclosure, one or more examples of which are illustrated in the figures. The drawings are schematic drawings which are not drawn to scale. Some elements in the drawings may have dimensions which are exaggerated for the purpose of highlighting aspects of the present disclosure and/or for the sake of clarity of presentation. To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation. Generally, only the differences with respect to individual embodiments are described.

Each example is provided by way of explanation of the disclosure and is not meant as a limitation of the disclosure. Further, features illustrated or described as part of one embodiment can be used on or in conjunction with other embodiments to yield yet a further embodiment. It is intended that the description includes such modifications and variations.

Embodiments described herein relate to a microfluidic device for producing one or more combinatorial microcompartment(s) .

A "microfluidic device" may be understood as a miniaturized system for controlling, directing and/or manipulating amounts of fluid, liquid or aqueous samples, e.g. within a microliter scale or a nanoliter scale. These devices may be made by fabricating channels and compartments in a substrate material, 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 fluidic operations, such as mixing, pumping, separation, and reaction, and they may be used in a variety of applications, including chemical analysis, cell culture, drug discovery, and medical diagnostics. The small size and precise control of microfluidic devices allow for efficient and cost-effective experimentation, as well as the ability to perform experiments and assays that would not be possible using traditional, larger- scale fluidic systems.

A "microcompartment" may be understood as a liquid, fluidic or aqueous sample which is dispersed within a continuous phase, e.g. a surrounding carrier phase or carrier fluid such as oil or water. In this respect, a microcompartment may be viewed as a miniaturized reaction or assay vessel. The fluid, liquid or the aqueous sample inside of a microcompartment is immiscible with the fluid or liquid of the carrier phase.

The microcompartments can be in the form of surfactant droplets, e.g. emulsions with volumes in the pico-liter range. A surfactant droplet may be a, e.g. spherical droplet of a surfactant solution surrounded by a continuous phase, e.g. oil or water. Surfactants are surface-active compounds that can reduce the surface tension between two immiscible liquids, allowing, e.g. formation of stable droplets in the continuous phase. In a surfactant droplet, the surfactant molecules are adsorbed at the interface between the droplet and the continuous phase, creating a stabilized interface that prevents the droplet, and also the material inside, from merging with the continuous phase. These droplets are also referred to as microfluidic droplets.

The microcompartments can also be (microfluidic) plugs, e.g. with volumes in the nanoliter range, that completely fdl a target channel or tubing. The plugs may be separated by the carrier phase or carrier fluid, e.g. oil or water. In this case, there is no need for stabilizing surfactants.

The microcompartments can also be flow segments, e.g. a portion or section within the microfluidic channel or tubing, where the fluid flow has a specific and distinct behavior, e.g. exceeding microliter volumes.

A "combinatorial microcompartment" may be understood as a microcompartment comprising at least two sample species or substances, e.g. sample species (A) and sample species (B), which may be chemically and/or biologically distinct. As such, a combinatorial microcompartment may be referred to as including a mixture of different species compartmentalized within the carrier phase.

Possible sample species include molecule 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.

Chemically and/or biologically distinct may refer to substances, entities or materials that have different chemical/biological properties and compositions. This may be understood in that they may differ in their molecular structure, chemical behavior, and/or biological activity. The distinction can be based on differences in the composition of individual molecules, the type 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 causing a specific cellular response. Biologically distinct entities include different types of cells, cells coming from different donors, cell libraries expressing different variants of a given protein and tissue slices

A microfluidic device according to embodiments discussed herein may include a first sample species reservoir comprising a first sample species (A) and at least a second sample species reservoir comprising a second sample species (B).

A "reservoir" or a "sample species reservoir" may be understood as a storage facility or container used to store, contain or hold volumes of a particular sample species, e.g. containing an aqueous sample, fluid or liquid. The applied pressure on the reservoir may also regulate a flow or outflow of these sample species. Typical volumes of such sample species reservoirs are in the range of a few picoliters to a few milliliters, particularly a few nanoliters to a few microliters, more particularly a few microliters to a few milliliters or also even higher/lower. A sample species reservoir in accordance with an embodiment of the present invention can be made of a rigid or a non-rigid material, however, a rigid material is preferred. Preferred materials for a sample species reservoir are plastic materials like polypropylene or polycarbonate, and glass. A "sample species" stored within a sample species reservoir may be understood as a fluid, liquid or aqueous sample comprising, e.g. water, which may further include dissolved or suspended substances, e.g. of a particular chemical/biological species, e.g. a substance or material having a chemically and/or biological distinct property, characteristic, compound and/or composition, e.g. (A), (B), (C) etc. The sample species may also comprise a carrier phase, e.g. oil and water. The sample, sample species and/or the carrier phase may be stored in the form of a fluid or liquid, wherein these terms may be used interchangeably herein. Carrier phase fluids or -liquids are usually selected from oils, but in certain applications of the invention may include aqueous sample liquids.

A "target channel" in context of the present invention shall be understood as the channel into which two, three or more fluids are combined, and wherein the combined fluids are further transported, for example to any assay area, detection area, storage reservoir, or any other means for which the combination of fluids is performed. The term should be understood as a channel or tube, e.g. of a microfluidic chip, into which samples according to the invention are combined. The target channel may be configured for holding, guiding or supporting the target channel fluid such as a carrier phase. The "carrier phase", e.g. within the target channel, may be regarded as a fluid or liquid that flows through the chip, e.g. continuously, in order to move or transport the sample through the chip. The carrier phase can be a liquid, such as water or oil and its properties, such as viscosity, surface tension, and pH, can be adjusted to optimize the transport of the samples.

The term "injection channel" shall refer to the one or more channels from which fluids are injected into the target channel to combine the one or more fluids in accordance with the invention. The injection channel in some instances of the invention may also be referred to as a sample injection channel. The term “sample” shall in this case refer to the one or more fluids to be injected into the target channel.

Although it is mentioned that the first sample species reservoir comprises a first sample species (A) that may be different from a second sample species (B) comprised in the second sample species reservoir, it may also be possible to have both sample species reservoirs to comprise the same sample species, e.g. (A) or (B).

The microfluidic device may comprise a first sample injection channel and a second sample injection channel. A "sample injection channel" may be understood as a (fluidic) channel, pathway or tubing which is used to transport or deliver a fluid, liquid or aqueous ample, for example a particular sample species to, e.g. the target channel. The carrier phase may also be injected using one or more sample injection channels.

Sample injection channels may be made from glass, silicone or plastics. The diameter of a sample injection channel ranges from a few tens of micrometers to several millimeters. For example, in a microfluidic device used for high-throughput screening applications, the sample injection channel may have a diameter of several micrometers. However, larger diameters may be beneficial. For example, when working with cells, larger channels diameters may be used to avoid clogging of the channels, e.g. by allowing the cells to pass through without getting trapped or stuck. The first sample injection channel comprises 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 comprises 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.

At the injection channel "reservoir end", the sample injection channel may be connected valveless to the sample species reservoir. However, the sample species reservoir may be pressurized and the pressure may be released by "external valves", e.g. valves connected to a pressure source (s. below).

In general, at least two sample injection channels connected to a respective first sample species reservoir comprising a first sample species and second sample species reservoir comprising a second sample species may be used to generate the combinatorial microcompartment recited herein. For creating a microcompartment, also only one sample injection channel may be sufficient.

The sample injection channel junction end of the first sample injection channel and/or the second sample injection channel are valvelessly connected to the target channel and comprise a hydrodynamic resistor.

The sample injection channel "junction end" may define an end of the sample injection channel where the same encounters, intersects, merges or is connected to the target channel. A "valveless" connection may be understood as a connection that does not comprise or use a valve, e.g. for controlling the flow of fluids. In a microfluidic device with a valveless connection between a sample injection channel and a carrier phase, the sample may be introduced into the carrier phase through a small opening or constriction at the sample injection channel junction end. The pressure difference between the sample and the carrier phase, e.g. along with the geometry of the channel and the fluidic resistance of the channels, may determine the flow rate and mixing of the aqueous sample and carrier phase. Valveless connections may have improved reliability, lower cost, and reduced complexity. They can also be easier to fabricate and to maintain compared to other devices that use valves.

The term "valveless" or "valvelessly" thus preferably means that no valve is used for closing or opening an inlet into a channel. Preferably, no valve is associated with the sample species reservoirs and/or with injecting sample species from the sample species reservoirs as described herein, e.g. a respective inlet is not closed or opened using a valve. In addition, according to a further preferred embodiment, no valve is used for providing resistances in the hydrodynamic resistors and/or for generating a non-injection pressure (P_m) and an injection pressure (Pi) as described herein. According to some embodiments, no valve actively regulates the feeding of any entity such as a particle, barcode oligonucleotide or other element or fluid to the injection channel(s). In other words, all reagents are injected by applying pressure to the reagent reservoirs only. Thus, according to a preferred embodiment in accordance with the present invention, the microfluidic device does not have valves in between the sample species reservoirs and the target channel. According to a further preferred embodiment, there is no valve on the whole of the microfluidic-device. It will be appreciated that the term "valve less" does not exclude the presence of valves outside of the microfluidic device or chip, e.g. to release a pressure after the respective elements or components have been injected or fed to the target channel or any other part of the microfluidic device. It will also be appreciated that according to an embodiment, valves not involved in the process of feeding elements or components from the sample species reservoirs and via the injection channels are not excluded by the term "valveless". The opposite of valveless with respect to the feeding of an entity, barcode oligonucleotide or other element or fluid to the injection channel(s) is "valve-operated", which means that the respective channel can be closed or opened using a valve.

A "hydrodynamic resistor" may be understood as a device that provides a hydrodynamic resistance to fluid flowing in the sample injection channel in order to control, regulate the flow of fluids in a microfluidic system. A pressure drop may be created across the resistor, which resists the flow of fluid and regulates the flow rate of the fluid. Hydrodynamic resistors may be implemented as narrow channels, constrictions or constricted regions, or regions of increased fluidic resistance within a microfluidic device creating a pressure drop or pressure increments, e.g. an amount by which pressure is increased or decreased during a time period, which generates hydrodynamic/flow resistance.

Hydrodynamic resistors may be implemented as a narrow channel, e.g. having a smaller diameter. This is because the resistance offered by the channel is roughly inversely proportional to the fourth power of the diameter of the channel. In other words, if the diameter of the channel is reduced by a factor of 2, the resistance of the channel increases by a factor of 16. As a result, the smaller the diameter of the channel, the higher the resistance, and the more effective the channel is at regulating pressure. Therefore, hydrodynamic resistors may be implemented as channels of small diameter, as this offers a higher resistance and better pressure regulation than shorter, wider channels.

Hydrodynamic resistors may serve as a pressure regulator or pressure controller which contributes in preventing backflow and ensures stable pressure in a microfluidic device. For example, when pressure is applied to a reservoir in a microfluidic device, a pressure pulse may be created that can lead to rapid pressure changes due to sudden flow rate changes, e.g. overshooting and overregulation of pressures in sample injection channels. This may result in undesired flow into one or another direction and disruption of the system. By using a hydrodynamic resistor, the pressure pulse may be attenuated or damped and a response time between applying a certain pressure and having an effect at the droplet generation point, e.g. where the droplet is produced, is increased, allowing for more stable and precise pressure regulation. This is also important for channels which are noninjecting, as the system may heavily overregulate without hydrodynamic resistors, causing pressure pulses and undesired flow in either direction. Thus, the hydrodynamic resistor may maintain stability and precision in a microfluidic device by regulating or controlling pressure and preventing backflow.

A hydrodynamic resistor may be a specific channel geometry, e.g. constrictions, narrowing's, bottlenecks, bending's; porous materials; membranes, e.g. with controlled pore size; hydrogels, e.g. with controlled swelling properties and/or capillary tubing, e.g. with controlled internal diameter and length.

According to a preferred embodiment, a hydrodynamic resistor is implemented as a specific channel geometry in a portion of a channel, more preferably as a plurality of turns or bendings in a section of the respective channel. According to a particularly preferred embodiment, the hydrodynamic resistor is a sequential arrangement of a plurality of turns or bendings in the channel of between about 170 and 190°, more preferably of about 180°, as exemplified e.g. in Fig. 8A. The plurality of turns or bendings of a hydrodynamic resistor preferably comprises between 4 and 40 tums/bendings, more preferably between 6 and 30, 8 and 20, 10 and 18, or 12 and 16 tums/bendings. According to a particularly preferred embodiment, in the sequential arrangement of a plurality of turns or bendings in the channel, the tums/bendings are evenly distributed along the respective channel section being implemented as the hydrodynamic resistor. It will be appreciated that an individual channel may comprise more than just one hydrodynamic resistor such as two, three, four, five, six, seven, eight, nine, ten or more hydrodynamic resistors.

