Analysis of cells and / or organelles in hydrogel cages

Abstract

The present invention relates to a microfluidic device (10) comprising: - a first wall (12) including a first substrate (14) on which a plurality of closed patterns (16) are grafted; - a second wall (18) facing the first wall (12) and including a second substrate (20); - a plurality of nucleic acids (22) grafted on the first substrate (14) or the second substrate (20), wherein each nucleic acid (22) comprises a barcode encoding the location of the nucleic acid on the first substrate (14) or the second substrate (20), wherein at least the plurality of closed patterns (16) or the second substrate (20) is made of an actuable hydrogel capable of swelling between a contracted state and a swollen state, wherein in the swollen state the closed patterns (16) are in contact with the second substrate (20).

Description

[0001] This invention relates to an apparatus for bioanalysis, particularly for analyzing cells and / or organelles. The invention also relates to a method for manufacturing said apparatus and various applications of said apparatus in bioanalysis.

[0002] Single-cell multi-omics analysis is a rapidly growing field. The market size was $300 million in 2018 and is projected to reach $1.6 billion by 2022 (BioInformatics, LLC). Single-cell analysis is an indispensable tool for overcoming the cellular heterogeneity of healthy and diseased tissues. In fact, analyzing a single set of cells cannot reveal the heterogeneity of its composition. For example, tumors typically contain multiple types of tumor cells: for instance, cancer tissue from a breast cancer patient may contain both HER2-positive and HER2-negative cells, the former being more susceptible to HER2-positive cells than the latter, which are more susceptible to HER2-negative cells. (The last sentence appears to be incomplete and possibly refers to the use of the therapeutic antibody trastuzumab.) The former responds to the treatment, while the latter does not. Furthermore, tumors typically contain a wide range of stromal cells and tumor-infiltrating immune cells. Analysis of the entire tumor can never reveal this granularity.

[0003] “Omics” analyses (genomics, epigenomics, and transcriptomics analyses) using barcoded next-generation sequencing can now be performed at high throughput on a single-cell scale. Single cells are isolated in compartments, and the nucleic acids released from the cells are labeled with nucleic acid sequence barcodes, which are unique to each compartment and therefore to each cell. The labeled nucleic acids are pooled and analyzed via next-generation sequencing. Subsequent bioinformatics analysis allows reads to be assigned to single cells, as reads from the same cell carry the same barcode. Similarly, phenotypic analyses using microscopy, flow cytometry, and mass spectrometry can now also be performed at high throughput on a single-cell scale. CITE-seq further allows for the combination of analysis of subfractions of the proteome displayed on the cell surface with transcriptomics analysis via barcoded next-generation sequencing. However, high-throughput single-cell omics data from barcoded next-generation sequencing have not yet been mapped onto phenotypic data from imaging.

[0004] Currently, there is a strong need in the research and clinical communities to better understand complex and heterogeneous biological systems. This requires obtaining phenotypic (protein expression, protein secretion, glycosylation, post-translational modifications, metabolism, cell morphology, and cellular response to external stimuli) and omics information of single cells, as well as phenotypic and omics information of thousands of cells in biological samples.

[0005] To answer certain research or clinical questions, it may be necessary to analyze only the subpopulations of interest. Reducing the number of cells to be sequenced allows for focusing on rare events, complex cell populations, or providing tools for competitive diagnostic purposes. While cell subpopulations can be pre-enriched prior to single-cell sequencing, for example by fluorescence activated cell sorting (FACS) or using magnetic beads, there is currently no integrated system that allows for monitoring of cells and selective single-cell sequencing of cell subpopulations with one or more specific phenotypes. Furthermore, pre-enrichment using FACS or magnetic beads is not well-suited for analyses of small numbers of cells (e.g., from tumor biopsies) and does not allow for cell-based time monitoring or cell sorting based on the detection of secreted molecules (e.g., antibodies and cytokines). Existing technology

[0006] Current systems allow for the isolation of single cells in compartments prior to next-generation sequencing, the release of nucleic acids from the cells, and the tagging of them with nucleic acid sequence barcodes. These nucleic acid sequence barcodes are unique to each compartment and therefore unique to each cell. However, there is currently no system that allows for the analysis of large numbers of cells (>10,000) and the direct combination of cell phenotypes determined by optical imaging with their omics characteristics (genome, epigenome, transcriptome, etc.), nor is there a system for selecting cells of interest and sequencing them individually.

[0007] Existing technology only allows :

[0008] 1. Barcode-coded single-cell sequencing of large numbers of cells (>10,000) without imaging, fluorescence measurement, or droplet sorting, wherein single cells are compartmentalized in a droplet together with individual beads carrying nucleic acid sequence barcodes that are unique to each bead;

[0009] 2. Single-cell analysis of small numbers of cells (≤800) without sorting or selective sequencing of cells with specific phenotypes is performed using valve microfluidics, wherein single cells are compartmentalized in microfabrication chambers separated by pneumatic valves, time-lapse imaging is performed by microscopy, and then single-cell sequencing is performed (Fluidigm C1, FluidigmPolaris).

[0010] 3. Single-cell sequencing of a small number of cells (<2,000) without imaging, fluorescence measurement or cell sorting, wherein the single cells are compartmentalized in microplate wells for single-cell sequencing.

[0011] When using droplet microfluidics, valve microfluidics, or microplate wells, cell subpopulations can be pre-enriched prior to compartmentalization using fluorescent labeling and fluorescence-activated cell sorting (FACS) of small groups (<50) of cell markers (typically cell surface markers), or using magnetic beads labeled with ligands targeting the cell surface markers. However, there is no one-to-one mapping of fluorescence data to sequencing data for each cell analyzed. With microplate wells, however, individual cells can be sorted into individual wells via FACS prior to single-cell sequencing, where the fluorescence spectra of the cells are mapped one-to-one with the omics data. Furthermore, other systems such as the CellenONE system and the Berkeley Lights optofluidic system allow for individual cell imaging followed by selective allocation of cells into microplate wells.

[0012] However, there is currently no available technology that allows for phenotypic monitoring after compartmentalization of >10,000 cells and the selection of a subpopulation for sequencing analysis.

[0013] In particular, there are currently no available technologies that allow combining omics analysis (genomics, epigenomics, transcriptomics, and proteomics) with phenotypic analysis via optical imaging.

[0014] D'Eramo L. et al., Microsystems & Nanoengineering, 2018, 4, 17069, doi:10.1038 teaches a microfluidic device including a microfluidic actuator based on a temperature-responsive hydrogel. However, the disclosed device does not include an array of cages with walls composed of temperature-responsive hydrogels combined with a nucleic acid array comprising multiple nucleic acids grafted onto one of a substrate, wherein each nucleic acid contains a barcode encoding the location of that nucleic acid on the substrate. Therefore, the disclosed device cannot be used for barcoded single-cell sequencing.

[0015] Advantages of the present invention

[0016] The microfluidic device according to the invention allows for the analysis of single cells or small cell populations, particularly at high throughput (over 10,000 cells) and at a highly competitive cost, where phenotypic data from optical imaging is mapped one-to-one to single-cell omics data from barcoded next-generation sequencing. It also allows for selective sequencing of cell subpopulations based on phenotypic data from optical imaging.

[0017] In particular, the microfluidic device according to the invention allows for the isolation of single cells or organelles within microcompartments (cages) made of an activatable hydrogel, thereby enabling visualization, labeling, and bioanalysis of the cellular and genetic material found in each isolated cell. The use of the expandable gel provides the ability to introduce and isolate groups of cells or organelles in the microcompartments as needed, such that single cells or organelles are arranged within the cages while providing a bio-sealing between the cages. The use of barcoded nucleic acids immobilized within the cages allows for the labeling (“barcoded”) of any nucleic acids released from or associated with the cell or organelle within the cage.

[0018] This new technology allows for high-throughput analysis and monitoring of single-cell phenotypes via optical imaging prior to cell lysis and sequencing. This enables a one-to-one mapping of cell phenotypes (protein expression, protein secretion, glycosylation, post-translational modifications, metabolism, cell morphology, and cellular responses to external stimuli) to single-cell omics data from barcoded sequencing. However, this system also offers many other advantageous features, including:

[0019] Imaging allows for quality control of experiments, providing information on the fraction of cell-laden cages, cell types, cell viability, and the efficiency of cell lysis.

[0020] Imaging allows for the exclusion of sequencing data from cages with an incorrect number of cells during bioinformatics analysis; for example, data from cages containing only single cells can be retained.

[0021] -In one embodiment of the invention, sequencing analysis is performed only on cell subpopulations identified based on phenotypic data from optical imaging;

[0022] - Unlike droplet-based compartmentalized systems, this system offers a degree of flexibility that current competing technologies cannot achieve due to the ability to open, partially open, or close the cages, allowing reagents to be added or removed at different steps during analysis.

[0023] - The analytics are user-friendly and fully automated.

[0024] Combining single-cell omics and phenotypic analysis through imaging allows for a better understanding of the complexity of biological systems. This invention will also enable more precise patient diagnosis and stratification, as well as improved diagnostic and therapeutic accuracy. Summary of the Invention

[0025] This invention relates to a microfluidic device, which includes:

[0026] - A first wall, comprising a first substrate, wherein a plurality of closed patterns are grafted onto the first substrate.

[0027] - A second wall, which faces the first wall and includes a second substrate.

[0028] - Multiple nucleic acids, grafted onto a first substrate or a second substrate, wherein each nucleic acid contains a barcode encoding the location of the nucleic acid on the first substrate or the second substrate.

[0029] At least one of the closed patterns or the second substrate is made of an actuable hydrogel that is capable of swelling between a contracted state and a swollen state, in which the closed pattern contacts the second substrate.

[0030] The present invention also relates to a method of manufacturing an apparatus according to the invention, the method comprising:

[0031] 1) Provide a first substrate,

[0032] 2) Graft multiple closed patterns onto the surface of the first substrate.

[0033] 3) Provide a second substrate,

[0034] 4) Grafting multiple nucleic acids onto the surface of a first substrate or the surface of a second substrate, wherein each nucleic acid contains a barcode encoding the location of the nucleic acid on the first substrate or the second substrate.

[0035] 5) The first substrate and the second substrate are positioned by placing a sealing pattern and nucleic acid between the first substrate and the second substrate.

[0036] 6) Bond the first substrate and the second substrate together.

[0037] The present invention also relates to a method for performing cell or organelle analysis, the method comprising:

[0038] a) Provide a microfluidic device according to the invention, and a preparation of a cell or organelle;

[0039] b) Optionally, associate all or part of the cells or organelles with a common labeled nucleic acid sequence or with multiple different labeled nucleic acid sequences;

[0040] c) Injecting cells or organelles in suspension form into a microfluidic device while the hydrogel is in a contracted state;

[0041] d) Change the conditions to actuate the hydrogel to a swollen state, thereby trapping cells or organelles in cages formed by the first and second walls of the microfluidic device, and in the closed pattern of the swollen hydrogel;

[0042] e) Optionally use optical imaging to analyze the captured cells or organelles and / or the molecules they secrete;

[0043] f) Optionally, the grafted nucleic acid is released from the surface of the first or second substrate of the microfluidic device within the cage;

[0044] g) Optionally, lyse the captured cells or organelles to release the nucleic acids of the cells or organelles within the cage;

[0045] h) Associate the barcode of the nucleic acid with the released cellular or organelle nucleic acid and / or labeled nucleic acid sequence to form barcoded nucleic acid;

[0046] i) Change the conditions to actuate the hydrogel to a contractile state;

[0047] j) If no release occurs in f), the grafted nucleic acid is released from the first or second substrate of the microfluidic device;

[0048] k) Recover and sequence barcoded nucleic acids. Detailed Implementation

[0049] This invention first relates to a microfluidic device, which includes:

[0050] - A first wall, comprising a first substrate, wherein a plurality of closed patterns are grafted onto the first substrate.