The term "entity" as used herein refers to a substance, compound or particle capable of being transported in the channels of a microfluidic device according to the invention. The term encompasses for example chemical or biological substances such as pharmaceuticals, molecule drugs or compounds, proteins or enzymes, and biomolecules such as sugars, lipids and hormones. According to a preferred embodiment, the entity is a drug such as a chemical substance and/or pharmaceutical. The term "entity" also includes a particle in its widest sense. The term "particle" as used herein includes any particle, man-made or natural, which incorporates, envelopes, is attached to, consists of or is in any other way associated with DNA and/or RNA. In a preferred embodiment, it is a biological particle, preferably a cell, a non-cellular life form, or a DNA and/or RNA carrier, or a DNA and/or RNA. The particle can also be a transcriptome of a cell or a DNA amplificate from a cell. Most preferably, the particle is a cell. The cell can be any prokaryotic or eukaryotic cell. Preferably, it is a eukaryotic cell, e.g. a yeast cell, plant cell or animal cell. Animal cells include insect, nematode, fish and mammalian cells. More preferably it is mammalian cell, e.g. a mouse, rat, monkey or human cell. For example, it can be a random cell of a heterogeneous cell population (e.g. from a tissue) or it can be a specifically selected cell, selected, e.g. by FACS. Also, it can be a cell a from cell line or a homogeneous culture, for example of a primary cell, wherein "primary" means derived directly from a tissue or organism and not manipulated to have altered properties, e.g. to divide indefinitely. Other examples for cells are developing cells, stem cells or cancer cells. Examples of non-cellular life form are viruses, viroids, cosmids, plasmids, phagemids and the like. Examples of DNA and/or RNA carriers are proteins such as histones or ribosomes. Non-biological particles, such as beads, are also envisaged. A "bead" (also termed "microbead") is a uniform polymer particle with a diameter of up to 1 micrometre, preferably of 0.5 to 500 pm, and with a surface to which nucleic acids can bind or be coupled. The beads referred to herein are usually polyethylene or polystyrene beads or beads made of gel matrices.

The term "barcode oligonucleotide" refers to an oligonucleotide having at least one so-called variable region, the nucleotide sequence of which is unique for this oligonucleotide compared to other barcode oligonucleotides used. In a preferred embodiment, at least two such barcode oligonucleotides are used. Alternatively, a barcode oligonucleotide may have at least two variable regions, the combined nucleotide sequences of which are unique for this oligonucleotide compared to other barcode oligonucleotides used. The term "variable" does not mean that the sequence of a particular oligonucleotide can change, but that there are oligonucleotides which are identical in structure and sequence with the exception of the sequence of the variable regions, i.e. the variable regions are different between oligonucleotides that are otherwise identical in structure and sequence. If the barcode oligonucleotide comprises more than one variable region, each of these variable regions is from separate and combinable components, which can be assembled in a combinatorial fashion to create different barcode oligonucleotides. A barcode further preferably comprises at least one priming region or alternatively one or more transposable elements.

A "component" of a barcode oligonucleotide is thus an oligonucleotide itself, which has one variable region. A "set of components" of a barcode oligonucleotide is a plurality of oligonucleotides which makes up exactly one barcode oligonucleotide (meaning one identity, not one molecule), i.e. it is a complete set of components. The components of one set combine by annealing into a preferably linear barcode oligonucleotide by virtue of their annealing regions, wherein only one linear combination is possible. Thus, within one set of components, the annealing regions are different between the components, but their variable regions can be identical, although they are more likely to be different as well.

The term "co-localizing" refers basically to putting two or more entities, such as a particle or a drug, and a barcode oligonucleotide or components thereof, together, preferably into the same microfluidic droplet. This can be achieved as described below, for example, by generating a microfluidic droplet from an aqueous fluid containing these entities, by fusing droplets separately, each containing one of these entities, or by injecting e.g. a continuous aqueous phase into pre-formed microfluidic droplets. The "fusing" of two droplets results in one microfluidic droplet comprising the contents of the two origin droplets. Thus, for example, the droplet resulting from such a fusion comprises the contents of the microfluidic droplet comprising the particle, and of the microfluidic droplet(s) comprising the barcode oligonucletotide or components thereof. Such a fusion can be achieved by one-to-one fusion, e.g. 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). 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-l-4614-3265-4_2, # Springer Science+Business Media, LLC 2012, Chapter 2.

The term "immiscible" as used in the context of a fluid or liquid refers to a fluid or liquid immiscible with the fluid or liquid the entity such as a particle and the sample species such as a barcode oligonucleotide or components thereof are comprised in the target channel. The immiscible liquid is preferably a hydrophobic liquid, preferably an oil. The oil phase should have a viscosity that is close to that of water and/or be inert with respect to the biological reagents contained in them. Several oils can be used, such as low-viscosity silicone oils, or dimethicone, silicone oils, hydrocarbon oils. Preferred oils for use in the context of the invention are fluorocarbon oils (or flourinated oils), because even low-viscosity versions of these oils do not swell PDMS. Surfactants are useful for reducing the surface tension of the oil-water interface and minimizing droplet coalescence, and can thus be present in the immiscible fluid or liquid. Surfactants utilized in droplet-based microfluidics normally consist of a hydrophilic head group and hydrophobic tail. The amphiphilic character of these molecules allows them to assemble at the oil-water interface of the droplet, thereby lowering its interfacial tension and enhancing stability. Surfactants with non-ionic head groups are preferred, as they minimize the adsorption of macromolecules such as proteins and DNA to the droplet interface, minimally impacting the methods of the invention. Suitable fluorosurfactants that can be readily synthesized in the lab are known in the art and described, e.g., in 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, e.g. from Sphere Fluidics Limited, UK. Additives to the aqueous phase can also enhance biocompatibility by increasing the retention of small molecules in the droplets and minimizing adsorption at the oil- water interface. Different oils can be mixed to optimize the properties of the emulsion for a particular application and methods for easily characterizing the properties of the combination that has been selected are known in the art (Kaltenbach et al. 2012 Lab Chip 12, 4185).

The term "microfluidic droplet" as used herein refers to an aqueous microcompartment of a certain size that encapsulates an aqueous liquid. The size of the microfluidic droplet can for example be expressed as the diameter of the droplet. The diameter is generally less than 1 mm, such between about 10 pm and 900 pm, between about 20 pm and 800 pm, between about 20 pm and 700 pm, between about 20 pm and 600 pm, between about 20 pm and 500 pm, between about 20 pm and 400 pm, and preferably between 30 and 350 pm, between 40 and 300 pm, between 40 and 250 pm, between 40 and 200 pm or between 40 and 100 pm (wherein each narrower range is preferred to the foregoing broader ranges and "between" includes the values mentioned). Other forms of microfluidic droplets include elongated droplets, which can be described as having a cylindrical shape such as a sausage shape, i.e. they are longer than wide. Such elongated droplets, also referred to as "plugs" herein, may have a length of a few millimeters, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, and up to 10 mm, preferably a length of between about 0.2 mm and 6 mm, more preferably between about 0.5 mm and 5.5 mm, even more preferably between about 1.0 mm and 5 mm, and a diameter of between about 300 pm and 900 pm, preferably of between about 400 pm and 800 pm, more preferably of between about 500 pm and 700 pm. According to a preferred embodiment, a plug has a diameter of about 300 pm and a length of between about 1 mm and 3 mm. According to a preferred embodiment, a plug has such dimensions that it completely fdls the target channel. In such embodiments, individual plugs may be separated by the carrier phase or carrier fluid, e.g. oil or water. Alternatively, the size of the microfluidic droplet can also be defined by volume. For example, it is usually less than 1 microlitre (pl). Preferably, it is less than 900 nanolitres (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, less than 150, less than 100 or less than 50 nl. In a preferred embodiment, it is between 0.05 and 150 nl, preferably between 0.05 and 125 nl, between 0.05 and 100 nl, between 0.05 and 80 nl, or between 0.05 and 4 nl (wherein each narrower range is preferred to the foregoing broader ranges and "between" includes the values mentioned). According to a preferred embodiment, the microfluidic droplet is a plug having a volume of about 500 nl.

The term "to generate microfluidic droplets" refers to creating a stream of monodispersed droplets in an immiscible phase. This can be achieved by means of a droplet generator. Microfluidic droplet generators work by combining two or more streams of immiscible fluids and generating a shear force on the discontinuous phase causing it to break up into discrete 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 may be used to create the microfluidic droplet used in accordance with the present invention. Indeed, more than 200 microencapsulation or compartmentalization methods have been identified in the literature (Finch, C. A. (1993) Encapsulation and controlled release. Spec. Publ.-R. Soc. Chem. 138, 35). These include membrane enveloped aqueous vesicles such as lipid vesicles (liposomes) (New, R. R. C., Ed. (1990). Liposomes: a practical approach. The practical approach series. Edited by Rickwood, D. & Hames, B. D. Oxford: Oxford University Press) and non-ionic surfactant vesicles (van Hal, D. A., Bouwstra, J. A. & Junginger, H. E. (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 herein incorporated by reference. Preferably, the microfluidic droplets or microcompartments of the present invention are formed from emulsions; heterogeneous systems of two immiscible liquid phases with one of the phases dispersed in the other as droplets of microscopic size (Becher, P. (1957) Emulsions: theory and practice. Reinhold, New York; Sherman, P. (1968) Emulsion science. Academic Press, London; Lissant, K.J., ed Emulsions and emulsion technology. Surfactant Science New York: Marcel Dekker, 1974; Lissant, K.J., ed. Emulsions and emulsion technology. Surfactant Science New York: Marcel Dekker, 1984), all of which are herein incorporated by reference. Emulsions may be produced from any suitable combination of immiscible liquids. Preferably the emulsion of the present invention has an aqueous phase (containing a particle and other components) as the phase present in the form of droplets and a hydrophobic, immiscible liquid (preferably an oil) as the surrounding matrix in which these droplets are suspended. Such emulsions are termed 'water-in-oil'. This has the advantage that the aqueous phase is compartmentalized in discrete droplets. The external phase, preferably being a hydrophobic oil, generally is inert. The emulsion may be stabilized by addition of one or more surface-active agents (surfactants). These surfactants act at the water/oil interface to prevent (or at least delay) separation of the phases. Many oils and many emulsifiers can be used for the generation of water-in-oil emulsions; a recent compilation listed over 16,000 surfactants, many of which are used as emulsifying agents (Ash, M. and Ash, I. (1993) Handbook of industrial surfactants. Gower, Aidershot), incorporated herein by reference. Suitable oils are mentioned above.

FIG. 1 shows a microfluidic device (100) for producing one or more combinatorial microcompartment(s) according to embodiments described herein.

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

However, at least two sample injection channels (114; 116) connected to a respective first sample species reservoir (104) comprising a first sample species (A) and second sample species reservoir (106) comprising a second sample species (B) may be used to generate the combinatorial microcompartment within the carrier phase recited herein.

As can be seen in Fig. 1, the microfluidic device (100) includes a first sample injection channel (114) comprising 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), and at least a second sample injection channel (116) comprising a sample injection channel reservoir end (116A) connected to the second sample species reservoir (106) and a sample injection channel junction end (116B) connected to the target channel (102). The sample injection channel junction end (114B; 116B) of the first sample injection channel (114) and/or the second sample injection channel (116) are valvelessly connected to the target channel (102) and comprise at least one hydrodynamic resistor.

The microfluidic device (100) shown in Fig. 1 further includes third sample injection channel

(118) comprising a sample injection channel reservoir end (118A) connected to the third sample species reservoir (108) and a sample injection channel junction end (118B) connected to the target channel (102), and a fourth sample injection channel (120) comprising a sample injection channel reservoir end (120A) connected to the fourth sample species reservoir (110) and a sample injection channel junction end (120B) connected to the target channel (102). However, the same are optional. The sample injection channel junction end (118B; 120B) of the third sample injection channel (118) and/or the fourth sample injection channel (120) are also valvelessly connected to the target channel (102) and comprise at least one hydrodynamic resistor

With a view on Fig. 1, it can be seen that the first sample injection channel (114) and the second sample injection channel (116) are separately connected with their respective sample injection channel junction ends (114B; 116B) to the target channel (102). The same correspondingly applies to the third and fourth sample injection channels (118; 120). To this end, the first sample injection channel (114) and the second sample injection channel (116) may also be viewed as individual or isolated channels or pathways, allowing to keep, e.g. species (A) which may be comprised in the first sample species reservoir (104) and species (B) which may be comprised in the second sample species reservoir (106), separated from each other. As such, this may prevent mixing up the different sample species.

Although not shown in Fig. 1, the one or more combinatorial microcompartment may flow into an outlet channel or read-out channel. A "read-out channel" may be regarded as a channel used to measure or detect the properties of the fluid and, particularly, one or more (combinatorial) microcompartment(s) created therein, being analyzed, e.g. to collect information about the different samples or sample species (e.g. cells) within the (combinatorial) microcompartment. To do so, fluorescence, luminescence, electrochemistry, and/or mass spectrometry may be used.

The microfluidic device (100) shown in Fig. 1 may further comprise one or more sensors (114C; 116C; 118C; 120C). As can be seen in Fig. 1, the first sample injection channel (114) is connected to a first sensor (114C) and the second sample injection channel (116) is connected to the second sensor (116C). The same may correspondingly apply to the third and fourth sample injection channels (118; 120) and the third and fourth sensors (118C, 120C) shown in Fig. 1.

The one or more sensors (114C; 116C; 118C; 120C) may be configured to monitor data related to a fluid, liquid or aqueous sample, e.g. a respective sample species, flowing within a respective sample injection channel (114, 116; 118; 120). For example, the data may include flow data or a flow rate indicating the volume of fluid, liquid or aqueous sample flowing or passing through a respective sample injection channel (114, 116; 118; 120) per unit time or also pressure data, e.g. a pressure of the fluid liquid or aqueous sample flowing or passing through a respective sample injection channel (114, 116; 118; 120) per unit time. Exemplary injection flow rates may be in the range of 5 to 40 pl/min, particularly between 8 and 17 pl/min. Preferred pressures may be selected from 10 mbar to 10 bar, particularly between 100 mbar to 5 bar, more particularly between 300 mbar to 2000 mbar. The one or more sensors (114C; 116C; 118C; 120C) may be pressure sensors and/or flow sensors. According to a preferred embodiment, the feeding of the sample species such as e.g. barcode oligonucleotides or set of components thereof from the sample species reservoir (104, 106) to the target channel (102) is controlled by the one or more sensors or detection means (114C; 116C; 118C; 120C). In particular, the time point for applying a non-inj ection pressure P_m or an injection pressure Pi can be controlled by the one or more sensors or detection means. Control by time can also be based on the time point an entity such as a particle is fed into the target channel, the distance it needs to travel to arrive at and leave the injection channel as well as the flow speed of the particle. If for example a plurality of particles is to be combined with a sample species in the same microfluidic droplet, the control by time can be arbitrary or based on the frequency or density of the particle in the target channel. Detections means can be placed, for example, at the beginning and/or at the end of respective injection channels, and/or above or in the vicinity of the sample species reservoirs, and trigger applying a non-injection pressure P_m or an injection pressure Pi upon detecting a particle. A combination of the two is also possible if one or more detection means are placed in a distance to the beginning and/or to the end of respective injection channels. In such a case, the control can be based on the time the particle is detected, the distance it needs to travel to arrive at and leave the series of oligonucleotide inlets as well as the flow speed of the particle. Detection means for detecting particles in a microfluidic channel are well known in the art and include light sensors, for example photomultiplier tubes, CMOS or CCD cameras, detection electrodes, or flow sensors, such as air flow sensors. Generally, the detection means is suitable for detecting a particle and/or a label attached to the particle, in particular a fluorescent label, and may use fluorescence or laser spectroscopy, imaging, impedance or magnetic measurements for detection.

In a particularly preferred embodiment, one or more detection means or sensors are within 10 mm to 50 mm, preferably within 1 mm to 30 mm and more preferably within 200 pm to 500 pm upstream of a first sample injection channel junction end (114B), wherein upstream means the direction from where the carrier phase flows the particle(s) through the target channel. Thus, a detection means or sensor that is arranged upstream of a sample injection channel junction end means that the detection means or sensor is located on the target channel in the direction from where the carrier phase flows the particle(s) through the target channel. Detection means or sensors can also be located upstream of more than just the first sample injection channel junction end (114B), such as upstream of every second sample injection channel junction end or even upstream of every sample injection channel junction end. Another embodiment is also included, wherein the detection means is downstream of where microfluidic droplets are generated in the target channel.

The microfluidic device (100) shown in Fig. 1 may further comprise one or more pressure control devices (114D; 116D; 118D; 120D). A first pressure control device (114D) may be connected to the first sample species reservoir (104) and at least a second pressure control device (116D) may be connected to the second sample species reservoir (106). The same may correspondingly apply to the third and fourth pressure control devices (118D; 120D) and the third and fourth sample species reservoirs (108, 110).

The pressure control devices (114D; 116D; 118D; 120D) may be connected to a pressure source (122) which may be used to generate a pressure in the microfluidic device (100). An example of a pressure source (122) may be syringe pumps, pneumatic pumps, and piezoelectric pumps.

The pressure control devices (114D; 116D; 118D; 120D) can be used to provide both positive and negative pressures, and can be programmed to operate in a variety of ways, such as with constant pressure, constant flow rate, or stepwise pressure changes. The pressure control devices (114D; 116D; 118D; 120D) are configured to apply different pressures to the sample species reservoirs (104; 106; 108; 110) including a pressure used for injection, or "injection pressure Pi " and a pressure used for non-injection, or "non-injection pressure P_m".

For example, the first pressure control device (114D) may be configured to apply a first injection pressure Pi to the first sample species reservoir (104) to cause injection of the first sample species (A) via the first sample injection channel (114), and the second pressure control device (116D) may be configured to apply a second injection pressure Pi to the second sample species reservoir (106) to cause injection of the second sample species (B) via the second sample injection channel (116). The first injection pressure Pi and the second injection pressure Pi may be identical or different.

An "injection pressure" (Pi) may be understood as a pressure being applied for injecting, e.g. a (positive) pressure which drives the fluid, liquid or the aqueous sample from a sample species reservoir (104; 106; 108; 110) through a sample injection channel (114; 116; 118; 120) to the target channel (102). The injection pressure may be applied, e.g. directly, at a respective sample species reservoir (104; 106; 108; 110) by the pressure control devices (114D; 116D; 118D; 120D). An example injection pressure Pi may be within the range of 10 mbar to 10 bar, particularly between 20 mbar to 7 bars, 100 mbar to 5 bar, 60 mbar to 4 bar, more particularly between 125 mbar and 2000 mbar, or 300 mbar to 2000 mbar. Such injection pressures result in an injection flow rate in the range of 1 to 500 pl/min, particularly between 1 to 100 pl/min, more particularly between 8 to 17 pl/min. However, these exemplary flow rates may be adjusted or varied, e.g. depending on the length of the tubing/channels etc. connected to the outlet.

The pressure (Pi or P_m) can be generated by respective means known to the skilled person e.g. by (external) pressure sources such as (external) mechanical pumps such as syringe pumps, pneumatic pumps and piezoelectric pumps, or by (integrated) micropumps such as mechanical micropumps. Micropumps are preferably 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 permeation micropumps; more preferably, the active micropumps are independently selected from the group consisting of syringe micropumps, pneumatic membrane micropumps and Braille pin micropumps. The pressure control devices (114D; 116D; 118D; 120D) may be communicatively coupled to the respective sensors (114C; 116C; 118C; 120C) of the sample injection channels (114; 116; 118; 120) in order to adjust pressures (e.g. one or more injection pressure(s) Pi or one or more non-injection pressure(s) P_m) on the basis of flow data monitored by a respective sensor (114C; 116C; 118C; 120C) of a sample injection channel (114; 116; 118; 120).

For example, the first pressure control device (114D) may be communicatively coupled to the first sensor (114C) of the first sample injection channel (114) and the second pressure control device (116D) may be communicatively coupled to the second sensor (116C) of the second sample injection channel (116), wherein the first and second injection pressures Pi are applied on the basis of the monitored flow data of the liquid in the first sample injection channel (114) and the second sample injection channel (116) respectively.

Needless to say that the third pressure control device (118D) may be communicatively coupled to the third sensor (118C) of the third sample injection channel (118) and the fourth pressure control device (120D) may be communicatively coupled to the fourth sensor (120C) of the fourth sample injection channel (120), wherein third and fourth injection pressures Pi are applied on the basis of the monitored flow data of the liquid in the third sample injection channel (118) and the second sample injection channel (120) respectively.

When an injection pressure Pi is applied, e.g. on one or more sample species reservoir(s) (104; 106; 108; 110), one or more sample species are injected via one or more sample injection channel(s) (114; 116; 118; 120), e.g. into the target channel (102). During injection via one sample injection channel, e.g. a first sample injection channel (114), on or more other sample injection channel(s), e.g. a second sample injection channel (116) and/or a third sample injection channel etc., may be also injecting or non-injecting. In the latter case, "non-injecting" is to be understood in that substantially no flow of fluid, liquid or aqueous samples is to be exiting the corresponding sample injection channel, e.g. at the sample injection channel junction end. A sample injection channel that is not injecting a sample species, e.g. at a first time, may be referred to as a "non-injecting channel". Consequently, a sample injection channel that is injecting a sample species, e.g. at the first time or at a second time being different from the first time, may be referred to as an "injecting channel".

According to embodiments of the present invention, a pressure is also applied for noninjection channels. Such a pressure may be referred to as a "non-injection pressure" (P_m). For example, a non-injection pressure may be in the range of within the range of 1 mbar to 5600 mbar or 10 mbar to 10 bar, particularly between 100 mbar to 5 bar, 50 mbar to 3200 mbar, more particularly between 300 mbar to 2000 mbar or 100 mbar to 1600 mbar. Such non-injection pressures P_m result in substantially zero flow rates of the fluid, liquid or aqueous sample, for example in a flow rate below approximately 30 pl/h, particularly below approximately 20 pil/h .

The first and second pressure control devices (114D; 116D) are configured to maintain a noninjection pressure P_m on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116). It is preferable that the non-injection pressure P_m being lower than, e.g. the first and second injection pressures Pi and following the relation: ?! > P_m preferably refers to the pressure of the environment or the ambient pressure acting on the device, when no other pressure such as an injection pressure Pi and a non-injection pressure P_m are applied, and in particular to the pressure acting on the sample species reservoirs. Each of the pressure control devices (114D; 116D; 118D; 120D) are further configured to increase the non-injection pressure P_m overtime.

For example, the non-injecting pressure P_m may be increased gradually or continuously, e.g. with a steady increase; stepwise, e.g. in discrete steps with a period of stability between each step; pulsed, e.g. in a series of short, sharp pulses with a period of stability between each pulse and/or random, e.g. varied randomly over time. As such, the first and second pressure control devices (114D; 116D) are configured to increase the non-injection pressure P_m on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116) over time. The same applies to the third and fourth pressure control devices (118D; 120D).

As shown in Fig. 1, the target channel (102) may be included in a microfluidic chip (124) which may be used to transport or move fluid (including respective aqueous samples or compartments) from one location to another within the chip (124).

The microfluidic device (100) shown in Fig. 1 may be used, for example, in the field of cancer therapy and/or for combinatorial drug screening. Particularly, the microfluidic device (100) according to embodiments described herein allows a high level of automation with different options for the primary readout, such as fluorescence spectroscopy, sequencing, imaging and the like.

In addition, the one or more (combinatorial) microcompartments generated, e.g. using the microfluidic device (100) shown in Fig. 1, can be used for high throughput screening applications. While about 20,000 cells are needed in conventional systems, e.g. using a microtiter plate, to test a single treatment option on cancer cells only about 100 cells are needed in the microfluidic device (100).

Furthermore, compared to a Braille valve microfluidic system in which cells are continuously injected either to the droplet maker or to the waste, a valve less setup consumes cells only during the production of droplets (no cells go to the waste). Therefore, the overall cell consumption is about 3 times less.

As discussed in further detail below, the microfluidic device (100) allows to reduce or prevent microfluidic backflow, e.g. unintended flows of liquid, fluid or aqueous samples from one sample injection channel (114; 116; 118; 120) or one combinatorial microcompartment (200A; 200B) into one or more other sample injection channel (114; 116; 118; 120) or another combinatorial microcompartment (200A; 200B). To this end, cross-contamination between, e.g. neighboring channels, microcompartments and/or combinatorial microcompartments can be a reduced and the liquids, fluids or aqueous samples can be kept separate. As such, unintentional mixing of samples may be reduced or avoided. Also, due to the fact that the microfluidic device (100) includes at least one hydrodynamic resistor, overshooting and overregulation of pressures in sample injection channels due to sudden flow rate changes and/or pressure changes are balanced out, reduced, prevented or minimized.