[0051] - A second wall, which faces the first wall and includes a second substrate.

[0052] - Multiple nucleic acids, grafted onto a first substrate or a second substrate, each nucleic acid containing a barcode encoding the location of the nucleic acid on the first substrate or the second substrate.

[0053] At least one of the closed patterns or the second substrate is made of an actuable hydrogel that is capable of swelling between a contracted state and a swollen state, in which the closed pattern contacts the second substrate.

[0054] The present invention also relates to a method for manufacturing a microfluidic device as defined above.

[0055] The present invention also relates to a method for performing biological analysis, particularly cellular or organelle analysis, using a microfluidic device as defined above.

[0056] All embodiments and preferred features detailed below are independently applicable to all purposes of the invention, particularly to devices, manufacturing methods and their various applications.

[0057] equipment

[0058] In the swollen state, the closed pattern comes into contact with the second substrate of the device. The device therefore includes a plurality of cages, each cage being defined by sidewalls made of the closed pattern and endwalls formed by the first and second substrates.

[0059] In the contracted state, the closed pattern and the second substrate are no longer in contact. The gap between the closed pattern and the second substrate allows fluid and cells to circulate freely within the device.

[0060] Between the contracted and swollen states, the device according to the invention undergoes several intermediate states in which the actuable hydrogel swells only partially. In these configurations, the gap between the closed pattern and the second substrate remains. However, relative to the contracted state, the height of the gap is sufficiently reduced such that cells trapped in the cages are retained within the cages. These intermediate configurations can generally be used to allow selective passage of fluids rather than cells.

[0061] Therefore, each closed pattern defines a cell trapping site, where closing and opening are triggered by an external stimulus. In a preferred embodiment, the external stimulus is a change in pH, light intensity, temperature, or current intensity. In a very preferred embodiment, the external stimulus is a change in temperature.

[0062] The microfluidic device comprises at least two closed patterns. Preferably, the microfluidic device comprises a large number of closed patterns, typically 100, 1,000, 10,000, 100,000, etc.

[0063] The first and second walls are made of a rigid material capable of withstanding temperature fluctuations ranging from -20°C to 100°C. According to a first embodiment, the walls (the first and / or the second wall) are made of a unique and homogeneous material. The walls are therefore composed of a substrate.

[0064] According to the second embodiment, the wall further includes a support material on which the substrate is fixed / coated. Typically, the wall consists of a support material made of glass or polydimethylsiloxane, covered with a substrate layer.

[0065] The device according to the invention may equivalently comprise two single-layer walls, two multi-layer walls, or one single-layer wall and one multi-layer wall.

[0066] The first substrate is typically made of a material selected from: silicon, quartz, glass, polydimethylsiloxane, thermoplastics (such as cyclic olefin copolymers and polycarbonates), preferably glass and polydimethylsiloxane.

[0067] Preferably, the first substrate is made of polydimethylsiloxane.

[0068] According to one embodiment, at least a portion of the surface of the first substrate is structured and / or functionalized.

[0069] In the context of this invention, "structured" means that the surface of the substrate is irregular. The surface of the substrate can be porous or microstructured. In particular, it can include microscopic striations, columnar structures, etc.

[0070] The substrate can be structured using any known method. For example, standard soft lithography can be mentioned, which is well documented.

[0071] In the context of this invention, "functionalization" means fixing chemical functional groups onto the surface of a substrate. Typically, the surface of the first substrate is functionalized with chemical groups selected from hydroxide groups, silanol groups, and mixtures thereof (preferably silanol groups).

[0072] The structuring and / or functionalization of the substrate allows for the facilitation of closed patterns and / or grafting of nucleic acids onto its surface.

[0073] According to a preferred embodiment, the first wall is made of a structured and / or functionalized polydimethylsiloxane plate, preferably a structured and functionalized polydimethylsiloxane plate.

[0074] Closed patterns can have a variety of shapes. Preferably, closed patterns are rectangles, squares, circles, or hexagons.

[0075] Preferably, the closed pattern is covalently grafted onto the first substrate.

[0076] According to one particular embodiment, the second substrate is made of hydrogel, and the closed pattern is made of a non-swellable material.

[0077] Preferably, according to this particular embodiment, the second wall comprises a non-swellable support material on which a swelling hydrogel is deposited.

[0078] The non-swellable support material can be structured and / or functionalized. The structuring and / or functionalization of the non-swellable material is carried out in a manner similar to that described above in the context of the first substrate.

[0079] Therefore, according to this embodiment, the closed pattern is non-swellable, and it is the swelling of the second substrate that allows the cage to close.

[0080] Preferably, according to this embodiment, the closed pattern is made of a material selected from: silicon, quartz, glass, polydimethylsiloxane, thermoplastic materials (such as cyclic olefin copolymers and polycarbonate), preferably selected from glass or polydimethylsiloxane.

[0081] Preferably, the height of the closed pattern ranges from 0.1 μm to 100 μm, and more preferably from 1 μm to 30 μm.

[0082] Preferably, the thickness of the wall of the closed pattern ranges from 0.1 μm to 500 μm, and more preferably from 1 μm to 20 μm.

[0083] Advantageously, according to this embodiment, the thickness of the second substrate, measured in the swollen state in contact with the closed pattern, ranges from 1 μm to 500 μm, preferably from 1 μm to 100 μm.

[0084] Advantageously, still according to this embodiment, the thickness of the second substrate containing hydrogel, measured in a dry state, ranges from 0.5 μm to 150 μm, preferably from 0.5 μm to 50 μm.

[0085] According to a preferred embodiment, the closed pattern is made of hydrogel, and the second substrate is made of a non-swellable material.

[0086] Therefore, according to this embodiment, the second substrate is non-swellable, and the closed pattern swells to close the cage-like structure.

[0087] Preferably, according to this preferred embodiment, the second substrate is made of a material selected from: silicon, quartz, glass, polydimethylsiloxane, thermoplastics (such as cyclic olefin copolymers and polycarbonate), preferably selected from glass or polydimethylsiloxane. Advantageously, when the hydrogel pattern is in contact with the second wall, the height of the hydrogel pattern, measured in the swollen state, ranges from 0.1 μm to 500 μm, preferably from 1 μm to 250 μm, and more preferably from 1 μm to 100 μm.

[0088] Advantageously, the height of the hydrogel pattern measured in the dry state ranges from 0.1 μm to 150 μm, preferably from 0.5 μm to 100 μm, and more preferably from 0.5 μm to 50 μm.

[0089] Preferably, when the hydrogel pattern is in contact with the second wall, the wall separation of the hydrogel pattern measured in the swollen state ranges from 0.1 μm to 100 μm, more preferably from 1 μm to 10 μm.

[0090] Preferably, the wall resolution of the hydrogel pattern measured in a dry state ranges from 0.1 μm to 100 μm, more preferably from 0.5 μm to 5 μm.

[0091] In the context of this invention, "hydrogel" refers to a polymer material that, when placed in an aqueous medium (particularly water) and under certain physical and / or chemical conditions, swells by absorbing and retaining the aqueous phase.

[0092] In this context, "hydrogel" refers to a gel comprising a polymer matrix forming a three-dimensional network that is capable of swelling in the presence of water under specific physicochemical conditions. Specifically, the swelling of a hydrogel can be initiated and / or modulated by thermal, optical, chemical, or electrical stimulation.

[0093] For example, the swelling (or shrinkage) of a hydrogel can be initiated and / or regulated by changes in the temperature, pressure, or pH of the medium in which it is placed.

[0094] Preferably, the hydrogel is a temperature-responsive swelling hydrogel. In the context of this invention, a "temperature-responsive swelling hydrogel" refers to a hydrogel that induced swelling or contraction upon temperature change. Temperature-responsive swelling hydrogels typically exhibit a rapid change in water solubility with temperature.

[0095] Within a specific temperature range, hydrogels are water-soluble and absorb large amounts of water.

[0096] Conversely, by changing the temperature of the medium, the hydrogel becomes no longer water-soluble. The hydrogel then releases water and contracts.

[0097] In the context of this invention, "swollen state" refers to a state of hydrogel in which the closed pattern contacts the second substrate, such that the device includes a plurality of hermetically sealed cages.

[0098] In the context of this invention, "shrinkage state" refers to a state of the hydrogel in which the closed pattern and the second substrate are not in contact: a gap exists between the closed pattern and the second substrate, allowing fluid and cells to circulate freely within the microfluidic device. The "shrinkage state" differs from the "dry state" as defined below in that the hydrogel is not completely water-free. In the shrinkage state, the hydrogel remains at least partially hydrated.

[0099] In the context of this invention, "dry state" refers to a state in which the hydrogel is almost entirely free of water. Typically, the hydrogel is in a dry state during the fabrication of microfluidic devices, particularly during the grafting of hydrogel patterns during the coating of the second wall with a hydrogel substrate.

[0100] The temperature at which the water solubility of a hydrogel changes drastically is designated as the critical dissolution temperature (CST).

[0101] Preferably, the critical dissolution temperature (CST) of the hydrogel is in the range of 4°C to 98°C, more preferably 20°C to 50°C, and even more preferably 25°C to 40°C.

[0102] According to the first variant, the critical dissolution temperature (CST) of the hydrogel is the lower critical dissolution temperature (LCST). Above the LCST, the hydrogel is in a shrinking state, while below the LCST, it is in a swollen state.

[0103] According to the second variant, the critical dissolution temperature (CST) of a hydrogel is the upper critical dissolution temperature (UCST). Above the UCST, the hydrogel is in a swollen state, and below the UCST, it is in a shrinking state.

[0104] The polymer matrix constituting the hydrogel is typically selected from homopolymers, copolymers, and terpolymers of acrylic acid, alkyl (meth)acrylates, alkyl (meth)acrylamide, low-poly(meth)acrylates, sulfobetaine (meth)acrylates, and N-acryloylglycine, preferably from homopolymers, copolymers, and terpolymers of alkyl (meth)acrylamide and any mixtures thereof, and more preferably the hydrogel comprises poly(N-isopropylacrylamide).

[0105] The polymer can be selected from LCST polymers, UCST polymers, and mixtures thereof.

[0106] Similar to what has been described above in the context of hydrogels:

[0107] - The term "LCST polymer" indicates a thermally responsive polymer with a lower critical solution temperature, and

[0108] - The term "UCST polymer" indicates a thermally responsive polymer with an upper critical dissolution temperature.

[0109] The overall behavior of the hydrogel (UCST and / or LCST behavior) depends on the nature and amount of the different polymers present in the hydrogel.

[0110] When the polymer is selected from UCST polymers, it is preferably selected from homopolymers, copolymers and terpolymers of acrylic acid, alkyl (meth)acrylates, alkyl (meth)acrylamides, low-polyethylene (meth)acrylates, sulfobetaine (meth)acrylates, N-acryloylglycine and mixtures thereof.

[0111] Preferably, the UCST polymer is a terpolymer of methacrylamide, acrylamide and allyl methacrylate.

[0112] When the polymer is selected from LCST polymers, it is preferably selected from homopolymers, copolymers and terpolymers of acrylic acid, alkyl (meth)acrylates, alkyl (meth)acrylamides, low-polyethylene (meth)acrylates and mixtures thereof, more preferably from homopolymers, copolymers and terpolymers of alkyl (meth)acrylamides, and even more preferably the LCST polymer is poly(N-isopropylacrylamide).