Fig. 2A shows a schematic view of a first sample injection channel (114) and a second sample injection channel (116) connected to the target channel (102). Fig. 2A shows injection of a first sample species (A) and a second sample species (B) without using a hydrodynamic resistor (not shown).

As can be seen in Fig. 2A, a first sample injection channel (114) is connected with its sample injection channel junction end to the target channel (102), e.g. of a microfluidic chip (124). A second sample injection channel (116) is connected with its sample injection channel junction end to the target channel (102), e.g. of the microfluidic chip (124). In Fig. 2A, the first sample injection channel (114) is an injecting channel, i.e. currently injecting the first sample species (A) in the target channel (102), e.g. during a first time, while the second sample injection channel (116) is a non-injecting channel, i.e. currently not injecting the second sample species (B), e.g. during the first time. This can be seen in the resulting microcompartments (202) of Fig. 2A, which, as an example, only include sample species (A).

As can be further seen in Fig. 2A, the first sample species (A) does not only end up in the target channel (102), e.g. to form microcompartments (202) therein, but also in the second sample injection channel (116), which is intended for injection of the second sample species (B). Accordingly, this backflow results in significant cross-contamination, e.g. of the created microcompartments (202), particularly, when the second sample injection channel (116) becomes an injecting channel, e.g. during a second time being different from the first time, i.e. when it is used for injecting the second sample species (B), since remaining traces of the first sample species (A) will be co-injected therewith.

Although not illustrated in Fig. 2A, such a backflow and/or cross contamination may not only flow into a (neighboring) sample injection channel which is currently non-injecting, e.g. the second sample injection channel (116), but even also into the second sample species reservoir (106). The same correspondingly applies to the other non-injecting sample injection channels present in the microfluidic device (100) shown in Fig. 1.

As can be further seen in the embodiment of Fig. 2A, the generated microcompartments (202) are not equally sized and have a non-uniform shape, e.g. the microcompartments (202) are bulky, deformed or distorted, which is caused by pressure differences related to the backflow seen in Fig. 2A and/or pressure/flow rate differences related to overshooting or overregulating due to sudden flow rate changes. With respect to a combinatorial microcompartment (s. for example combinatorial microcompartment (200A; 200B) of Fig. 2C), this may also result in a cross-contamination combinatorial microcompartment, i.e. where the two species are mixed.

Fig. 2B shows a schematic view of a first sample injection channel (114) and a second sample injection channel (116) which are valvelessly connected to the target channel (102) as in Fig. 2A. Fig. 2B shows injecting the first sample species (A) and the second sample species (B) using a hydrodynamic resistor according to embodiments of the present disclosure (not shown).

In the embodiment of Fig. 2B, the first sample injection channel (114) is valvelessly connected with its sample injection channel junction end to the target channel (102), e.g. of a microfluidic chip (124). Likewise, the second sample injection channel (116) is valvelessly connected with its sample injection channel junction end to the target channel (102), e.g. of the microfluidic chip (124). The sample injection channel junction end (114B; 116B) of the first sample injection channel (114) and/or the second sample injection channel (116) include at least one hydrodynamic resistor (not shown).

In Fig. 2B, the first sample injection channel (114) is an injecting channel, i.e. currently injecting the first sample species (A) in the target channel (102), e.g. during a first time, while the second sample injection channel (116) is a non-injecting channel, i.e. currently not injecting the second sample species (B), e.g. during the first time. This can be seen in the resulting microcompartments (202) of Fig. 2B, which, as an example, only include sample species (A).

As can be seen in Fig. 2B, the first sample species (A) does not end up in the second sample injection channel (116), which is intended for injection of the second sample species (B). This is due to the fact that a non-injection pressure P_m is maintained on the second sample species reservoir (106) of the sample injection channel (116), which is currently non-injecting, e.g. during the first time and the use of hydrodynamic resistor in connection with the valveless connection. As a result, "backflow" of sample species (A) in the second sample injection channel (116) is avoided. This reduces or prevents cross-contamination, particularly, when the second sample injection channel (116) becomes an injecting channel, e.g. during a second time being different from the first time, i.e. when it is used for injecting the second sample species (B), since no remaining traces of the first sample species (A) will be co-injected therewith. Also, pressure and/or flow rate differences related to overshooting or overregulating due to sudden flow rate changes can be balanced out, reduced, prevented or minimized.

In addition, when compared to the generated microcompartments (202) of Fig. 2A, the generated microcompartments (202) of Fig. 2B, are more equally sized and have a more uniform shape, e.g. continuous, which is a result of backflow being reduced or avoided, i.e. due to maintaining a non-injection pressure P_m on the sample species reservoir (106) of non-injecting second sample injection channel (116) in combination with using a hydrodynamic resistor.

It should be noted that although Figs. 2A and 2B depict generation of a microcompartment (202), these embodiments are not so limited, i.e. the first and second sample injection channels (114; 116) may likewise be used for generation of a combinatorial microcompartment (200A; 200B) as shown in Fig. 2C, e.g. including mixtures of the first sample species (A) and the second sample species (B).

Fig. 2C shows a schematic view of different microcompartments created within a carrier phase of a target channel (102) according to embodiments of the present disclosure, e.g. generated maintaining a non-injection pressure P_m on the sample species reservoir (106) of non-injecting second sample injection channel (116) and using at least one hydrodynamic resistor according to embodiments of the present disclosure.

In particular, Fig. 2C shows two combinatorial microcompartments (200A; 200B) comprising at least two sample species, e.g. of a different composition and a microcompartment (202) comprising only one sample species. The first combinatorial microcompartment (200A) includes the first sample species (A) and the second sample species (B). The second combinatorial microcompartment (200B) includes a plurality of sample species (A-D), including the first sample species (A), the second sample species (B), a third second sample species (C) and a fourth second sample species (D). The 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 there are at least two sample species included.

The two combinatorial microcompartments (200A; 200B) and the microcompartment (202) of Fig. 2C have been produced, e.g. by the microfluidic device (100) shown in Fig. 1 and therefore have a uniform shape and/or are equally sized. As can be further seen with respect to the combinatorial microcompartments (200A; 200B), no cross-contamination between the different sample species (A- D) is present within the created combinatorial microcompartments (200A; 200B). Rather, the sample species (A-D) are clearly separated, divided or distinct from each other.

Fig. 3 shows a flow diagram of a method (300) according to embodiments described herein. As shown in Fig. 3, the method (300) is for providing one or more combinatorial microcompartments (200A; 200B) each comprising at least two sample species (A; B). The method comprises injecting (302), into a continuous flow of a carrier phase of a target channel (102), a first sample species (A) from a first sample species reservoir (104) via a first sample injection channel (114) to create a microcompartment (202), and injecting (304), into the created microcompartment (202), at least one second sample species (B) from a second sample species reservoir (106) via a second sample injection channel (116) to create a combinatorial microcompartment (200). The method further comprises providing (306) a resistance within the first in the first injection channel (114) and/or the second injection channel (116) using at least one hydrodynamic resistor. According to a preferred embodiment, the first sample species and the at least one second sample species are barcode oligonucleotides or set of components thereof. Preferably, the barcode oligonucleotides or set of components thereof of the first sample species are different from the barcode oligonucleotides or set of components thereof of the at least one second sample species.

The resistance may be a hydrodynamic resistance. The resistance may be provided during i) injecting (302) of the first sample species (A) and/or injecting (304) the second sample species (B) and ii) non-injecting of the first sample species (A) and/or non-injecting the second sample species (B).

The first sample species (A) and the second sample species (B) may be injected by applying an injection pressure Pi to the respective first species reservoir (104) and the second species reservoir (106), while a non-injection pressure P_m being lower than the injection pressure Pi may be maintained on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116).

The method may optionally include the step of repeating (306) of the steps (302; 304) to create another combinatorial microcompartment in order to create a sequence of combinatorial microcompartments within the carrier phase of a target channel (102).

In the method according to Fig. 3, injecting the second sample species (B) may occur sequentially or concomitantly to injecting the first sample species (A). That is, during a first time, both the first sample species (A) and the second sample species (B) may be injected. Alternatively, the first sample species (A) may be injected at a first time and the second sample species (B) may be injected at a second time being different than the first time, or vice versa.

In the method of Fig. 3, the first sample species (A) does not end up in the second sample injection channel (116), which is intended for injection of the second sample species (B). This is due to the fact that the non-injection pressure P_m is maintained on the second sample species reservoir and a resistance within the first injection channel (114) and/or the second injection channel (116) using at least one hydrodynamic resistor is provided. As a result, "backflow" of sample species (A) from the first sample injection channel (114) into the second sample injection channel (116) is reduced or avoided. Also, pressure and/or flow rate differences related to overshooting or overregulating due to sudden flow rate changes can be balanced out, reduced, prevented or minimized. Furthermore, this avoids cross-contamination, e.g. when the second sample injection channel (116) is used for injecting the second sample species (B), as no remaining traces of the first sample species (A) will be coinjected therewith.

Fig. 4A shows a pressure profile during sample injection without maintaining a non-injection pressure P_m on the non-injecting sample species reservoir. The x-axis of Fig. 4A shows a time in seconds, while the y-axis of Fig. 4A shows a pressure in mbar.

In the diagram of Fig. 4 A, it can be clearly seen that, without maintaining a non-injection pressure P_m irregular pressure profiles or pressure peaks follow or occur, which indicate a high probability of undesired backflow and, hence, cross contamination. A (combinatorial) microcompartment created therefrom may be less uniform and not equally sized (s. for example the microcompartments created in Fig. 2A). This may also cause overshooting or overregulating due to sudden flow rate changes.

Fig. 4B on the other hand, shows a pressure profile during sample injection maintaining a non- injection pressure P_m and using a hydrodynamic resistor according to embodiments of the present disclosure. The x-axis of Fig. 4B shows a time in seconds, while the y-axis of Fig. 4B shows a pressure in mbar.

As can be seen, the pressure profiles of pressure peaks are rather uniform when compared to the measurements shown in Fig. 4A, resulting in the formation of equally sized and non-contaminated (combinatorial) microcompartments (s. for example Fig. 2B or Fig. 2C). With respect to microcompartments (s. for example Fig. 2C), this may also result in reduction or prevention of crosscontamination as well as to a reduction in overshooting or overregulating due to sudden flow rate changes.

Note that in Fig. 4B, the linear increase in pressure is due to increasing back pressure in, e.g. the target channel (102) (s. Fig. 1) with increasing sample numbers.

Fig. 5A shows a flow rate profde during sample injection without maintaining a non-injection pressure P_m. The x-axis of Fig. 5A shows a time in seconds, while the y-axis of Fig. 5A shows a flow rate in pl/min.

It is desirable to have the flow rate profiles shown in Fig. 5 A to be as similar to each other as possible, because doing so will result in the (combinatorial) microcompartments being created therefrom to be uniform and/or equally sized. This also reduces cross contamination.

It should be noted that in the diagram of Fig. 5A, the flow rates on the y-axis include positive values and negative flow rate values. As can be seen, for the second sample species B (which corresponds to sample 2 in the diagram of Fig. 5A), a negative (smaller than zero) flow rate is observed. Such a negative flow rate indicates undesired back flow and cross-contamination between neighboring sample injection channels as mentioned above (s. for example Fig. 2A).

As can be seen in Fig. 5 A, "backflow" indicated by the negative flow rates between neighboring sample injection channels is observed causing significant cross-contamination between sample species (A) (which corresponds to sample 1 in the diagram of Fig. 5A) and sample species (B) (which corresponds to sample 2 in the diagram of Fig. 5A).

Fig. 5B on the other hand shows a flow rate profile maintaining a non-injection pressure P_m and using a hydrodynamic according to embodiments of the present disclosure. The x-axis of Fig. 5 A shows a time in seconds, while the y-axis of Fig. 5A shows a flow rate in pl/min.

As can be seen in Fig. 5B, negative flow rates related to backflow between neighboring channels is significantly reduced (substantially zero) by which cross-contamination between neighboring sample injection channels is avoided. To this end, more uniform flow rates are achieved resulting in the formation of equally sized and non-contaminated (combinatorial) microcompartments (s. for example Fig. 2B and Fig. 2C). With respect to combinatorial microcompartments, this may also result in reduction or prevention of cross-contamination (s. for example Fig. 2C).