[0113] Preferably, the LCST polymer is poly(N-isopropylacrylamide).

[0114] Advantageously, the polymer comprises one or more UCST or LCST polymers, preferably composed of them.

[0115] Advantageously, the microfluidic device also includes at least one inlet and at least one outlet, thereby allowing reactants to be introduced into and removed from the device, respectively.

[0116] Preferably, the heating device is integrated into the device according to the invention.

[0117] According to one embodiment, each cage includes an independent heating device. This embodiment is particularly advantageous because it allows each cage to be opened and closed independently.

[0118] For example, the localized heating device can consist of nanoparticles that are heated upon irradiation with light (plasma effect). The nanoparticles can be deposited, for example, between a hydrogel and the walls coating the hydrogel, or dispersed within a polymer matrix of the hydrogel. Preferably, the nanoparticles are selected from metallic nanoparticles and plasma nanoparticles, and preferably include gold, graphene, silver, copper, and titanium nitride.

[0119] In another example, micro-resistors are used for localized heating; for example, micro-resistors comprising chromium / gold bilayer or TiO2 structures.

[0120] The microfluidic device also includes multiple nucleic acids grafted onto a first substrate or a second substrate, wherein each nucleic acid contains a sequence barcode encoding the location of the nucleic acid on the first substrate or the second substrate.

[0121] Advantageously, nucleic acids are grafted onto the hydrogel while it is in a swollen state to place it in a cage-like structure.

[0122] More advantageously, nucleic acids are grafted onto a first substrate inside the closed pattern or onto a second substrate opposite the closed pattern.

[0123] Preferably, when the closed pattern is made of hydrogel, nucleic acids are grafted onto the surface of the second substrate. Preferably, when the second substrate is made of hydrogel, nucleic acids are grafted onto the surface of the first substrate.

[0124] The grafted nucleic acid can be RNA or DNA, preferably DNA. The grafted nucleic acid can be single-stranded, double-stranded, or partially double-stranded.

[0125] The grafted nucleic acid is preferably 60 to 100 nucleotides in length.

[0126] Grafted nucleic acids can be attached to the substrate directly at the 3' or 5' end or via a adapter.

[0127] According to one implementation, grafted nucleic acids sharing the same barcode have multiple sequences. According to another implementation, grafted nucleic acids sharing the same barcode have the same sequence.

[0128] According to one implementation scheme, all or part of the grafted nucleic acid hybridizes with another nucleic acid or multiple nucleic acids to form partial or complete double-stranded DNA, double-stranded DNA / RNA, or double-stranded RNA.

[0129] According to one implementation scheme, the grafted nucleic acid comprises one or any combination of the following sequences:

[0130] 1) Restriction sites or lightly cleavable sites used for nucleic acid release.

[0131] 2) Sequences complementary to the amplification primers used for further amplification.

[0132] 3) The T7 RNA polymerase promoter sequence for further in vitro transcription (IVT),

[0133] 4) Hybridization sites, linkage sites, or recombination sites used for nucleic acid labeling, and

[0134] 5) The sequence of random nucleotide residues that function as unique molecular identifiers (UMIs).

[0135] Preferably, the grafted nucleic acid includes at least i) a sequence barcode encoding the location of the nucleic acid on the first substrate or the second substrate, and ii) a restriction site or a photocuttable site, and optionally also includes iii) a primer sequence, and / or a T7 sequence and / or a hybridization, ligation or recombination site.

[0136] According to one embodiment, the grafted nucleic acid of the microfluidic device contains a constant sequence, i.e., a sequence present in all grafted nucleic acids. The grafted nucleic acid of the microfluidic device can hybridize with DNA containing a sequence complementary to all or part of the constant sequence of the grafted nucleic acid. One or more different DNAs containing sequences complementary to all or part of the constant sequence can hybridize with the grafted nucleic acid.

[0137] The microfluidic device according to the invention may also include a structure capable of trapping cells or organelles. Such a structure is typically selected from publications such as: Vigneswaran N. et al., 2017, Microfluidic hydrodynamictrapping for single cell analysis: mechanisms, methods and applications, Anal. Methods, 9, 3751-3772.

[0138] Preferably, the structure capable of capturing cells or organelles is located on a first substrate inside the closed pattern or on a second substrate opposite to the closed pattern.

[0139] Preferably, each cage includes at least one structure capable of capturing cells or organelles.

[0140] According to a particular implementation, multiple ligands are directly or indirectly, covalently or non-covalently grafted onto the first substrate (14) and / or onto the second substrate (20), opposite to the closed pattern.

[0141] Advantageously, nucleic acids are grafted into cages when the ligands are in a swollen state.

[0142] In particular, when grafted onto the first substrate (14), the ligand is typically grafted within a closed pattern (16).

[0143] Alternatively, when grafted onto the second substrate (20), the ligand faces the closed pattern.

[0144] The ligands can all be grafted onto the same substrate. Alternatively, some ligands are grafted onto the first substrate (14), while other ligands are grafted onto the second substrate (20).

[0145] Preferably, when directly grafted onto the first substrate (14) or the second substrate (20), a plurality of ligands are covalently grafted onto the first substrate (14) or the second substrate (20).

[0146] According to a more specific embodiment, multiple ligands are indirectly grafted: multiple ligands are grafted onto an intermediate structure, which is directly grafted onto a first substrate (14) or a second substrate (20). Therefore, according to this specific embodiment, there is no direct bonding between the multiple ligands and the substrates (14, 20).

[0147] Preferably, when indirectly grafted onto the first substrate (14) or the second substrate (20), a plurality of ligands are non-covalently grafted onto the first substrate (14) or the second substrate (20).

[0148] According to the first implementation plan, multiple ligands are conjugated to nucleic acids and associated with at least some of the grafted nucleic acids through hybridization (22).

[0149] According to another example, multiple ligands are non-covalently grafted onto an adhesive coating previously coated on a first substrate (14) or a second substrate (20). As an adhesive coating, streptavidin coatings are particularly noteworthy.

[0150] In these implementations, preferably, each ligand is independently selected from the group consisting of an antibody, an antibody fragment, agglutinin, and an aptamer.

[0151] The ligand is typically selected to bind one or more analytes that are secreted or released by the lysis of cells or organelles trapped in cages formed by the first wall (14) and the second wall (20) of the microfluidic device (10), and in the closed pattern (16) of the hydrogel in a swollen state.

[0152] Manufacturing method

[0153] The present invention also relates to a method for manufacturing a microfluidic device as defined above.

[0154] Preferably, the manufacturing method includes the following steps:

[0155] 1) Provide a first substrate,

[0156] 2) Graft multiple closed patterns onto the surface of the first substrate.

[0157] 3) Provide a second substrate,

[0158] 4) Grafting multiple nucleic acids onto the surface of a first substrate or the surface of a second substrate, wherein each nucleic acid contains a barcode encoding the location of the nucleic acid on the first substrate or the second substrate.

[0159] 5) The first substrate and the second substrate are positioned by placing a sealing pattern and nucleic acid between the first substrate and the second substrate.

[0160] 6) Bond the first substrate and the second substrate together.

[0161] Grafting of closed patterns can be performed using any known method.

[0162] When the closed pattern is made of a non-swellable material, the grafting of the closed pattern is usually performed using soft lithography.

[0163] According to a specific implementation, the first substrate and the closed pattern are prepared together in one and only step.

[0164] When the closed pattern is made of hydrogel, the grafting of the closed pattern is usually performed by photopatterning, preferably under UV (ultraviolet) radiation. The photopatterning method involves grafting the polymer matrix of the hydrogel onto the surface of a first substrate, and simultaneously through the crosslinking of the polymer matrix of the hydrogel.

[0165] Preferably, the polymer is covalently cross-linked.

[0166] More preferably, the crosslinking of the polymer is carried out in the presence of a crosslinking agent selected from dithiol molecules, such as dithioerythritol.

[0167] Hydrogel patterning is typically performed using standard photolithography techniques or by using direct laser writing equipment.

[0168] These techniques are specifically disclosed in the following literature: Chollet, B., D'Eramo, L., Martwong, E., Li, M., Macron, J., Mai, TQ, Tabeling, P. and Tran, Y., 2016. Tailoring patterns of surface-attached multiresponsive polymer networks. ACS Applied Materials & Interfaces, 8(37), pp. 24870-24879.

[0169] Nucleic acid grafting is usually performed via dot-matrix synthesis or in situ light-guided synthesis, as detailed in the following references: DeRisi, J. et al. Use of a cDNA microarray to analyze gene expression. Nat. Genet 14, 457–460 (1996); and Fodor, SP et al. Light-directed, spatially addressable parallel chemical synthesis. Science (80-.). 251, 767–773 (1991).

[0170] Advantageously, during step 5), when the hydrogel is in a swollen state, the first and second substrates are positioned in a manner that allows nucleic acids to be located inside the cage.

[0171] More advantageously, nucleic acids are grafted onto a first substrate inside the closed pattern or onto a second substrate opposite the closed pattern.

[0172] The bonding step can be performed using any known method.

[0173] According to the first embodiment, the bonding step is performed by oxygen plasma treatment.

[0174] Preferably, the oxygen plasma treatment is carried out at room temperature, typically at a temperature ranging from 5°C to 50°C, more preferably from 10°C to 40°C, and even more preferably from 15°C to 30°C.

[0175] Preferably, the duration of the oxygen plasma treatment ranges from 10 seconds to 2 minutes, more preferably from 30 seconds to 1 minute.

[0176] Preferably, according to the first embodiment, the method further includes a preparation step of depositing a mask on the nucleic acid before step 6), the mask being capable of protecting the nucleic acid during exposure to oxygen plasma.

[0177] Masks are typically made of tape, and preferably of a material selected from plastic film, paper, cloth, foam or foil coated with adhesive.

[0178] After plasma treatment, the mask is usually removed by stripping.

[0179] According to the second embodiment, the bonding step is performed by applying pressure to the surface of the device.

[0180] Preferably, according to this embodiment, pressure on the device surface is achieved by applying negative pressure to external microfluidic channels surrounding the main design.

[0181] According to a third embodiment, the bonding step is performed by using a crosslinking composition comprising at least one polymer and optionally at least one crosslinking agent.

[0182] According to this third implementation scheme, the bonding steps are as follows:

[0183] a) Connect the first wall and the second wall together.

[0184] b) Depositing a composition comprising at least one polymer and at least one crosslinking agent between the two walls to fill the gap between the first wall and the second wall, and

[0185] c) Crosslinking at least one polymer, preferably self-crosslinking.

[0186] Preferably, the polymer is selected from polyepoxides.

[0187] The method may also include:

[0188] - An intermediate step between step 1) and step 2) of structuring and / or functionalizing the surface of the first substrate, and / or

[0189] - An intermediate step between step 3) and step 4) to structure and / or functionalize the surface of the second substrate.

[0190] When the substrate is made of hydrogel, the functionalization of the substrate can generally be carried out according to the schemes described in the following literature: Chollet, B.D'eramo, L., Martwong, E., Li, M., Macron, J., Mai, TQ, Tabeling, P. and Tran, Y., 2016. Tailoring patterns of surface-attached multiresponsive polymer networks. ACS applied materials & interfaces, 8(37), pp. 24870-24879.

[0191] If the structure is not made of hydrogel, it can usually be functionalized according to the scheme detailed in the following literature: Beal, John HL et al., “A rapid, inexpensive surface treatment for enhanced functionality of polydimethylsiloxane microfluidic channels.” Biomicrofluidics, Vol. 6, 336503. July 30, 2012.

[0192] When the substrate is made of hydrogel, the structuring of the substrate can generally follow the schemes described in the following literature: Chollet, B.D'eramo, L., Martwong, E., Li, M., Macron, J., Mai, TQ, Tabeling, P. and Tran, Y., 2016. Tailoring patterns of surface-attached multiresponsive polymer networks. ACS Applied Materials & Interfaces, 8(37), pp. 24870-24879.

[0193] When the substrate is made of a non-expandable material, the substrate structuring can usually follow standard photolithography schemes, especially standard photolithography techniques.

[0194] According to a particular embodiment, the method may also include an additional step prior to depositing the hydrogel material, which consists of depositing a layer of nanoparticles (preferably a patterned chromium / gold bilayer) on the surface of the substrate.

[0195] The deposition of patterned layers can be performed, for example, using standard photolithography techniques.

[0196] According to a particular implementation, the method further includes at least one of the following steps:

[0197] a) grafting multiple ligands (directly) onto the surface of the first substrate (14) and / or the surface of the second substrate (20), and / or

[0198] b) Grafting multiple ligands (indirectly) onto the surface of the first substrate (14) and / or onto the surface of the second substrate (20).

[0199] Step a) can be performed at any time in the manufacturing process defined above. In particular, step a) can be performed before or after the grafting of the closed pattern (16) and the grafting of the nucleic acid (22).

[0200] According to the first embodiment, indirect grafting of ligands is performed by associating multiple ligands with multiple graft nucleic acids (22) through hybridization, wherein the multiple ligands are conjugated with nucleic acids complementary to at least a portion of the graft nucleic acids (22).

[0201] According to the first embodiment, step b) is preferably performed after the grafting of nucleic acid (22). Step b) can continue until the conditions are changed to actuate the hydrogel to a swollen state, thereby trapping cells or organelles in cages formed by the first wall (14) and the second wall (20) of the microfluidic device (10), and in the closed pattern (16) of the swollen hydrogel.

[0202] According to the second embodiment, the indirect grafting of the ligand includes i) coating an adhesive coating on at least a portion of the surface of the first substrate (14) and / or the second substrate (20), and ii) grafting the ligand onto the adhesive coating.

[0203] Step i) can be performed before or after grafting the closed pattern (16) or before or after grafting the nucleic acid (22).

[0204] Step ii) is preferably performed after the deposition of the adhesion coating. Step ii) can continue until the conditions are changed to actuate the hydrogel to a swollen state, thereby trapping cells or organelles in cages formed by the first wall (14) and the second wall (20) of the microfluidic device (10), and in the closed pattern (16) of the swollen hydrogel.

[0205] The method for manufacturing a microfluidic device further includes one or more of the following steps:

[0206] 1) Hybridize DNA containing sequences complementary to all or part of constant sequences present in graft nucleic acids, particularly in all or part of all or part of the graft nucleic acids;

[0207] In particular, one or more different DNAs containing sequences complementary to all or part of the constant sequence can be used;

[0208] 2) Extend hybrid DNA by polymerization (e.g., using Maxima, SuperScript RT, Phusion, or Q5 polymerase);

[0209] 3) Link the grafted nucleic acid, especially the grafted DNA, to one or more DNA sequences;

[0210] And / or

[0211] 4) Release all or part of the grafted nucleic acid from the surface of the first or second substrate by cleavage (e.g., by photocleavage or by cleavage catalyzed by a nuclease), the grafted nucleic acid possibly having been previously modified by hybridization, extension or ligation according to 1), 2) or 3).

[0212] In some implementations, the hybrid DNA is combined with the grafted nucleic acid to form a double-stranded DNA containing restriction sites for endonucleases.

[0213] The method may also include a further step of fixing structures capable of capturing cells or organelles.

[0214] This supplementary step is typically achieved using standard photolithography techniques.

[0215] application

[0216] The microfluidic device of the present invention can be used in methods for analyzing cells or organelles, and has the possibility of combining phenotypic information from optical imaging with omics information for single cells or organelles (or for, for example, two or more interacting cells), and this is simultaneously for thousands of cells.

[0217] Methods for performing cell or organelle analysis include:

[0218] a) Provide preparations of microfluidic devices and cells or organelles according to the present invention;

[0219] a) Optionally, associate all or part of the cells or organelles with a common marker nucleic acid sequence or with multiple different marker nucleic acid sequences;

[0220] b) Injecting cells or organelles in suspension form into a microfluidic device while the hydrogel is in a contracted state;

[0221] c) Change the conditions to actuate the hydrogel to a swollen state, thereby trapping cells or organelles in cages formed by the first and second walls of the microfluidic device, and in the closed pattern of the swollen hydrogel;

[0222] d) Optionally use optical imaging to analyze the captured cells or organelles and / or the molecules they secrete;

[0223] e) Optionally, the grafted nucleic acid is released from the surface of the first or second substrate of the microfluidic device within a cage;

[0224] f) Optionally, lyse the captured cells or organelles to release the nucleic acids of the cells or organelles within the cage;

[0225] g) Associate the barcode of the nucleic acid with the released cellular or organelle nucleic acid and / or labeled nucleic acid sequence to form barcoded nucleic acid;

[0226] h) Change the conditions to actuate the hydrogel to a contractile state;

[0227] i) If no release occurs in f), the grafted nucleic acid is released from the first or second substrate of the microfluidic device;

[0228] j) Recover and sequence barcoded nucleic acids; and

[0229] k) Optionally, the barcoded sequencing data is mapped onto the data obtained from optical imaging in e).

[0230] In step c), the injection of cells or organelles in suspension into a microfluidic device while the hydrogel is in a contracted state is typically performed by setting a temperature, pressure, or pH—depending on the properties of the actuable hydrogel—to induce a contracted state in the hydrogel. For example, if the microfluidic device contains a lower critical solution temperature (LCST) responsive hydrogel, the temperature of the microfluidic device is increased above the LCST to cause the hydrogel to contract. For temperature-responsive hydrogels containing poly(N-isopropylacrylamide) (PNIPAM) or composed thereof, the hydrogel fully expands at ≤28°C, fully contracts at ≥36°C, and partially expands between these temperatures (see [link to relevant documentation]). Figure 7The temperature response allows the cage to fully open at 37°C for cell or organelle loading (D'Eramo et al., Microsystems & Nanoengineering (2018) 4, 17069). For example, if the microfluidic device contains an upper critical dissolution temperature (UCST) temperature-responsive hydrogel, the temperature of the microfluidic device is lowered below the UCST to cause the hydrogel to shrink. For temperature-responsive hydrogels containing P(MA-AM-AMA) or composed thereof, the hydrogel fully shrinks at ≤10°C, fully expands at ≥50°C, and partially expands between these temperatures, allowing the cage to fully open at 10°C for cell or organelle loading. According to one embodiment, in step d), a single cell or organelle is trapped in the cage. According to another embodiment, two (or more) interacting cells are trapped in a cage, such as plasma cells and reporter cells; cytotoxic T cells (or CAR T cells) and target cells (e.g., tumor cells); T cells and antigen-presenting cells.

[0231] To actuate a hydrogel to a swollen state, temperature, pressure, or pH is varied depending on the properties of the actuable hydrogel, causing it to swell and contact a second substrate. For example, if a microfluidic device contains a lower critical solution temperature (LCST) responsive hydrogel, the temperature of the microfluidic device is lowered below the LCST to allow the hydrogel to swell. For temperature-responsive hydrogels containing poly(N-isopropylacrylamide) (PNIPAM) or composed thereof, the temperature is typically set to ≤28°C, where the hydrogel fully swells (D'Eramo et al., Microsystems & Nanoengineering (2018) 4, 17069). For example, if a microfluidic device contains an upper critical solution temperature (UCST) responsive hydrogel, the temperature of the microfluidic device is raised above the UCST to allow the hydrogel to swell. For temperature-responsive hydrogels containing P(MA-AM-AMA) or composed thereof, the hydrogel fully swells at ≥50°C.

[0232] The method may further include altering the surrounding conditions of the cells or organelles between step d) and step h). Altering the surrounding conditions includes circulating an aqueous phase containing, for example, salts, detergents, proteins, and / or nucleic acid sequences within a microfluidic device. Altering the surrounding conditions also includes exchanging molecules (such as salts) through the hydrogel that passes through the closed cage (where the cage also contains structures capable of trapping cells or organelles) by fully opening the cage (or partially opening the cage).

[0233] According to one implementation, the method further includes, for example, the steps after step d) and before step e), but are not mandatory:

[0234] e1) To bind one or more analytes secreted or released by the captured cells or organelles to ligands directly or indirectly grafted onto the surface of the first substrate (14) and / or the surface of the second substrate (20);

[0235] e2) Detect one or more analytes bound to the grafted ligand by binding to a labeled second ligand or a labeled ligand specific to one or more analytes bound to it.

[0236] According to the first embodiment, in step e2, detection is performed directly using one or more fluorescently labeled second ligands.

[0237] According to the second embodiment, in step e2, detection is performed indirectly using one or more second ligands that identify nucleic acid markers by ligands, the ligands identifying nucleic acids specific to one or more analytes bound to the graft ligands, wherein the sequence of the ligands identifying nucleic acids allows for the identification of the ligands and one or more analytes bound to the graft ligands.

[0238] According to this second embodiment, the method may further include amplifying the sequence of the nucleic acid identified by the ligand. The amplification preferably consists of linear amplification, more preferably using at least one polymerase and at least one restriction enzyme or nickase.

[0239] According to the second embodiment of the method, in step h), the method may further include associating the barcode of the nucleic acid (22) with a ligand-identified nucleic acid to form a barcoded nucleic acid.

[0240] According to one embodiment, the common marker DNA sequence or multiple different marker DNA sequences provided in step b) are used in a DNA-toolbox reaction (or dynamic DNA reaction network) for phenotypic sorting of cells or organelles, thereby initiating the release of grafted nucleic acids in step f) or j). The principle of the DNA-toolbox reaction is described, for example, in international patent applications WO2017141068 and WO2017141067.

[0241] According to one embodiment, in step g), the captured cells or organelles are lysed by osmotic shock. This can be readily performed by those skilled in the art by circulating a hypotonic or hypertonic aqueous phase in a microfluidic device. For this operation, the cage can remain closed.

[0242] According to one embodiment, step h) includes hybridizing the barcode-containing nucleic acid with complementarity to the released cellular or organelle nucleic acid and / or labeled nucleic acid sequence, the nucleic acid which may remain grafted onto or be released from the surface of the first or second substrate of the microfluidic device. Specifically, when the barcode-containing nucleic acid is DNA, step h) [or the method between steps i) and j) may additionally include extending the DNA containing the barcode hybridized to the released cellular or organelle nucleic acid (or labeled nucleic acid sequence) using a DNA polymerase to produce a complementary strand of the released cellular or organelle nucleic acid (or labeled nucleic acid sequence) containing the barcode. Nucleic acids may include, for example, a 3' region of oligod(T) or oligod(T)VN sequences used for hybridization to mRNA (for mRNA sequencing), a 3' region of a sequence complementary to a specific RNA (for targeted RNA sequencing) or DNA (for targeted DNA sequencing), a 3' region of a random sequence such as d(N)6 (for RNA or DNA sequencing), a 3' region with three ribo(G) nucleotides used for reverse transcriptase template conversion (for RNA sequencing), or a 3' region complementary to a nucleotide sequence introduced by recombination, for example after “tag-up” catalyzed by Tn5 transposase. The latter can be used for epigenomic analysis, such as genomic DNA sequencing, or DNA methylation (using Methyl-seq or bisulfite sequencing) or chromatin structure (using transposase-accessible chromatin sequencing, ATAC-Seq), or for RNA sequencing after tagging the RNA-DNA duplex formed after the synthesis of the first-strand cDNA or the double-stranded DNA formed on the RNA released from the cell or organelle after the synthesis of the first-strand cDNA and the second-strand cDNA.