Fig. 5C shows another flow rate profile of a microfluidic chip maintaining a non-injection pressure P_m, but without using hydrodynamic resistors. The x-axis of Fig. 5C shows a time in seconds, while the y-axis of Fig. 5C shows a flow rate in pl/min.

Fig. 5C further shows how the flow rate changes over time when three samples (Sample 1-3) are being injected. At the beginning of injection, a sudden flow rate change occurs, e.g. overshooting, where the flow rate briefly exceeds a desired or target flow rate before settling back or returning to the target flow rate. That is, in Fig. 5C, the overshoot peaks represent instances where the flow rate exceeds the desired flow rate. These overshoots or peaks can have negative impacts on the accuracy and consistency of flow rates in microfluidic systems, leading to backflow and cross contamination.

In order to measure cross-contamination, as it is the case in Figs. 6A, 6B and 6C, a microcompartment was generated using samples of different fluorophores for injection, such as a green, an orange and a blue fluorescent dye. A fluorescent dye is a type of molecule that absorbs light at one wavelength and re-emits it at a longer wavelength, producing a visible fluorescence in different colors, e.g. depending on the fluorophore used. These dyes may be used to visualize specific structures within a microcompartment or track the movement of molecules within cells. In this case, the fluorescence dyes can be used to detect cross-contamination within a (combinatorial) microcompartment. Fluorescent dyes can be excited with a specific light source to produce a bright and easily detectable fluorescence signal, which may be indicative of cross-contamination.

Fig. 6A shows a measurement of cross contamination in a combinatorial microcompartment comprising at least two sample species (A; B) created without maintaining a non-inj ection pressure Pm.

In the example of Fig. 6A, cross-contamination is monitored while injecting a green, an orange and a blue fluorescent dye in a microcompartment to create a combinatorial microcompartment (200A; 200B), i.e. comprising at least two sample species. In the embodiment of Fig. 6A, the combinatorial microcompartment is created without maintaining a non-injection pressure P_m. The x- axis of Fig. 6A shows a time in seconds, while the y-axis of Fig. 6A shows a fluorescence intensity in arbitrary units, e.g. a unit of measurement that is not based on any internationally recognized standard, but rather on an arbitrary scale chosen by the experimenter including values ranging from 0 to 6 A.U.

For fluorescence analysis Z-factors may be used. A Z-factor may be a statistical measure used to evaluate the quality of an assay or, in this case, a sample species within a combinatorial microcompartment. The Z-factor may therefore correspond to a measure indicating cross contamination between at least two species in the combinatorial microcompartment, as crosscontamination alters the fluorescence readings and, e.g. decreases the Z-factor. Generally, Z-factors of 0.5 to 1.0 are considered good (e.g. having no or reduced cross-contamination), while values lower than 0.5 or even negative Z-factor values indicate cross-contamination between species within the combinatorial microcompartments.

As can be seen in the measurements shown in Fig. 6A, the fluorescence response is rather non- uniform indicating cross-contamination between the fluorescent dyes. The Z-factor measured for the green fluorescence dye was -4.03, the Z-factor measured for the orange fluorescence dye was -0.726 and the Z-factor measured for the blue fluorescence dye was -1.462.

In the measurements shown in Fig. 6A, the Z-factors measured in a combinatorial microcompartment created without maintaining a non-injection pressure P_m are negative, indicating significant cross contamination between within the combinatorial microcompartments. In the embodiment of Fig. 6B, cross contamination is monitored while injecting a green, an orange and a blue fluorescent dye in a microcompartment to create a combinatorial microcompartment (200A; 200B), i.e. comprising at least two sample species. In the embodiment of Fig. 6B, the combinatorial microcompartment was created maintaining a non-injection pressure P_m and using a hydrodynamic resistor according to embodiments of the present disclosure. The x-axis of Fig. 6B shows a time in seconds, while the y-axis of Fig. 6B shows a fluorescence intensity in arbitrary units, e.g. a unit of measurement that is not based on any internationally recognized standard, but rather on an arbitrary scale chosen by the experimenter including values from 0 to 1 AU.

As can be seen in the measurements shown in Fig. 6B, the fluorescence response is rather uniform indicating significantly reduced cross-contamination between the fluorescent dyes when compared to measurements shown in Fig. 6A. The Z-factor measured for the green fluorescence dye was 0.612, the Z-factor measured for the orange fluorescence dye was 0.74 and the Z-factor measured for the blue fluorescence dye was 0.714.

In the measurements shown in Fig. 6B, the Z-factors measured in a combinatorial microcompartment created by maintaining a non-injection pressure P_m and using a hydrodynamic resistor according to embodiments of the present disclosure are not only positive values, but also within the range of 0.5-1.0 indicating that cross contamination within the combinatorial microcompartments is significantly reduced or avoided, when compared to the measurement shown in Fig. 6A.

The combinatorial microcompartment measured in Fig. 6B was created using the microfluidic device (100) shown in Fig. 1 and/or the method shown in Fig. 3.

Fig. 7A shows flow rate data (i.e. backflow and overshoot) recorded with a microfluidic chip without using a hydrodynamic resistor while Fig. 7B shows flow rate data (i.e. backflow and overshoot) recorded with a microfluidic chip using a hydrodynamic resistor both while maintaining a non-injection pressure P_m according to embodiments of the present disclosure.

Fig. 8A shows a particularly preferred embodiment of a microfluidic chip according to the present invention, comprising hydrodynamic resistors implemented as bendings or turns of about 180° in the respective channels. Fig. 8B shows a microfluidic device without hydrodynamic resistors.

When comparing both, the recorded backflow and/or the recorded overshoot, it can be seen that with a microfluidic chip that incorporates a hydrodynamic resistor, the occurring backflow and/ or overshoot is significantly lower as compared to a microfluidic chip that does not incorporate a hydrodynamic resistor.

According to a further aspect, the present invention provides a method for co-localizing an entity with a barcode oligonucleotide or set of components thereof in a microfluidic device. The barcoding of cellular mRNAs with unique identifiers, such as incorporation of unique nucleotide sequences during cDNA synthesis, is widely used in genomic applications (A. E. Saliba, A. J. Westermann, S. A. Gorski, J. Vogel, Single-cell RNA-seq: advances and future challenges. Nucleic Acids Research 42, 8845 (2014)), and a respective method is described e.g. in WO 2016/207441, which is herein incorporated by reference in its entirety.

The microfluidic device to be used in the method for co-localizing an entity with a barcode oligonucleotide or set of components thereof is preferably a device as disclosed herein. With respect to Fig. 1, the device preferably comprises a target channel (102), which preferably comprises a target channel fluid such as a carrier phase, a first sample species reservoir (104) comprising a first sample species (A) in form of a first barcode oligonucleotide or set of components thereof, and at least a second sample species reservoir (106) comprising a second sample species (B) in form of a second barcode oligonucleotide or set of components thereof. The carrier phase is preferably an immiscible phase, more preferably an immiscible fluid. The first and the at least one second barcode oligonucleotide or set of components thereof can be the same or different. According to a preferred embodiment, the first and the at least one second barcode oligonucleotide or set of components thereof are different from each other. It will be appreciated that the device may comprise more than just the first and second sample species reservoir with the first and second barcode oligonucleotide or set of components thereof, such as a plurality of three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, or more than 30 such as 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, or 200 sample species reservoirs with a respective number of barcode oligonucleotides or set of components thereof. In the embodiment of Fig. 1, a third and a fourth sample species reservoir (108, 110) are shown. According to a preferred embodiment, the respective barcode oligonucleotides or sets of components thereof in the plurality of sample species reservoirs are different from each other.

The device preferably further comprises a first sample injection channel (114) comprising 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 device likewise comprises at least a second sample injection channel (116) comprising a sample injection channel reservoir end (116A) connected to the second sample species reservoir (106), and a sample injection channel junction end (116B) connected to the target channel (102). It will be appreciated that the device may comprise more than just the first and second sample injection channel comprising a sample injection channel reservoir end and a sample injection channel junction end, such as three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, or more than 30 such as 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 comprising a sample injection channel reservoir end and a sample injection channel junction end. In the exemplary embodiment of Fig. 1, a third and a fourth sample injection channel (118, 120) with respective injection channels (118, 120) are shown. The sample injection channel junction ends (114B, 116B) of the first sample injection channel (114) and of the at least one the second sample injection channel (116) are valvelessly connected to the target channel (102). The term "valvelessly" is to be understood as defined herein above. The term preferably means that no valve is provided at the junction of the channels, and more preferably that no valve is provided anywhere between the sample injection channels and their junctions to the target channel and to the respective sample species reservoir.

The injection channel according to one embodiment is an integral channel. According to an alternative embodiment, the injection channel can be made of two or more different sections. In one such embodiment, the injection channel has a first section that is connected to the sample species reservoir, e.g. a tubing, and a second section that is connected to the target channel, e.g. a microchannel. The first section and the second section can be interconnected by way of an adapted. In cases where the first section and the second section have a different diameter, the adapter may have a tapering shape allowing a connection of two channel sections having a different diameter and/or being made of different materials.

According to a particular preferred embodiment, each sample injection channel comprises at least one hydrodynamic resistor. A hydrodynamic resistor is as defined herein and is preferably implemented as a specific channel geometry in a portion of a channel, more preferably as a plurality of turns or bendings in a section of the respective channel. According to a particularly preferred embodiment, a hydrodynamic resistor is a sequential arrangement of a plurality of turns or bendings in the channel of between about 170 and 190°, more preferably of about 180°, as exemplified e.g. in Fig. 8A. The plurality of turns or bendings of a hydrodynamic resistor preferably comprises between 4 and 40 tums/bendings, more preferably between 6 and 30, 8 and 20, 10 and 18, or 12 and 16 tums/bendings. According to a particularly preferred embodiment, in the sequential arrangement of a plurality of turns or bendings in the channel, the tums/bendings are evenly distributed along the channel section that serves as the hydrodynamic resistor. According to a further preferred embodiment, the distance from one turn or bending in the hydrodynamic resistor to the following next turn or bending in the hydrodynamic resistor is essentially the same. It will be appreciated that an individual channel may comprise more than just one hydrodynamic resistor such as two, three, four, five, six, seven, eight, nine, ten or more hydrodynamic resistors.

The method for co-localizing an entity with a barcode oligonucleotide or set of components thereof in a microfluidic device comprises as a first step the feeding of the entity into a target channel. The target channel is preferably a target channel (102) of one of the devices disclosed herein. The entity is preferably a substance such as a drug or a particle preferably comprising a nucleic acid, preferably DNA and/or RNA. According to a preferred embodiment, the particle is a cell. According to a further preferred embodiment, the particle a drug.

In a second step, the method comprises passing the entity past a first sample injection channel and at least one second sample injection channel, wherein one of the sample injection channels feeds a barcode oligonucleotide or set of components thereof from a first or at least one second sample species reservoir to the target channel via the respective sample injection channel and respective sample injection channel junction ends. This is done by applying an injection pressure (Pi) to the respective one of the first and at least one second sample species reservoirs, thereby injecting the barcode oligonucleotide or set of components thereof into the target channel. The first and the at least one second sample injection channel, the first and the at least one second sample species reservoir, and the respective injection channel junction ends are preferably as defined herein. Thus, according to a preferred embodiment, the second step comprises passing the entity past the first sample injection channel (114) and the at least one second sample injection channel (116), wherein one of the sample injection channels feeds a barcode oligonucleotide or set of components thereof from the sample species reservoir (104, 106) to the target channel (102) via the respective sample injection channel (114, 116) and the respective sample injection channel junction end (114B, 116B) by applying an injection pressure (Pi) to the respective one of the first and the at least one second sample reservoirs (104, 106), thereby injecting the barcode oligonucleotide or set of components thereof into the target channel (102).

In an optional third step, the method comprises repeating the second step. This repetition is not limited to a single repeat but may involve a plurality of repeats, which may be chosen depending on individual needs. When repeating the second step, according to a preferred embodiment, the other one of the first sample injection channel and the at least one second sample injection channel feeds a barcode oligonucleotide or set of components thereof from the respective other one of the first and the at least one second sample species reservoir to the target channel. Thus, according to a preferred embodiment, when in the second step the barcode oligonucleotide or set of components thereof was fed from the first sample species reservoir via the first sample injection channel into the target channel, the barcode oligonucleotide or set of components thereof in the third step is fed from the at least one second sample species reservoir via the at least one second sample injection channel into the target channel. The third step can be repeated multiple times with or without alternating feeding of barcode oligonucleotides or set of components thereof from the first and the at least one second sample species reservoir, depending on the circumstances and individual needs. A specific order of repeats of method step (ii) with feedings of the barcode oligonucleotides or set of components thereof from the same or different sample species reservoir can be set-up to satisfy individual needs. It will therefore be appreciated that the present invention is not limited to any specific order of feedings from any specific sample species reservoir but may be accordingly adapted. This also applies to the number of sample species reservoir and respective injection channels.