[0243] According to another embodiment, the nucleic acid containing the barcode is DNA, and may be fully or partially double-stranded, and step h) includes ligating the barcode-containing DNA to DNA released by the cell or organelle. For example, the barcode can be ligated to genomic DNA, such as after restriction digestion (for genomic DNA sequencing or DNA methylation analysis), or after digestion with micrococcal nucleases (for metagenomic analysis using MNase-seq or ChIP-seq).

[0244] According to another embodiment, the barcode-containing nucleic acid is DNA and may be fully or partially double-stranded, and step h) includes recombination of the barcode-containing DNA with DNA released from a cell or organelle. For example, the barcode can be recombinated with genomic DNA for genomic DNA sequencing, or for epigenomic analysis of DNA methylation (using Methyl-seq or bisulfite sequencing) or chromatin structure (using transposase-accessible chromatin sequencing, ATAC-Seq). Alternatively, the barcode-containing nucleic acid is recombinated with an RNA-DNA duplex formed on RNA released from a cell or organelle after the synthesis of a first-strand cDNA, or with a double-stranded DNA formed on RNA released from a cell or organelle after the synthesis of both first-strand cDNA and second-strand cDNA (for RNA sequencing). In a preferred embodiment, the oligonucleotide comprises a chimeric end (ME) sequence, which is recombinated with DNA catalyzed by Tn5 transposase.

[0245] According to one embodiment, the method further includes, between step d) and step h), releasing the barcode-containing nucleic acid, for example by an adjacent linkage assay or adjacent extension assay, when cellular or organelle material (e.g., surface molecules, secretory molecules, or lysis products) is present in the cage.

[0246] The present invention is further illustrated by the following figures and embodiments. Attached Figure Description

[0247] Figure 1 This is a schematic diagram of the closed pattern of the device according to the present invention.

[0248] The microfluidic device 10 according to the invention includes a plurality of closed patterns 16 arranged in a tabular form. The closed patterns 16 are in the form of squares, but can be equivalently in the form of rectangles, circles, or even hexagons. The closed patterns 16 have a thickness e and a height h.

[0249] Figure 2 This is a schematic diagram of a microfluidic device according to a first embodiment of the present invention, wherein the pattern is made of an actuable hydrogel.

[0250] The microfluidic device 10 includes a first wall 12 comprising a first substrate 14 to which a plurality of closure patterns 16 are grafted. A second wall 18 facing the first wall 12 includes a second substrate 20. A plurality of nucleic acids 22 are grafted onto the second substrate 20. The closure patterns 16 are made of an actuable hydrogel capable of swelling between a contracted state and a swollen state, in which the closure patterns 16 are in contact with the second substrate 20. The microfluidic device 10 also includes an inlet 24 and an outlet 26, thereby allowing reactants to be introduced into and removed from the device 10, respectively.

[0251] In scheme A, the closed pattern 16 is in a contracted state. The gap 28 between the closed pattern 14 and the second substrate 20 allows fluids and cells present inside the device to circulate freely inside the device 10.

[0252] Under specific physicochemical conditions, the closed pattern 16 begins to absorb water and swell. The closed pattern 16 thus elongates until it contacts the second substrate 20.

[0253] In embodiment B, the closed pattern 16 is in a swollen state and is in contact with the second substrate 20. The device 10 therefore includes a plurality of cages 30, each cage 30 being defined by sidewalls and endwalls, the sidewalls being made of one of the closed patterns 16, and the endwalls being formed by a portion of the first substrate 14 and the second substrate 20.

[0254] Figure 3 This is a schematic diagram of a microfluidic device according to a second embodiment of the present invention, wherein the second substrate is made of an actuable hydrogel.

[0255] The microfluidic device 10 includes a first wall 12 comprising a first substrate 14 on which a plurality of closed patterns 16 are grafted. A second wall 18 facing the first wall 12 includes a second substrate 20. A plurality of nucleic acids 22 are grafted onto the first substrate 14. The second substrate 20 is made of an actuable hydrogel capable of swelling between a contracted state and a swollen state, in which the closed patterns 16 are in contact with the second substrate 20. The microfluidic device 10 also includes an inlet 24 and an outlet 26, thereby allowing reactants to be introduced into and removed from the device 10, respectively.

[0256] In scheme A, the second substrate 20 is in a contracted state. The gap 28 between the closed pattern 14 and the second substrate 20 allows fluids and cells present inside the device to circulate freely within the device 10.

[0257] Under specific physicochemical conditions, the second substrate 20 begins to absorb water and swell. The thickness of the second substrate 20 thus increases until it contacts the closed pattern 16.

[0258] In embodiment B, the second substrate 20 is in a swollen state and is in contact with the closed pattern 16. The device 10 therefore includes a plurality of cages 30, each cage 30 being defined by sidewalls and endwalls, the sidewalls being made of one of the closed patterns 16, and the endwalls being formed by a portion of the first substrate 14 and the second substrate 20.

[0259] Figure 4 This is a schematic diagram of the main stages of a method for performing cell or organelle analysis according to the present invention. The stages are labeled in ascending order from top to bottom.

[0260] Stage 1: At temperature A, the dissociated cells are injected into the microfluidic device, thereby causing the cage to shrink.

[0261] Phase 2: The cage swells by changing the temperature of the microfluidic device to temperature B, allowing cells to be captured. Cells are then lysed by changing the surrounding buffer (e.g., using a low-salt buffer). Uncaptured cells are washed away.

[0262] Phase 3: Still at temperature B, change the buffer again to allow nucleic acids to hybridize to the grafted oligonucleotides at the bottom of each cage.

[0263] Phase 4: The cages are contracted by changing the temperature of the microfluidic device to temperature A. This allows for the injection and incubation of the reaction mixture to associate the specific barcodes present on each cage on the grafted oligonucleotide with the hybrid nucleic acid released from the cell.

[0264] Phase 5: Still at temperature A, inject a fresh reaction mixture to release the grafted or hybridized barcoded cDNA and / or tag from the microfluidic device. The recovered sample is then purified, amplified, and sequenced.

[0265] Figure 5 This is a schematic diagram of a series of operations performed in an exemplary method for cell or organelle analysis according to the present invention, based on transcriptome analysis. The gray rectangles represent hydrogel cages containing cells (circular) and capture primers (strips). The method, from top to bottom, includes: a- closing the cages after cell capture; b- lysing cells by osmotic shock and hybridizing mRNA to markers within the cages; c- opening the cages, RT (in the case of transcriptome); d- cleaving and recovering cDNA.

[0266] Figure 6 This represents a chip comprising 200 cages. A magnified view of the cages from top to bottom shows trapping (top right row), lysis caused by osmotic shock (induced by circulation of distilled water between cages, second row), and the release of lysed cellular material (last row).

[0267] Figure 7 The curves show the swelling ratio of the PNIPAM hydrogel synthesized in Example 1 as a function of temperature in four different aqueous solutions. Curve 1 was obtained in pure water, curve 2 in phosphate buffer, curve 3 at pH=2, and curve 4 at pH=9.

[0268] Figure 8 This illustrates the microarray structure of DNA strands in the microfluidic device of Example 1. Figure 8'a' represents a cross-section of the microarray included in the microfluidic device of Example 1. Arrows indicate the locations of DNA strands. Each DNA spot consists of a set of DNA strands. Figure 8 b represents an example of the composition of the DNA strands constituting the DNA array included in the microfluidic device of Example 1. All DNA strands in the DNA spots are identical. Between each DNA spot, the DNA strands differ due to their spot-specific barcodes. Up to one million spot-specific barcodes have been designed.

[0269] Figure 9 This is a partial representation of a positioning grid based on fluorescent tags. The shape and color define a unique location.

[0270] Figure 10 The expression represents the swelling ratio of the poly(methacrylamide-acrylamide-allyl methacrylate) hydrogel synthesized in Example 3 in phosphate buffer as a function of temperature.

[0271] Figure 11 This shows the structure of the DNA micropores in the device used in Example 4. Figure 11 'a' represents a cross-section of the microarray included in the microfluidic device of Example 4. Arrows indicate the location of DNA strands. Each DNA spot consists of a set of DNA strands. Vertical black bars represent walls made of PDMS, forming cage-like walls. Figure 11 b represents an example of the composition of the DNA strands constituting the DNA array included in the microfluidic device of Example 4. All DNA strands in the DNA spots are identical. Between each DNA spot, the DNA strands differ due to their spot-specific barcodes. Up to one million spot-specific barcodes have been designed.

[0272] Figure 12 This illustrates the composition of the biotinylated oligonucleotides used for specific labeling of cell subpopulations in Example 5. Cells were labeled with subpopulation-specific antibodies carrying antibody-specific barcodes. The antibodies were conjugated with streptavidin (e.g., using the Streptavidin Conjugation Kit-Lightning-Link from abcam-ab102921). Prior to cell labeling, the biotinylated oligomers as shown in this figure were added to the antibody solution at a 12:1 ratio.

[0273] Figure 13 This illustrates the microarray structure of DNA strands in the microfluidic device of Example 6. Figure 13 a is a schematic diagram of the DNA array preparation in Example 6. The procedure involves reversing the oligonucleotides within each DNA spot of the DNA array by hybridizing each graft strand to an adjacent graft strand and then cleaving each graft strand at the 5' of the hybridization site using a nick restriction enzyme. Figure 13Figure b represents a cross-section of the microarray included in the microfluidic device of Example 6. Arrows indicate the location of the DNA strands. Each DNA spot consists of a set of DNA strands. Vertical black bars represent walls made of PDMS, which form cage-like walls. Sub-figure c shows an example of the composition of the DNA strands constituting the DNA array included in the microfluidic device of Example 6. All DNA strands in the DNA spots are identical. Between each DNA spot, the DNA strands differ due to their spot-specific barcodes. Up to one million spot-specific barcodes have been designed.

[0274] Figure 14 This illustrates the microarray structure of DNA strands in the microfluidic device of Example 7. Figure 14 'a' represents a cross-section of the microarray included in the microfluidic device of Example 7. Arrows indicate the location of DNA strands. Each DNA spot consists of a set of DNA strands. Figure 14 b represents an example of the composition of the DNA strands constituting the DNA array included in the microfluidic device of Example 7. All DNA strands in the DNA spots are identical. Between each DNA spot, the DNA strands differ due to their spot-specific barcodes. Up to one million spot-specific barcodes have been designed.

[0275] Figure 15 This illustrates the microarray structure of DNA strands in the microfluidic device of Example 8. Figure 15 'a' represents a cross-section of the microarray included in the microfluidic device of Example 8. Arrows indicate the location of DNA strands. Each DNA spot consists of a set of DNA strands. Figure 15 b represents an example of the composition of the DNA strands constituting the DNA array included in the microfluidic device of Example 8. All DNA strands in the DNA spots are identical. Between each DNA spot, the DNA strands differ due to their spot-specific barcodes. Up to one million spot-specific barcodes have been designed.

[0276] Figure 16 This is an identification of DNA array spots and cages as part of the microfluidic device of Example 8. This identification is based on the superposition of bright-field and fluorescence images (excited at 650 nm and emitted at 670 nm) from the microfluidic device. The bright-field image allows for the identification of cages. The fluorescence image, indicated by black circles A, B, and C, enables the identification of certain spots on which fluorescent DNA probes have previously hybridized. The relative positions of black circles A, B, and C define unique locations on the chip, allowing the original organization of the DNA array to be mapped onto the superimposed image. Finally, due to this identification, spot-specific barcodes can be associated with each cage.