According to a particularly preferred embodiment of the invention, the method further comprises applying a non-injection pressure (P_m) during a non-injection phase to the first sample species reservoir (104) and/or to the at least one second sample species reservoir (106). The non- injection pressure P_m is preferably larger than the pressure of the environment (P atmosphere). The pressure of the environment or ambient pressure refers to the pressure acting on the device, when no other pressure such as an injection pressure Pi and a non-injection pressure P_m are applied, and in particular to the pressure acting on the sample species reservoirs. It may thus be the same as the pressure surrounding the device. However, if the pressure within the device acting on the components of the device and in particular on the sample species reservoirs differs from that of the pressure surrounding the device, the pressure of the environment refers to the pressure acting on the sample species reservoirs. According to a preferred embodiment, Pi is larger than P_m, and P_m is larger than P_atmos_Phere. According to a particularly preferred embodiment, the ratio between ?! and P_m is between about 1.25 and about 2.5, preferably between about 1.5 and about 2.25, between about 1.75 and about 2.0, most preferably of about 2.0.

According to a preferred embodiment, P_m is between about 5-5600 mbars and Pi is between about 10-7000 mbars, more preferably P_m is between about 50-3200 mbars and Pi is between about 60- 4000 mbars, most preferably P_m is between about 100-1600 mbars and Pi is between about 125-2000 mbars According to the present invention, all pressures indicated are above (i.e. in addition to) ambient pressure or P_atmos_Phere, in other words the pressure values indicated are gauge pressure values.

In accordance with the invention, when an injection pressure Pi is applied to one sample species reservoir, at the same time a non-injection pressure P_m is applied to the other sample species reservoirs so as to prevent any sample species from the injecting sample species reservoirs and the injecting channels to enter the non-injecting channels. It will be understood that depending on the respective practical use of the device and the method, the injection pressure Pi is applied to more than just one sample species reservoir simultaneously in order to allow for controlled mixing of barcode oligonucleotide or set of components thereof from different sample species reservoirs. However, in most cases, an injection pressure Pi is applied to only a single sample species reservoir, while at the same time, i.e. for as long as the injection pressure Pi is applied to one sample species reservoir, a noninjection pressure P_m is applied to all other sample species reservoirs.

According to a preferred embodiment of the invention, the non-injection pressure P_m on each sample species reservoir of each non-injecting sample injection channel is increased over time. If the non-injection pressure P_m is increased overtime, it nevertheless does not exceed the injection pressure Pi, and the ratio between Pi and P_m is preferably maintained to be between about 0.25 and about 2.5.

According to a preferred embodiment of the invention, the injection pressure (Pi) and the non- injection pressure (P_m) are generated by pressurizing the first sample species reservoir (104) and/or the at least one second sample species reservoir (106). This is preferably done under the control of a pressure control devices such as pressure control devices (114D, 116D, 118D, 120D) as exemplarily shown in Fig. 1. A preferred pressure control device is microfluidic flow regulator such as a Microfluidic Flow Control System (MFCS).

According to one embodiment of the invention, the feeding of the barcode oligonucleotide or set of components thereof from the sample species reservoir to the target channel is controlled by one or more detection means or sensors. The detection means or sensors are as defined above and are exemplarily shown with reference numerals 114C, 116C, 118C, and 120C, respectively, in Fig. 1. It will be appreciated that the arrangement shown in Fig. 1 is just one example and thus not limiting the invention. Accordingly, detection means or sensors can be placed at other locations in the device alternatively or additionally, for example, at the beginning and/or at the end of respective injection channels, on the target channel, or at or in the vicinity of the sample species reservoirs, such as for example between the sample species reservoir and a connection of the samples species reservoir to the outside of the microfluidic device (e.g. to a fill line for filling the sample species reservoir with the sample species). Detection means for detecting particles (also referred to as particle detection means) and other components in a microfluidic channel are well known in the art and include light sensors, for example photomultiplier tubes, CMOS or CCD cameras, or detection electrodes. Generally, the detection means is suitable for detecting a particle and/or a label attached to the particle, in particular a fluorescent label, and may use fluorescence or laser spectroscopy, imaging, impedance or magnetic measurements for detection. According to a preferred embodiment, the detection means is a sensor, preferably a flow sensor, such as a highly sensitive microfluidic flow sensor based on e.g. microelectromechanical systems (MEMS) technology. A further preferred detection means is e.g. a high precision thermal flow sensor which is particularly useful for monitoring the flowrate of liquids and cells. A further sensor useful in the context of the present invention is for example a coriolis mass flow sensor. According to a preferred embodiment, the sensor is a flow sensor detecting fluids that are fed into the sample species reservoirs. For example, such flow sensor detects the amount of gas, such as air, that follows the amount of sample species fed from the sample species reservoir into the respective injection channel. In other words, for each volume of sample species fed from the sample species reservoir into the injection channel and subsequently into the target channel, a respective volume of fluid such as air enters the sample species reservoir. The flow sensor is arranged such that it is capable of detecting this volume of fluid entering the sample species reservoir. In addition or alternatively, the device may comprise detection means or sensors that are arranged at one end and/or at the other end of respective injection channels, i.e. at the injection channel junction to the sample species reservoir and/or at the injection channel junction to the target channel, and/or on one or more of the injection channels themselves. Arrangements of detection means or sensors at or on or near the sample species reservoirs, the injection channels and/or the junctions of the injection channels are also referred to as an upstream arrangement or upstream detection. Preferred means for upstream detection are flow sensors such as the ones described herein.

In addition to or as an alternative to the detection means or sensors and specifically the upstream arrangement described above, the device may comprise detection means or sensors that are arranged at or on the target channel. Such an arrangement of detection means or sensors is referred to herein as a downstream arrangement. Particularly preferred detection means or sensors for the downstream arrangement or downstream detection are particle detection means, in particular particle detection means allowing detection of solid particles such as e.g. cells. Particularly preferred detection means for downstream detection are those capable of imaging or of detecting fluorescence signals. According to a preferred embodiment, the barcode oligonucleotide or set of components thereof fed into the targeting channel while an entity passes the sample injection channel junction end(s) is predetermined or recorded.

For controlling the feeding of the barcode oligonucleotide or set of components thereof from the sample species reservoir to the target channel, the detection means or sensors preferably trigger applying the non-injection pressure P_m or the injection pressure Pi on respective ones of the sample species reservoir upon detecting an entity. According to a preferred embodiment, the entity is detected in the target channel (102).

Applying a non-injection pressure P_m or an injection pressure Pi on the sample species reservoirs can also or alternatively be triggered by time. Control by time can be based on the time point the entity is fed into the target channel, the distance it needs to travel to arrive at and leave the sample injection channel junction end, as well as the flow speed of the entity.

According to a preferred embodiment of the invention, the method for co-localizing an entity with a barcode oligonucleotide or set of components thereof in a microfluidic device comprises repeating all steps one or more times with a further entity after a first entity is fed into the targeting channel prior to this further entity. For each entity passing the respective sample injection channel junction end, a different sample reservoir is pressurized with an injection pressure P while the other sample reservoir(s) are pressurized with a non-injection pressure P_m. Preferably, the barcode oligonucleotide or set of components thereof fed into the targeting channel while an entity passes the sample injection channel junction end(s) is predetermined or recorded.

According to an embodiment in which a plurality of entities is co-localized with barcode oligonucleotides or components thereof, a preferred embodiment comprises repeating all steps one or more times while entities are constantly fed into the target channel, wherein different sample species reservoirs are pressurized subsequently with an injection pressure Pi preferably in a time-dependent manner. Preferably, the order of the barcode oligonucleotide or set of components thereof fed into the target channel is recorded.

The method preferably generates microfluidic droplets comprising the entity and the barcode oligonucleotide or components thereof. According to a preferred embodiment, the method further comprises fusing of microfluidic droplets comprising the entity and the barcode oligonucleotide or components thereof with a further microfluidic droplet, such as a microfluidic droplet comprising a further entity such as a cell. Alternatively, the further entity can be injected into the microfluidic droplets comprising the entity and the barcode oligonucleotide or components thereof.

The microfluidic droplets are created, handled and/or controlled in the microfluidic device as described herein. The generation of microfluidic droplets is based on the manipulation of continuous liquid flow through microfabricated channels. In the present invention, actuation of the liquid flow is implemented by e.g. (external) pressure sources, (external) mechanical pumps, (integrated) mechanical micropumps applying the non-injection pressure P_m or the injection pressure Pi on the sample species reservoirs.

Microfluidic droplets can be generated by generating independent compartments using e.g. two-phase microfluidics, in which aqueous droplets surrounded by an immiscible oil phase serve as closed vessels, as described e.g. in WO 2016/207441.

According to one embodiment, the method may additionally comprise the step of injecting additional components or elements into the microfluidic droplet comprising the entity and the barcode oligonucleotide or set of components thereof. The additional components or elements preferably comprise reagents such as a reaction mixture, in particular one or more of a ligation mix, a primer extension mix, a reverse transcription mix (RT), a PCR mix, an RT-PCR mix, a transposition mix and/or a lysis buffer. Such additional components or elements are particularly suitable in cases where the microfluidic droplet comprises the entity such as a drug, the barcode oligonucleotide or components thereof, and at least one cell. According to one embodiment, the injection is preferably performed by injecting an aqueous phase containing the respective additional components or elements into said microfluidic droplet.

If a plurality of entities is to be co-localized with the barcode oligonucleotide or set of components thereof in the same microfluidic droplet, the control by time can be arbitrary or based on the frequency or density of the particle in the co-localizing channel.

According to one embodiment of the invention, the method further comprises detecting the entity and the barcode oligonucleotide or components thereof. The entity is preferably detected with the respective barcode oligonucleotide or components thereof associated therewith using any suitable means known to the skilled person. For example, the entity and the barcode oligonucleotide or components thereof can be detected by way of sequencing.

According to one embodiment, if the entity is a cell, the cell is preferably phenotyped after it has been co-localized with the barcode oligonucleotide or set of components thereof, such as for example in the target channel or after the cell has left the target channel. The phenotyping is preferably performed while the cell co-localized with the barcode oligonucleotide or set of components thereof is in the target channel. Alternatively, the phenotyping can be performed when the cell co-localized with the barcode oligonucleotide or set of components has left the target channel. In cases where a microfluidic droplet comprising the entity and the barcode oligonucleotide or components thereof additionally comprises a cell as described herein above, and optionally additional elements or components in the form of (reaction) reagents, the phenotyping is carried out after an incubation that allows the reagents to perform the desired reaction, such as lysis and/or PCR. The skilled person may readily select respective incubation parameters for the desired reaction to take place. Phenotyping may comprise the detection of any biophysical or biochemical property of the cell, such as size, shape, morphology, staining, for example immunostaining, ligand binding etc. This can be achieved by applying imaging techniques, for example bright field or fluorescence imaging, spectroscopy including fluorescence spectroscopy etc. The phenotyoping allows, for instance, to correlate a certain cell phenotype, in particular of a single cell, with its transcriptome or a genetic aberration such as a mutation, or the effect a certain drug has on a specific cell or group of cells.

According to a further embodiment, the microfluidic device may additionally comprise one or more further injection channels connected to the target channel, which may feed additional components into the target channel.

According to one embodiment, the present invention also provides a method for determining the effect of a drug on a cell or on the transcriptome of a cell or on a DNA amplificate from a cell, the method comprising barcoding the drug or the transcriptome of a cell or a DNA amplificate from a cell using the method of the invention as described herein. The sequence of the barcode in the drug or the barcoded transcriptome or the barcoded DNA amplificate is preferably indicative of the drug to which the cell is exposed, or to the cell which is exposed to the drug.