[0277] Figure 17 This represents the spatial redistribution of 14,752 barcodes extracted from valid gene reads in the sequencing data of Example 8 within the microfluidic device. Each black circle represents a DNA spot from the DNA array whose barcode is associated with at least one cDNA in the sequencing data. Missing circles are DNA spots from the DNA array whose barcodes were not found to be associated with at least one cDNA in the sequencing data. The hexagonal shape represented by the location of the recovered barcodes corresponds to regions protected from oxygen plasma during the fabrication of the microfluidic device. Most of the spots in the protected regions have been identified in the sequencing data.

[0278] Figure 18 This indicates the length distribution of genes identified by sequencing in Example 8. Figure 18 'a' represents the length distribution of 20,465 genes annotated as protein-coding genes in the Genome Reference Consortium component GRCh38.p13 Gene Code 38, annotated by Ensembl version 104.38 (reference: http: / / may2021.archive.ensembl.org / Homo_sapiens / Info / Annotation). Gene length was determined as the sum of exon lengths. For each gene, the average size of alternative splicing variants was used. Figure 18 b represents the length distribution of the same database of only 14,507 unique genes identified from the valid gene and valid barcode reads presented in Example 8. The similarity of the size distribution in subplots a and b indicates that there was no size bias in gene recovery in Example 8.

[0279] Figure 19 This refers to the annotated UMAP (McInnes, L, Healy, J, UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction, ArXiv e-prints 1802.03426, 2018) used for clustering the triplicate replicates of Example 8 using two different cell lines. The RNA-seq described in Example 8 was repeated twice with Jurkat human T cells and once with Ramos human B cells. For each experiment, 15,000 barcodes were randomly grouped to form 200 superbarcodes to address the issue of shallow sequencing depth and a small number of cDNAs identified per barcode, which prevented clustering. Then, principal component analysis (PCA) was run using the Scanpy Python software (https: / / scanpy.readthedocs.io), followed by Leiden graph clustering (Traag et al., 2018, From Louvain to Leiden: guaranteeing well-connected communities) to detect clusters, which were then represented in two dimensions using UMAP graphs before annotation. The complete procedure can be found on the Scanpy website at the following link: https: / / scanpy-tutorials.readthedocs.io / en / latest / pbmc3k.html#Clustering-the-neighborhood-graph.

[0280] Two clusters were identified, one (cluster 1) consisting of data from two experiments using Jurkat cells (T cells) and the other (cluster 2) consisting of data from an experiment using Ramos cells (B cells), demonstrating that the method can be used to identify different (closely related) cell types.

[0281] Example

[0282] The oligonucleotides used in the examples have the sequences disclosed in Table 1.

[0283] Table 1: Oligonucleotide Structures

[0284]

[0285]

[0286] Example 1 - Total scRNA-seq in LCST cages with DNA arrays

[0287] Synthesis of thermo-actuated hydrogels

[0288] Synthesize olefin-functionalized poly(N-isopropylacrylamide) following the steps described in the supplemental information of the following literature: D'Eramo L. et al., Microsystems & Nanoengineering, 2018, 4, 17069, doi:10.1038.

[0289] The swelling properties of the obtained polymer were evaluated (as a function of temperature) in various aqueous solutions: pure water, phosphate buffer (pH 2 and pH 9). Results were... Figure 7 The information is provided in the text.

[0290] Fabrication of the first substrate

[0291] A first substrate made of polydimethylsiloxane (PDMS) containing microstructures and chambers is fabricated using standard soft lithography. The height of the structures and chambers depends on the target and can range from a few tenths of a micrometer to 100 micrometers.

[0292] Functionalization of the first substrate

[0293] After cleaning with isopropanol, the PDMS substrate was exposed to oxygen plasma for 50 seconds. Immediately after surface activation, an anhydrous toluene solution containing 3 vol% mercaptopropyltrimethoxysilane (ABCR Gelest) was brought into contact with the substrate in a reactor under nitrogen for 3 hours. After thiol modification of the surface, the substrate was rinsed with toluene and finally dried with a nitrogen stream.

[0294] The hydrogel film was optically patterned onto the first substrate.

[0295] Pre-formed functionalized pNIPAM (alkene-reactive type) was spin-coated onto a thiol-modified and microstructured PDMS substrate using a dithiol crosslinking agent. Small volumes of 100 μL butanol and methanol solutions (V / V = 1 / 1) containing 3% to 15% by weight of functionalized pNIPAM and 3% to 10% by weight of dithioerythritol (purchased from Sigma Aldrich, CAS No. 3483-12-3) crosslinking agent were deposited onto the substrate. Spin-coating conditions were fixed with angular velocities varying between 500 rpm and 3000 rpm and a spin-coating time of 30 seconds. The unfolded films were then dried in a 90°C oven for 5 minutes. The resulting layer thicknesses ranged from a few tenths of a micrometer to 15 micrometers.

[0296] A chromium mask exhibiting numerous microstructured cage-like structures was aligned with the chamber design and placed under a UV lamp for deep UV exposure (8 W, 250 nm wavelength). After exposure, the free polymer chains were rinsed off by washing the substrate in an ultrapure water bath for 5 minutes. The hydrogel-patterned substrate was then dried using a nitrogen stream.

[0297] Fabrication of the second substrate

[0298] The second substrate used was a glass slide dotted with DNA strands (purchased from Agilent and called Agilent Microarray Format).

[0299] It features up to a million unique spots, each grafted with different DNA strands, and the substrate provides a different barcode on each spot. Each spot contains millions of DNA strands.

[0300] DNA microarray structures such as Figure 8 As shown, each dot has a different barcode.

[0301] The localization system is integrated into the array design. Among the many unique spots, some carry specific sequences (two or more) for fluorescent tag capture. For example... Figure 9 As shown, they are arranged in a variety of shapes, including triangles, squares, and circles.

[0302] device shutdown

[0303] Bonding between the first and second substrates was achieved by treating with O2 plasma for 50 seconds. A protective layer was applied to the area of ​​interest to prevent activation by the oxygen plasma. After exposure termination, the PDMS substrate was placed on top of the DNA array, with the area of ​​interest facing the hydrogel structure. A curing step was performed by storing the chip in an oven at 70°C for at least 30 minutes.

[0304] Preparation of chips for specific capture: total scRNA-seq

[0305] 100 mM potassium acetate solution; 30 mM HEPES, pH 7.5; and 20 μM oligonucleotides A, B, C, D, and E were injected into the microfluidic chamber at 40 °C (with the cage open) to hybridize additional capture sequences to the immobilized DNA strand and perform the localization step. Flow was stopped, and the chip containing the mixed oligonucleotides was heated above 60 °C for 2 minutes, then cooled to room temperature for 10 minutes and washed with PBS solution at 37 °C.

[0306] Fluorescence imaging was performed at continuous wavelengths of 520 nm, 563 nm, 665 nm, and 704 nm in the main chamber to locate the fluorescence shape and spots relative to the cage.

[0307] Single-cell capture

[0308] Tests were conducted using different microfluidic geometries, and all of them showed a common feature: a large chamber that contained the hydrogel cage.

[0309] A cell suspension with a concentration of 10 million / ml was prepared using 1% Pluronic f68, 15% Optiprep, and 1% BSA in TBS. Cells were injected into the chip at a rate of 100 μl / h; the chip was heated at 37°C to open the cages. Once the cells had circulated around and above the cages, the flow was stopped, and the temperature was lowered to 20°C to close the cages. The cells were then trapped inside the cages.

[0310] To facilitate lysis, a low-salt aqueous solution is injected into the chamber through an additional inlet that is not blocked by the swollen hydrogel cage.

[0311] When the aqueous phase around the cage is altered with PBS, the RNA strands of the cells are then hybridized with the grafted capture sequence.

[0312] After stopping the flow for 10 minutes, a cleaning step is performed by opening the cage (raising the temperature to 37°C) and allowing PBS to flow through the chamber.

[0313] After thorough cleaning, a mixture containing 10 U / μl of reverse transcriptase (Thermo Scientific Maxima H+), 0.5 mM dNTPs, 1 U / μl of RiboLock RNase inhibitor, and 26 μM oligonucleotide F in 1x RT buffer was injected into the chamber. The chamber was then immobilized and flowed to reverse transcribe the captured strands at 55°C for 2 hours. The enzyme was immediately washed with PBS aqueous solution to stop RT.

[0314] A second mixture containing BmtI and Hi-T4 ligase was injected at 37°C into 1x CutSmart buffer supplemented with 1 mM ATP, and then stopped inside the chamber to ligate the barcoded strands to cDNA and isolate the cDNA from the DNA array. The internal volume was then collected by pushing the internal volume out with PBS solution.

[0315] The collection tube was then incubated at 65°C for 20 minutes to inactivate the previously injected enzyme.

[0316] After ExoI treatment, the collected samples were amplified by PCR using primers G and H.

[0317] The PCR products were then purified and quantified before sequencing.

[0318] Consider demultiplexing sequencing information from the barcodes at positions 30 to 44, the UMIs at positions 65 to 76, and the RNA transcripts starting at position 109 of Read1.

[0319] Example 2 - Targeted scRNA-seq in LCST cages with DNA arrays

[0320] The microfluidic device is the same as in Example 1.

[0321] Fabrication of chips for specific capture

[0322] Preparation is detailed in Example 1, where the 3' end of oligonucleotide B, poly(dT)VN, is replaced with a gene-specific sequence. A mixture of oligonucleotides targeting different genes or different parts of genes can be used.

[0323] Capture, lysis and library preparation

[0324] The capture, lysis, and hybridization steps are detailed in Example 1.

[0325] During reverse transcription, the 3' end of oligonucleotide F is replaced with a gene-specific sequence that targets a gene similar to oligonucleotide B but in the opposite manner, using the rGrG+G terminology. A mixture of oligonucleotides targeting different genes or different parts of a gene can be used.

[0326] The steps from release to sequencing are detailed in Example 1.

[0327] Demultiplexing of sequencing data is performed as described in Example 1.

[0328] Example 3 - Total scRNA-seq in a UCST cage with a DNA array

[0329] Synthesis of thermo-actuated hydrogels exhibiting UCST behavior

[0330] The olefin-functionalized UCST polymer was synthesized via free radical polymerization of methacrylamide (MA), acrylamide (AM), and allyl methacrylate (AMA) in a 90:5:5 molar ratio using 2,2'-azobis(2-methylpropanemidine) dihydrochloride (V50) as a thermal free radical initiator. MA (8 g, 94 mmol), AM (0.371 g, 5.2 mmol), AMA (0.659 g, 5.2 mmol), and V50 (0.071 g, 0.3 mmol) were mixed in 247 mL of water and 123 mL of formamide. The solution was deoxygenated for 1 hour under reflux at 55 °C by bubbling with nitrogen. This process was then carried out under reflux at 55 °C for 24 hours under nitrogen. The polymer solution was then dialyzed against pure water at 70 °C for four days. Finally, the olefin-functionalized UCST P (MA-AM-AMA) terpolymer was recovered by freeze-drying.

[0331] The swelling properties of the resulting polymer (as a function of temperature) were evaluated in phosphate buffer. Results were... Figure 10 The information is provided in the text.

[0332] Fabrication of the first substrate

[0333] Details are in Example 1.

[0334] Functionalization of the first substrate

[0335] Details are in Example 1.

[0336] The hydrogel film was optically patterned onto the first substrate.