The microfluidic device and the method for co-localizing an entity with a barcode oligonucleotide or set of components thereof can be used for example for barcoding a substance such as a drug to be tested on a cell or a culture of cells. In such applications, the method comprises the steps of:

(i) co-localizing the substance, such as a drug, with a barcode oligonucleotide or components thereof and optionally a cell in a microfluidic droplet using the method as described herein;

(ii) introducing a cell into the microfluidic droplet of step (i) if no cell is co-localized in step (i);

(iii) injecting or fusing a reaction mixture comprising reagents e.g. for reverse transcription and PCR, and/or optionally for lysing the cell, into the microfluidic droplet of step (ii);

(iv) incubating the microfluidic droplet of step (iii), allowing the reaction mixture to optionally lyse the cell and/or to carry out a reaction, such as e.g. reverse transcription and PCR, optionally annealing the barcode oligonucleotide to RNA or DNA of the lysed cell in the microfluidic droplet.

According to one embodiment, the method may further comprise the steps of:

(v) inactivating any enzymes in the microfluidic droplet and/or disrupting the microfluidic droplet, in any order; and

(vi) analyzing the content of the microfluidic droplet, optionally by sequencing.

If step (ii) is present, the cell is preferably introduced into the microfluidic droplet by way of fusing the microfluidic droplet comprising the substance and the barcode oligonucleotide or components thereof with a further microfluidic droplet comprising the cell. Alternatively, the cell can be injected into the microfluidic droplets comprising the substance and the barcode oligonucleotide or components thereof.

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

Any or all steps of the method described above can be performed on a microfluidic device as described herein, i.e. on a microfluidic device in accordance with the present invention. According to a preferred embodiment, at least step (i) and optionally step (ii) are carried out on a device in accordance with the invention.

The reaction mixture may contain buffer(s), nucleotides, culture and/or expression media, and /or enzymes such as reverse transcriptase, polymerase, ligase, RNAse and/or DNAse inhibitors. According to a preferred embodiment, the reaction mixture contains an RT-PCR mix for performing RT-PCR, and optionally a lysis buffer for lysing the cell and an RNAse inhibitor for preventing RNA degradation. According to one embodiment, the reaction mixture is selected from the group consisting of an RT mix, an RT-PCR mix, a PCR mix, a transposition mix. Respective components of and their concentrations in the reaction mixture can be readily selected by the skilled person depending on the intended purpose.

It will be appreciated that the method of barcoding a substance such as a drug as described herein is not limited to the barcoding of a single substance or drug, but that also more than one substance or drug, such as a combination of different substances and/or drugs can be used in the context of the method described herein. In other words, the method can be used for barcoding e.g. a substance or drug combination such as a combinatorial therapy of different drugs.

The device used for the method of barcoding a substance such as a drug as described herein is preferably a microfluidic device as described herein. In the present setting of barcoding a substance such as a drug, the sample species reservoirs preferably not only contain the barcode oligonucleotide and/or components thereof, but at least one of the sample species reservoirs preferably contains the substance drug to be barcoded. If more than just a single substance or drug is to be barcoded, the different substances or drugs may be contained in a single species reservoir (e.g. as a pre-mix), or they may be contained in more than one sample species reservoir either individually or in other pre-mixed combinations. The method and the device of the present invention allows the combination of different substances and drugs in different combinations. It will be appreciated that also other entities such as a cell or DNA and/or RNA can be supplied from respective sample species reservoirs into the target channel via respective injection channels.

According to a particularly preferred embodiment and in particular in accordance with the method of barcoding a substance such as a drug to be tested on a cell or a culture of cells as described herein, sample species reservoirs of the microfluidic device comprise barcode oligonucleotides or sets of components thereof, the substance(s) and/or drug(s) to be barcoded, and optionally the cells on which the substance(s) and/or drug(s) shall be tested. The method may also comprise a subsequent step of phenotyping the cells as described herein. The phenotyping is preferably performed after the cell has left the target channel, more preferably after the incubation in step (iv).

The injection of the reaction mixture is preferably performed in a T-junction channel as exemplarily shown in Fig. 13C.

The microfluidic device and the method for co-localizing an entity with a barcode oligonucleotide or set of components thereof can also be used for example for barcoding the transcriptome of a cell, for barcoding a DNA amplificate from a cell, or for barcoding the genome of a cell. In such applications, the method comprises the steps of

(i) co-localizing a cell with a barcode oligonucleotide in a microfluidic droplet using the method as described herein,

(ii) lysing the cell in the microfluidic droplet, and

(iii) annealing the barcode oligonucleotide to RNA or DNA of the lysed cell in the microfluidic droplet,

(iv) carrying out a reverse transcription (RT), RT-PCR, PCR, or a transposition reaction using the annealed barcode oligonucleotide as primer(s) or transposable elements, respectively, in the microfluidic droplet or in an aqueous phase in which the microfluidic droplet is disrupted, thereby generating a barcoded transcriptome, DNA amplificate or genome. The RT mix, RT-PCR mix, PCR mix, or transposition mix, respectively, is preferably comprised in the target channel, in a microfluidic droplet fused to a microfluidic droplet generated in the method of the invention, or is comprised in an aqueous phase in which the microfluidic droplet is disrupted. According to one embodiment, the method may further comprise

(v) inactivating any enzymes of the microfluidic droplet comprising the barcode oligonucleotide and disrupting the microfluidic droplet, in any order, and

(vi) analyzing the barcoded transcriptome, barcoded DNA amplificate, or barcoded genome.

In step (v), enzymes are preferably inactivated to prevent enzyme activity, such as polymerase or reverse transcriptase activity, using components (primers, DNA, or RNA) of the microfluidic droplet with primers, DNA, or RNA which was/were not comprised in the same microfluidic droplet (e.g. which was comprised in a different microfluidic droplet if droplets are pooled). Enzymes can be inactivated, for example, by changing the temperature (e.g. heat inactivation), the pH, the buffer composition or by adding enzyme-specific inhibitors to achieve conditions under which the enzyme does not function.

Analyzing the barcoded transcriptome, barcoded DNA amplificate, or barcoded genome preferably comprises sequencing, e.g. by next-generation sequencing. Preferably, the barcoded transcriptomes, barcoded DNA amplificates, and/or barcoded genomes are sequenced together. This allows performing only one analysis step, such as one sequencing reaction, for all cells to be analyzed, instead of performing a separate analysis for each cell to be analyzed. The present invention further provides a method for correlating the phenotype of a single cell with its transcriptome, with a DNA amplificate derived from the cell or with its genome, comprising barcoding the transcriptome of a single cell, barcoding a DNA amplificate from a single cell or barcoding the genome of a single cell using the method of the invention as described herein, wherein the cell is further phenotyped, and wherein the sequence of the barcode in the barcoded transcriptome, amplificate or genome indicates the phenotype of the cell from which the transcriptome, DNA amplificate or genome is derived. The phenotyping can be performed as described herein above.

As show herein, the methods and devices of the present invention simplify the system architecture by employing a single sample species reservoir for generating diverse concentration gradients of the sample species stored in each reservoir. Previously, a separate reservoir for each reagent concentration was needed, mitigating the risks associated with contamination and device malfunction. Furthermore, the present invention allows to adjust concentrations continuously over a wide range rather than having only distinct concentrations of reagents, pre-filled in the reservoirs. This also reduces the overall consumption of reagents and accelerates the preparation process.

The present invention further pertains to the following items.

Item 1: A microfluidic device (100), comprising

- a target channel ( 102) comprising a target channel fluid, such as a carrier phase,

- a first sample species reservoir (104) comprising a first sample species (A) and at least a second sample species reservoir (106) comprising a second sample species (B),

- a first sample injection channel (114) comprising 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), and

- at least a second sample injection channel (116) comprising a sample injection channel reservoir end (116A) connected to the second sample species reservoir (106) and a sample injection channel junction end (116B) connected to the target channel (102); wherein

- the sample injection channel junction end (114B; 116B) of the first sample injection channel (114) and/or the second sample injection channel (116) are valvelessly connected to the target channel (102) and comprise at least one hydrodynamic resistor.

Item 2: The microfluidic device (100) of item 1, wherein the device is configured such that the first sample species reservoir (104) and the at least one second sample species reservoir (106) are capable of being pressurized, preferably during both injection and non-injection phases.

Item 3: The microfluidic device (100) of item 1 or 2, wherein the first sample injection channel (114) and the second sample injection channel (116) are separately connected with their respective sample injection channel junction ends (114B; 116B) to the target channel (102).

Item 4: The microfluidic device (100) of any one of claims 1-3, wherein the micro fluidic device is for producing one or more combinatorial microcompartment(s) (200A; 200B) comprising at least two sample species (A; B) within the carrier phase, and wherein the first sample species (A) and the second sample species (B) are chemically distinct, wherein the combinatorial microcompartment (200A; 200B) is a combinatorial droplet or combinatorial plug.

Item 5: The microfluidic device (100) of any one of items 1-4, wherein the carrier phase is an immiscible phase, wherein one or more combinatorial microcompartments (200A; 200B) flow into an outlet channel or a read-out channel.

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

Item 7: The microfluidic (100) device of any one of items 1-6, comprising a first pressure control device (114D) connected to the first sample species reservoir (104) and at least a second pressure control device (116D) the second sample species reservoir (106), preferably wherein the first pressure control device (114D) and the at least one second pressure control device (116D) are connected to a pressure source (120).

Item 8: The microfluidic device (100) of item 7, wherein the first pressure control device (114D) is configured to apply a first injection pressure Pi to

- the first sample species reservoir (104) to cause injection of the first sample species via the first sample injection channel (114), and the second pressure control device (116D) is configured to apply a second injection pressure Pi to

- the second sample species reservoir (106) to cause injection of the second sample species via the second sample injection channel (116).

Item 9: The microfluidic device (100) of item 8, wherein the first pressure control device (114D) is communicatively coupled to the first sensor (114C) of the first sample injection channel (114) and the second pressure control device (116D) is communicatively coupled to the second sensor (116C) second sample injection channel (116), wherein the first and second injection pressures Pi are applied on the basis of the monitored flow data of the liquid in the respective sample injection channel (114; 116).

Item 10: The microfluidic device (100) of any one of items 7-9, wherein the first and second pressure control devices (114D; 116D) are configured to maintain a non-injection pressure P_m on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116), preferable wherein the non-injection pressure P_m being lower than the first and second injection pressures Pi and following the relation: Pi > P_m > Patmosphere, and optionally, wherein the first and second pressure control devices (114D; 116D) are configured to increase the non-injection pressure P_m on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116) overtime. Item 11 : The microfluidic (100) device of item 10, wherein the non-injection pressure applied to each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116) causes a flow of liquid in the respective sample injection channel (114; 116)

Item 12: A method (300) for providing one or more combinatorial microcompartment(s) (200A; 200B) comprising at least two sample species (A; B), the method comprising:

- injecting (302), into a continuous flow of a carrier phase of a target channel (102), a first sample species (A) from a first sample species reservoir (104) via a first sample injection channel (114) to create a microcompartment (202);

- injecting (304), into the created microcompartment (202), at least one second sample species (B) from a second sample species reservoir (106) via a second sample injection channel (116) to create a combinatorial microcompartment (200);

- providing a resistance within the first injection channel (114) and/or the second injection channel (116) using at least one hydrodynamic resistor.

Item 13: The method of item 12, wherein the first sample species (A) and the second sample species (B) are injected by applying an injection pressure Pi to the respective first species reservoir (104) and the second species reservoir (106), while a non-injection pressure P_m being lower than the injection pressure Pi is being maintained on each sample species reservoir (104; 106) of each noninjecting sample injection channel (114; 116).

Item 14: The method of item 13, further comprising:

- increasing the non-injection pressure P_m on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116) over time, wherein the injection pressure Pi and the non-injection pressure P_m follow the relation: Pi> Pni > Patmosphere.

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

Item 16: A microfluidic chip (122) including one or more combinatorial microcompartment(s) (200) comprising at least two sample species (A; B) produced by the method of any one of items 10- 15.

Item 17: A use of a microfluidic device for combining a first sample species (A) and a second sample species (B) into a target channel fluid, the use comprising the steps of applying a non-injection pressure Pni during non-injection phases to a first sample species reservoir (104) comprising a first sample species (A), and applying a Pni during non-injection phases to at least a second sample species reservoir (106) comprising a second sample species (B), applying an injection pressure Pi to at least one of the first and second sample reservoir (104, 106) during an injection phase and thereby injecting the sample species A and/or B into the target channel fluid, wherein ?! > characterized in that, the device used comprises:

- a target channel (102) comprising the target channel fluid,

Item 18: A method for co-localizing an entity with a barcode oligonucleotide or set of components thereof in a microfluidic device according to any one of items 1 to 11, said method comprising:

(i) feeding the entity into the target channel (102);

(ii) passing the entity past the first sample injection channel (114) and the at least one second sample injection channel (116), wherein one of the sample injection channels feeds a barcode oligonucleotide or set of components thereof from the sample species reservoir (104, 106) to the target channel (102) via the respective sample injection channel (114, 116) and the respective sample injection channel junction end (114B, 116B) by applying an injection pressure (Pi) to the respective one of the first and second sample reservoirs (104, 106), thereby injecting the barcode oligonucleotide or set of components thereof into the target channel (102);

(iii) optionally repeating step (ii), preferably wherein when repeating step (ii) the other of the first sample injection channel (114) and the at least one second sample injection channel (116) feeds a barcode oligonucleotide or set of components thereof from the sample species reservoir (104, 106) to the target channel (102).

Item 19: The method of item 18, the method further comprising applying a non-injection pressure (P_m) during a non-injection phase to the first sample species reservoir (104) and/or to the at least one second sample species reservoir (106), wherein P_m is larger than the pressure of the environment Wherein

Item 20: The method of item 18 or 19, wherein the injection pressure (Pi) and the non- injection pressure (P_m) are generated by pressurizing the first sample species reservoir (104) and the at least one second sample species reservoir (106). Item 21: The method of any one of items 18 to 20, wherein the feeding of the barcode oligonucleotide or set of components thereof from the sample species reservoir (104, 106) to the target channel (102) is controlled by one or more detection means or sensors.

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

Item 23: The method of any one of items 18 to 22, wherein the method further comprises generating microfluidic droplets comprising the entity and the barcode oligonucleotide or components thereof, or wherein the method further comprises fusing of microfluidic droplets comprising the entity and the barcode oligonucleotide or components thereof with a further microfluidic droplet, and/or the method further comprises injecting reagents into the microfluidic droplet.

Item 24: The method of any one of items 18 to 23, wherein the method further comprises detecting the entity and the barcode oligonucleotide or components thereof.

While the foregoing is directed to embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Examples

Example 1: Generation of microcompartments hosting different concentrations of reagents

This example illustrates how the pressure-controlled microfluidic device in accordance with the invention can generate microfluidic microcompartments with varying concentrations of the reagents stored in the species sample reservoirs. The experimental setup comprises pressurized reservoirs filled with carrier oil, the fluorescent dye Resazurin (7-hydroxy-10-oxidophenoxazin-10- ium-3-one, sodium), and FreeStyle™ 293 Expression Medium. Microfluidic microcompartments were generated by injecting the different reagents into the channel network as shown in Fig. 9A. By adjusting the pressure applied to the reservoirs, the relative flow rates of the aqueous samples (fluorescent dye and buffer) were changed dynamically, resulting in different final concentrations of the dye within the microfluidic microcompartments, while injecting oil at a constant flow rate.

In the present example, microcompartments with a concentration of ! and % of a fluorescent dye loaded into the reservoir were generated. For condition A: The microcompartments were generated by injecting red fluorescent dye at 8 pl/min and buffer at 24 pl/min. For condition B: The microcompartments were generated by injecting the red fluorescent sample at 24 pl/min and the buffer at 8 pl/min. For both conditions A and B, P_m was between 100 and 600 mbars, and Pi was between 125 and 2000 mbars. Fluorescence signals of the samples were determined subsequent to the generation of the microfluidic microcompartments by laser spectroscopy, using a setup as described in Panwar, J., Autour, A. & Merten, C.A. (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). Results are shown in Fig. 9B. The measured fluorescence signal of microcompartments generated for condition A was: 0.068 [a.u.], and the measured fluorescence signal of microcompartments generated for condition B was: 0.209 [a.u.]. The ratio of microcompartment fluorescence signal of condition B to A was 3.077 [a.u.], matching the resulting concentrations of the dye ([a.u.] = arbitrary unit, describing a quantitative signal which amplitude depends on amplification).

In an alternative set-up shown in Fig. 10A, the same method was used to generate microcompartments with concentrations corresponding to 0.0909 and 0.909 times of the fluorescent dye in the sample reservoir (with a stock concentration of 11 pM).

For condition C: The microcompartments were generated by injecting the red fluorescent liquid at 24 pl/min and the buffer at 2.4 pl/min. For condition D: The microcompartments were generated by injecting the red fluorescent liquid at 2.4 pl/min and the buffer at 24 pl/min. Results are shown in Fig. 10B. The measured fluorescence signal of microcompartments generated for condition C was 8.924 [a.u.], and the measured fluorescence signal of microcompartments generated for condition D was: 0.895 [a.u.]. The ratio of microcompartment fluorescence signals of conditions C to D was 9.969 [a.u.], matching the 10-fold difference as expected from the flow rates.

These results show that the method allows the generation of microfluidic microcompartments with a wide range of concentrations of the stock solutions present in the sample species reservoirs.

Example 2: Generation of microcompartments hosting different combinations of barcoding oligonucleotides

This example illustrates how the device can be used to generate microfluidic microcompartments for co-localizing cells with different combinations of barcoding oligonucleotides. The experimental setup comprises pressurized sample species reservoirs filled with carrier oil, a cell suspension, and drugs mixed with barcodes of known oligonucleotide sequence (Combi-seq barcodes 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, incorporated herein by reference). Microfluidic microcompartments were generated by injecting the different reagents into the channel network as shown in Fig 11. By varying the combination of the injected reagents, microcompartments with different combinations of drugs and barcoding oligonucleotides were generated. Specifically, 18 different combinatorial barcodes were generated according to table 1 below. Ligated barcodes were double stranded and had a ligation site (gcggc) in between the 5 ’-barcodes and the 3’ poly-T barcodes. A scheme of the barcode ligation is shown in Fig. 12. Table 1 shows the barcode elements used in this example. Table 1 : Barcode elements (Pred = prednisone; DMSO = dimethylsulfoxide; VCR = vincristine; Dauno = daunorubicin; ASP = asparaginase; Venet = venetoclax)

Fig. 13 A and B exemplarily show the microcompartments generated for Condition no. 6 (Fig. 13A) and Condition no. 11 (Fig. 13B) as detailed in Table 1 above in a schematic drawing. After generation, the microfluidic microcompartments were incubated in an incubator maintained at 37°C, 5% CO2 for 16 hours. After incubation, the microcompartments were injected into a T-junction channel for additional injection of reagents to ligate the two barcode fragments into a single combinatorial oligonucleotide, and to perform reverse transcription as shown in Fig. 13. The microcompartments were injected at a flow rate of 30 pl/min, and the reagent at 10 pl/min. The respective reagent composition is shown in Table 2. Table 2: Composition of the stock solution injected into pre-incubated microcompartments to initiate ligation of combinatorial barcode fragments, cell lysis and reverse transcription.

Subsequently, the microcompartments were incubated at room temperature for 40 minutes, followed by incubation at 50°C for 30 minutes, and finally at 85°C for 5 minutes. The microcompartments were then merged together into one aqueous solution for library preparation for sequencing as described in Mathur, L., Szalai, B., Du, N.H. et al., Nat Commun 13, 4450 (2022), https://doi.org/10.1038/s41467-022-32197-0, incorporated herein by reference) without cDNA purification with Cl dynabeads. Libraries were sequenced on a NextSeq 500 instrument (Illumina) with a sequencing depth of on average 16.9 million reads/sample. The number of reads obtained for each ligated barcode is listed in Table 3.

Table 3: Number of reads obtained for each ligated barcode

A large number of sequencing reads was obtained for all combinatorially generated barcodes, demonstrating that the desired on-demand sample compositions could be successfully generated using the method and device of the present invention, and that the desired barcodes were functional and could be used for amplifying cellular RNA and generating barcoded cDNA for all samples.

Claims

1. A microfluidic device (100), comprising

2. The microfluidic device (100) of claim 1, wherein the device is configured such that the first sample species reservoir (104) and the at least one second sample species reservoir (106) are capable of being pressurized, preferably during both injection and non-injection phases.

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

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

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

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

7. The microfluidic (100) device of any one of claims 1-6, comprising a first pressure control device (114D) connected to the first sample species reservoir (104) and at least a second pressure control device (116D) the second sample species reservoir (106), preferably wherein the first pressure control device (114D) and the at least one second pressure control device (116D) are connected to a pressure source (120).

8. The microfluidic device (100) of claim 7, wherein the first pressure control device (114D) is configured to apply a first injection pressure Pi to

9. The microfluidic device (100) of claim 8, wherein the first pressure control device (114D) is communicatively coupled to the first sensor (114C) of the first sample injection channel (114) and the second pressure control device (116D) is communicatively coupled to the second sensor (116C) second sample injection channel (116), wherein the first and second injection pressures Pi are applied on the basis of the monitored flow data of the liquid in the respective sample injection channel (114; 116).

10. The microfluidic device (100) of any one of claims 7-9, wherein the first and second pressure control devices (114D; 116D) are configured to maintain a non-injection pressure P_m on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116), preferable wherein the non-injection pressure P_m being lower than the first and second injection pressures Pi and following the relation: Pi> P_m> Patmosphere, and optionally, wherein the first and second pressure control devices (114D; 116D) are configured to increase the non-injection pressure P_m on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116) overtime.

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

12. A method (300) for providing one or more combinatorial microcompartment(s) (200A; 200B) comprising at least two sample species (A; B), the method comprising:

13. The method of claim 12, wherein the first sample species (A) and the second sample species (B) are injected by applying an injection pressure Pi to the respective first species reservoir (104) and the second species reservoir (106), while a non-injection pressure P_m being lower than the injection pressure Pi is being maintained on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116).

14. The method of claim 13, further comprising:

- increasing the non-injection pressure P_m on each sample species reservoir (104; 106) of each non-injecting sample injection channel (114; 116) over time, wherein the injection pressure Pi and the non-injection pressure P_m follow the relation: Pi> Pni >

15. The method of any one of claims 10-14, wherein injecting the second sample species (B) occurs sequentially or concomitantly to injecting the first sample species (A) and, wherein the carrier phase is an immiscible phase and, wherein the first sample species (A) and the second sample species (B) are chemically distinct, wherein the combinatorial microcompartment (200) is a combinatorial droplet or combinatorial plug comprising at least one prokaryotic or eukaryotic cell.

16. Microfluidic chip (122) including one or more combinatorial microcompartment(s) (200) comprising at least two sample species (A; B) produced by the method of any one of claims 10-15.

17. A use of a microfluidic device for combining a first sample species (A) and a second sample species (B) into a target channel fluid, the use comprising the steps of applying a non-injection pressure P_m during non-injection phases to a first sample species reservoir (104) comprising a first sample species (A), and applying a P_m during non-injection phases to at least a second sample species reservoir (106) comprising a second sample species (B), applying an injection pressure Pi to at least one of the first and second sample reservoir (104, 106) during an injection phase and thereby injecting the sample species A and/or B into the target channel fluid, wherein ?! > P_m > Patmosphere; characterized in that, the device used comprises:

- a target channel (102) comprising the target channel fluid,

18. A method for co-localizing an entity with a barcode oligonucleotide or set of components thereof in a microfluidic device according to any one of claims 1 to 11, said method comprising:

(i) feeding the entity into the target channel (102);

19. The method of claim 18, the method further comprising applying a non-injection pressure (P_m) during a non-injection phase to the first sample species reservoir (104) and/or to the at least one second sample species reservoir (106), wherein P_m is larger than the pressure of the environment (P atmo sphere), and wherein P i Pni Patmosphere-

20. The method of claim 18 or 19, wherein the injection pressure (Pi) and the non-injection pressure (P_m) are generated by pressurizing the first sample species reservoir (104) and the at least one second sample species reservoir (106).

21. The method of any one of claims 18 to 20, wherein the feeding of the barcode oligonucleotide or set of components thereof 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 of any one of claims 18 to 21, wherein the entity is a nucleic acid, a cell, or a drug.

23. The method of any one of claims 18 to 22, wherein the method further comprises generating microfluidic droplets comprising the entity and the barcode oligonucleotide or components thereof, or wherein the method further comprises fusing of microfluidic droplets comprising the entity and the barcode oligonucleotide or components thereof with a further microfluidic droplet, and/or the method further comprises injecting reagents into the microfluidic droplet.

24. The method of any one of claims 18 to 23, wherein the method further comprises detecting the entity and the barcode oligonucleotide or components thereof.

25. A method for barcoding a substance to be tested on a cell or a culture of cells, the method comprises the steps of:

(i) co-localizing the substance with a barcode oligonucleotide or components thereof and optionally a cell in a microfluidic droplet using the method of any one of claims 18 to 24;

(ii) optionally introducing a cell into the microfluidic droplet of step (i) if no cell is colocalized in step (i);