[0337] At a temperature of at least 40°C, a P(MA-AM-AMA) terpolymer (an olefin-reactive UCST polymer) was spin-coated onto a thiol-modified and microstructured PDMS substrate using a dithiol crosslinking agent. Several 100 μL volumes of acetic acid solution (V / V = 1 / 1) containing between 3% and 15% by weight of the P(MA-AM-AMA) polymer and between 3% and 10% by weight of dithioerythritol (purchased from Sigma-Aldrich, CAS No. 3483-12-3) crosslinking agent were deposited onto the substrate. Spin-coating conditions were maintained with angular velocities varying between 500 rpm and 3000 rpm and a spin-coating time of 30 seconds. The unfolded films were then dried in a water-saturated oven at 90°C for 5 minutes. The resulting layer thicknesses ranged from a few tenths of a micrometer to 15 micrometers.

[0338] A chromium mask exhibiting numerous microstructured cage-like structures was aligned with the chamber design and placed under a UV lamp for deep UV exposure (8 W, 250 nm wavelength). After exposure, the free polymer chains were rinsed off by washing the substrate in an ultrapure water bath for 5 minutes. The hydrogel-patterned substrate was then dried using a nitrogen stream.

[0339] Fabrication of the second substrate

[0340] Details are given in Example 1.

[0341] Fabrication of a chip for specific capture: scRNA-seq

[0342] The fabrication of the chip is detailed in Example 1. The only difference is the temperature of these steps: the oligonucleotide injection and rinsing were performed at 20°C.

[0343] Single-cell capture

[0344] The capture process is detailed in Example 1, in which cell injection is performed at 20°C. The cage is closed by heating the chip to above 37°C.

[0345] The cleavage and hybridization steps are detailed in Example 1.

[0346] In this case, the cleaning process is carried out at room temperature.

[0347] The final steps are detailed in Example 1.

[0348] Example 4 - Total scRNA-seq using an LCST membrane and a PDMS substrate containing speckled chains in micropores

[0349] Synthesis of thermo-actuated hydrogels

[0350] See Example 1 for details.

[0351] Fabrication of the first substrate

[0352] A clean and new glass slide (1 mm thick) was used as the first substrate.

[0353] Functionalization of the first substrate

[0354] The thiol modification of the substrate was carried out following the same procedure given in Example 1.

[0355] Photopatterning of hydrogel membranes

[0356] Apart from the mask design, this scheme is the same as the one detailed in Example 1, but it presents a different design: a large rectangle that matches the width and length of the microfluidic chamber.

[0357] Fabrication of the second substrate

[0358] The second substrate used is made of PDMS and constructed following standard soft lithography techniques. The cavity contains an array of micropores with depths ranging from a few micrometers to 100 micrometers.

[0359] Based on the detection of microalgae causing harmful algal blooms using capture oligonucleotide probes covalently immobilized on glass and poly(dimethylsiloxane) surfaces (Analytical and Bioanalytical Chemistry. 2013.10.1117 / 12.2034011), the substrate was functionalized to immobilize amine-modified DNA strands within the micropores.

[0360] Using a standard DNA dotting protocol, 5' amine-modified single DNA strands are deposited and then grafted into microwells, each with a different barcode.

[0361] The structure of DNA micropores is as follows: Figure 11 As shown.

[0362] The positioning system is integrated into the array design, following the same scheme as in Example 1.

[0363] device shutdown

[0364] The device is shut down in the same manner as in Example 1, where the microarray to be protected is known to be on a PDMS substrate.

[0365] Fabrication of a chip for specific capture: scRNA-seq

[0366] 100 mM potassium acetate solution; 30 mM HEPES, pH 7.5; and 100 μM oligonucleotides C, D, E, and I were injected into the microfluidic chamber at 40 °C (with the cage open). Flow was stopped, and the chip containing the mixed oligonucleotides was heated above 60 °C for 2 minutes, then cooled to room temperature for 10 minutes, and rinsed with PBS solution at 40 °C.

[0367] Fluorescence imaging for localization was performed in the same manner as in Example 1.

[0368] Single-cell capture

[0369] The capture, lysis, hybridization, and RT steps are detailed in Example 1.

[0370] A solution containing the enzyme BmtI in 1x CutSmart buffer was injected at 37°C, and then stopped inside the chamber to link the barcoded strand to cDNA and separate the cDNA from the surface of the chip. The internal volume was then collected by pushing the internal volume out with PBS solution.

[0371] The collection tube was then incubated at 65°C for 20 minutes to inactivate the previously injected enzyme.

[0372] After ExoI treatment, the collected samples were amplified by PCR using primers J and H.

[0373] The PCR products were then purified and quantified before sequencing.

[0374] Consider demultiplexing sequencing information from barcodes at positions 1 to 15, UMIs at positions 16 to 27, and RNA transcripts starting at position 60 of Read1.

[0375] Example 5 - scCITE-seq in an LCST cage with a DNA array

[0376] The microfluidic device is the same as in Example 1.

[0377] Fabrication of chips for specific capture

[0378] The preparation is detailed in Example 1, where oligonucleotide B is supplemented with 10% oligonucleotide K.

[0379] Cell markers

[0380] Cells are labeled with subset-specific antibodies carrying antibody-specific barcodes, such as... Figure 12 As shown.

[0381] The antibody was conjugated with streptavidin (e.g., using the Streptavidin Conjugation Kit from abcam-ab102921 - Lightning-Link). Prior to cell labeling, the antibody was conjugated as follows: Figure 6 The biotin-conjugated oligomer described in section b was added to the antibody solution at a ratio of 12:1.

[0382] Capture, lysis and library preparation

[0383] The capture, lysis, and hybridization steps are detailed in Example 1.

[0384] After thorough cleaning, a mixture containing 10 U / μl of reverse transcriptase (Thermo Scientific Maxima H+), 0.5 mM dNTPs, 1 U / μl of RiboLock RNase inhibitor, and 26 μM oligonucleotide F in 1x RT buffer was injected into the chamber. The chamber was then immobilized and flowed to reverse transcribe the captured strands at 55°C for 2 hours. The enzyme was immediately washed with PBS to stop RT.

[0385] A second mixture containing BmtI and Hi-T4 ligase was injected at 37°C into 1x CutSmart buffer supplemented with 1 mM ATP, and then stopped inside the chamber to ligate the barcoded strands to cDNA and isolate the cDNA from the DNA array. The internal volume was then collected by flowing PBS solution.

[0386] The collection tube was then incubated at 65°C for 20 minutes to inactivate the previously injected enzyme.

[0387] After ExoI treatment, the collected samples were amplified by PCR using primers G, H, and L.

[0388] The PCR products were then purified and quantified before sequencing.

[0389] As described in Example 1, the sequencing data was demultiplexed, and the antibody tag starting from position 97 of Read1 was observed.

[0390] Example 6 - Total scRNA-seq in LCST cages with bridge-shaped DNA arrays

[0391] Preparation of the first substrate

[0392] The solution is detailed in Example 1.

[0393] Fabrication of the second substrate

[0394] DNA arrays are detailed in Example 1, and this example includes... Figure 13 sequence shown.

[0395] device shutdown

[0396] Detailed in Example 1.

[0397] Fabrication of a chip for specific capture: scRNA-seq

[0398] A solution containing enzyme Nb.BstI in 1x CutSmart buffer was injected into the microfluidic chamber at 37°C (with the cage open) to perform the inversion step.

[0399] Stop the flow at 37°C for 10 minutes, and then rinse with PBS solution at 37°C.

[0400] A solution containing the enzyme Hi-T4 ligase in 1x CutSmart buffer supplemented with 1 mM ATP and 20 μM oligonucleotides M and N was injected into the microfluidic chamber at 37°C (with the cage open) to perform the extension step.

[0401] Stop the flow at 37°C for 10 minutes, and then rinse with PBS solution at 37°C.

[0402] Then, 100 mM potassium acetate solution, 30 mM HEPES, pH 7.5, and 20 μM oligonucleotides C, D, and E were injected into the microfluidic chamber at 40 °C to hybridize the fluorescent sequence to the immobilized DNA strand (localization step).

[0403] Stop the flow, heat the chip containing the mixed oligonucleotides at above 60°C for 2 minutes, then cool at room temperature for 10 minutes, and rinse with PBS solution at 37°C.

[0404] Fluorescence imaging for localization was performed in the same manner as in Example 1.

[0405] Capture, lysis and library preparation

[0406] The capture, lysis, and hybridization steps are detailed in Example 1.

[0407] After thorough cleaning, a mixture containing 10 U / μl of reverse transcriptase (Thermo Scientific Maxima H+), 0.5 mM dNTPs, 1 U / μl of RiboLock RNase inhibitor, and 26 μM oligonucleotide F in 1x RT buffer was injected into the chamber. The chamber was then immobilized and flowed to reverse transcribe the captured strands at 55°C for 2 hours. The enzyme was immediately washed with PBS to stop RT.

[0408] A second mixture containing the enzyme NheI in 1x CutSmart buffer was injected at 37°C, and then stopped inside the chamber to ligate the barcoded strands to cDNA and isolate the cDNA from the DNA array. The internal volume was then collected by pushing the internal volume out with PBS solution.

[0409] The collection tube was then incubated at 65°C for 20 minutes to inactivate the previously injected enzyme.

[0410] After ExoI treatment, the collected samples were amplified by PCR using primers O and H.

[0411] The PCR products were then purified and quantified before sequencing.

[0412] Consider demultiplexing sequencing information from the barcodes at positions 22 to 36, the UMIs at positions 63 to 74, and the RNA transcripts starting at position 107 of Read1.

[0413] Example 7 - scATAC-seq in UCST cages

[0414] Fabrication of the first substrate

[0415] The solution is detailed in Example 3.

[0416] Fabrication of the second substrate

[0417] DNA arrays are detailed in Example 3, which is included in this example. Figure 14 sequence shown.

[0418] device shutdown

[0419] Detailed in Example 3.

[0420] Fabrication of chips for specific capture: scATAC-seq

[0421] A solution containing the enzyme T4 polynucleotide kinase (NEB M0201) in 1X T4 polynucleotide kinase reaction buffer was injected into the microfluidic chamber at 20°C (with the cage open) and incubated at 37°C for 30 minutes to perform 5' phosphorylation of the strands anchored to the DNA array.

[0422] The chamber was then rinsed with PBS solution at 20°C.

[0423] 100 mM potassium acetate solution; 30 mM HEPES, pH 7.5 and 20 μM oligonucleotides C, D and E were injected into the microfluidic chamber at 20 °C to hybridize additional fluorescent sequences to the immobilized DNA strand (localization step).

[0424] Stop the flow, heat the chip with the mixed oligonucleotides at above 60°C for 2 minutes, then cool at room temperature for 10 minutes, and rinse with PBS solution at 20°C.

[0425] Fluorescence imaging for localization was performed in the same manner as in Example 1.

[0426] A solution containing enzyme phi29 in 1x phi29 DNA polymerase reaction buffer with 20 μm oligonucleotide A was injected into the microfluidic chamber at 20 °C (with the cage open) to generate double-stranded DNA aptamers (barcoded MEDS).

[0427] After incubating at 37°C for 10 minutes, the chamber was rinsed with PBS solution at 20°C.

[0428] Capture, lysis and library preparation

[0429] The capture and lysis are detailed in Example 3.

[0430] Inject a mixture containing enzyme BmtI in 1x CutSmart buffer at 37°C, then stop in the chamber and incubate at 37°C for 10 minutes to release dsDNA from the DNA array.

[0431] The mixture containing Nextera Tn5 transposase (TDE1) in 1x TD reaction buffer was then injected into the chamber at 37°C, and the flow was then fixed at 37°C for 30 minutes to allow transposition.

[0432] The internal volume was then collected by flowing the aqueous solution at 20°C.

[0433] After ExoI treatment, the collected samples were amplified by PCR using primers G and P.

[0434] The PCR products were then purified and quantified before sequencing.

[0435] Consider demultiplexing sequencing information from the barcodes at positions 30 to 44 and from the DNA transcript at position 64 of Read1.

[0436] References: Buenrostro, J., Wu, B., Litzenburger, U. et al. Single-cell chromatinaccessibility reveals principles of regulatory variation. Nature 523, 486–490 (2015)

[0437] Example 8 - Total RNA-seq from LCST cages with DNA arrays starting with purified RNA

[0438] Synthesis of thermo-actuated hydrogels

[0439] Details are in Example 1.

[0440] Fabrication of the first substrate

[0441] Details are in Example 1.

[0442] Functionalization of the first substrate

[0443] Details are in Example 1.

[0444] The hydrogel film was optically patterned onto the first substrate.

[0445] Details are in Example 1.

[0446] Fabrication of the second substrate

[0447] The second substrate used was a glass slide dotted with DNA strands (purchased from Agilent Technologies and known as the Agilent Microarray Format).

[0448] It features up to a million unique spots, each grafted with different DNA strands, and the substrate provides a different barcode on each spot. Each spot contains millions of DNA strands.

[0449] DNA microarray structures such as Figure 15 As shown, each dot has a different barcode.

[0450] The positioning system is integrated into the array design. Among the many unique spots, some carry specific sequences (two or more) of DNA oligonucleotides used to capture fluorescently labeled DNA through hybridization. Figure 9 As shown, they are arranged to form various shapes, including triangles, squares, and circles.

[0451] device shutdown

[0452] Details are in Example 1.

[0453] Preparation of chips for specific capture: Total RNA-seq

[0454] The oligonucleotides were designed in a computer and acquired from IDT, and were desalted in standard at concentrations of 25 μM to 100 μM in IDTE buffer (pH 8.0).

[0455] A solution of 100 mM potassium acetate, 30 mM HEPES (pH 7.5), and 10 μM oligonucleotides Q, R, and S was injected into the microfluidic chamber at 40 °C (with the cage open) to hybridize additional capture sequences to the immobilized DNA strand and perform the localization step. Flow was stopped, and the chip containing the mixed oligonucleotides was heated above 60 °C for 2 minutes, then incubated at room temperature for 10 minutes and washed at 40 °C with 1x SSC solution (Thermo Scientific #15413549).

[0456] Bright-field and fluorescence imaging of the main chamber was performed (excitation at 650 nm and emission at 670 nm). For example... Figure 16 The image shows the identification of DNA array spots and cage-like structures. This identification is based on the overlay of two images. The bright-field image allows for the identification of cage-like structures. Figure 16 The black circles A, B, and C in the image represent fluorescent patterns that allow for the identification of certain spots on which the fluorescent DNA probes have previously hybridized. The relative positions of these circles define unique locations on the chip, allowing the original organization of the DNA array to be mapped onto the overlay image. Finally, thanks to this identification, we can associate spot-specific barcodes with each cage.

[0457] Primer synthesis .

[0458] A mixture of Thermopol 1x (NEB#B9004S) containing sulfur fern DNA polymerase IV (NEB#M0327S) and Hi-T4 ligase (NEB#M2622S) supplemented with 1 mM ATP (NEB#P0756S) was injected at 40°C, followed by incubation at 50°C inside the chamber for 2 hours to polymerize and ligate barcoded primers. The internal volume was then washed with 1x SSC solution (ThermoScientific#15413549) at 40°C.

[0459] Cell culture

[0460] Jurkat human T lymphocytes were cultured in RPMI 1640 medium (Gibco#61870044) supplemented with 10% heat-inactivated fetal bovine serum (Gibco#10082147) and 1% penicillin-streptomycin (Gibco#15140122). TIB-152. Following ATCC recommendations, cells were seeded at 37°C and 5% CO2 in a 25cm² incubator. 2 Or 75cm 2 In culture flasks. When the cells reach 75% to 80% confluence, dilute them. After removing them from the cell culture, centrifuge the cells at 130 rpm for 5 minutes.

[0461] mRNA chain capture

[0462] Total RNA was purified from Jurkat cells using the Qiagen RNeasy mini-kit (Qiagen #74104) and injected with 1x SSC and 1 U / μl of an RNase inhibitor (Thermo Scientific #11581505) at 40°C. The microfluidic apparatus was then incubated at 65°C for 5 minutes, gradually lowered to 4°C, and subsequently incubated at 25°C for 5 minutes. The mRNA strand was then hybridized to the grafted capture sequence.

[0463] Barcoded cDNA Synthesis

[0464] The cleaning procedure was performed by opening the cage at 37°C and allowing RT buffer (ThermoScientific#EP0742) equivalent to 20 times the internal volume of the chamber to flow through the chamber for 3 minutes.

[0465] After cleaning, a mixture containing 10 U / μl of reverse transcriptase (Thermo Scientific #EP0742), 0.5 mM dNTPs (NEB #N0447S), and 1 U / μl of RNase inhibitor (Thermo Scientific #11581505) in 1x RT buffer was injected into the chamber. The flow was then fixed, and the captured strands were reverse transcribed at 50°C for 2 hours. The enzyme was immediately washed with 1x SSC solution to stop reverse transcription.

[0466] Inject 1x exonuclease I reaction buffer (NEB#M0293S) containing 2 U / μl of exonuclease I in a second mixture and incubate at 37°C for 30 minutes. Immediately wash the enzyme with 1x SSC solution to stop the reaction.

[0467] Distilled water (Invitrogen #10977023) free of DNase / RNase was injected into the chamber and heated to 98°C to dehybridize the synthesized cDNA. The internal volume was then collected by circulating additional water.

[0468] Add 10 μL of NEB2 buffer (NEB#M0212L) and 10 μL of 10 μM oligonucleotide T to 65 μL of recovered sample.

[0469] Incubate the solution at 95°C for 2 minutes and immediately transfer it to ice. Then, add 8 μL of 10 nM dNTP and 7 μL of Klenow exo-(NEB#M0212L).

[0470] The mixture was placed in a thermal circulator pre-cooled at 4°C, and the temperature was slowly raised to 37°C and held for 30 minutes to synthesize the second chain.

[0471] Then, follow the manufacturer's instructions to purify the cDNA using SPRIselect magnetic beads (Beckman #B23317).

[0472] The purified sample was then subjected to 15 cycles of 30-second extended two-step PCR amplification using primers G and H and Q5 high-fidelity DNA polymerase (NEB#M0491).

[0473] The PCR products were purified and quantified using SPRIselect magnetic beads (Beckman #B23317), and then sequenced using the Illumina sequencing platform.

[0474] Sequencing information was demultiplexed by retrieving the barcodes from positions 1 to 15 of the Index1 read, the UMIs from positions 1 to 12 of the Read1 read, and the RNA transcripts starting from position 1 of the Read2 read for each read.

[0475] Of the 1,504,957 complete sequencing reads, we identified 738,483 unique cDNAs, which were determined to be reads with unique barcodes, unique genomes, and unique UMIs. We have identified the mean of 102 reads per barcode and the median of 50 UMIs per barcode. The low number of reads per UMI indicates that the sequencing depth is not yet saturated.

[0476] Figure 17 This represents the spatial redistribution of 14,752 barcodes extracted from valid gene reads in the presented sequencing data within the microfluidic device. Each black circle represents a DNA spot from the DNA array whose barcode is associated with at least one cDNA in the sequencing data. Missing circles are DNA spots from the DNA array whose barcodes were not found to be associated with at least one cDNA in the sequencing data. The hexagonal shape, represented by the location of the recovered barcodes, corresponds to regions protected from oxygen plasma during microfluidic device fabrication. Most spots within the protected regions have been identified in the sequencing data.

[0477] 14,507 unique genes were extracted from the read segments that presented valid barcodes.

[0478] Figure 18 The figure shows the length distribution of genes identified by sequencing in Example 8. Subplot a shows the length distribution of 20,465 genes annotated as protein-coding genes in gene code 38 of component GRCh38.p13 of the Genome Reference Consortium, annotated by Ensembl version 104.38 (reference: http: / / may2021.archive.ensembl.org / Homo_sapiens / Info / Annotation). Gene length was determined as the sum of exon lengths. For each gene, the average size of alternative splicing variants was used. Sub-image b represents the length distribution of the same database of only 14,507 unique genes identified from valid gene and barcode reads presented in the sequencing data. The similarity of the size distribution in subplots a and b indicates that there is no size bias in gene recovery.

[0479] The treatment was repeated twice with Jurkat cells and Ramos human B lymphocytes. CRL-1923 cells were replicated once (cultured under conditions similar to Jurkat cells). Figure 19 This refers to the annotated UMAP (McInnes, L, Healy, J, UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction, ArXiv e-prints 1802.03426, 2018) used for clustering the triplicate replicates of Example 8 using two different cell lines. The RNA-seq described in Example 8 was repeated twice with Jurkat human T cells and once with Ramos human B cells. For each experiment, 15,000 barcodes were randomly grouped to form 200 superbarcodes to address the issue of shallow sequencing depth and a small number of cDNAs identified per barcode, which prevented clustering. Then, principal component analysis (PCA) was run using the ScanpyPython software (https: / / scanpy.readthedocs.io), followed by Leiden plot clustering (Traag et al., 2018, From Louvain to Leiden: guaranteeing well-connected communities), and the clusters were represented in two dimensions using UMAP plots before annotation. The complete procedure can be found on the Scanpy website at the following link: https: / / scanpy-tutorials.readthedocs.io / en / latest / pbmc3k.html#Clustering-the-neighborhood-graph.

[0480] Two clusters were identified, one (cluster 1) consisting of data from two experiments using Jurkat cells (T cells) and the other (cluster 2) consisting of data from an experiment using Ramos cells (B cells), demonstrating that the method can be used to identify different (closely related) cell types.

[0481] Example 9 - LCST cages with DNA arrays starting from a mixture of Jurkat and Ramos human cells Total scRNA-seq .

[0482] The procedure is the same as in Example 8 except that cells are injected, captured, and lysed in a chip as described in Example 1, instead of injecting purified RNA in a microfluidic device.

Claims

1. A microfluidic device (10) comprising: - a first wall (12) comprising a first substrate (14) on which a plurality of confinement patterns (16) are grafted, - a second wall (18) facing the first wall (12) and comprising a second substrate (20), - a plurality of nucleic acids (22) grafted on the first substrate (14) or on the second substrate (20), wherein each nucleic acid (22) comprises a barcode encoding the position of the nucleic acid on the first substrate (14) or on the second substrate (20), wherein at least the plurality of confinement patterns (16) or the second substrate (20) are made of an actuatable hydrogel capable of swelling between a contracted state and a swollen state in which the confinement patterns (16) and the second substrate (20) are in contact.

2. The microfluidic device (10) according to claim 1, wherein the actuatable swelling hydrogel is a temperature-responsive swelling hydrogel.

3. The microfluidic device (10) according to claim 2, wherein the temperature-responsive hydrogel has a critical solution temperature range of 4°C to 98°C.

4. The microfluidic device (10) according to claim 2, wherein the temperature-responsive hydrogel has a critical solution temperature range of 20°C to 50°C.

5. The microfluidic device (10) according to claim 2, wherein the temperature-responsive hydrogel has a critical solution temperature range of 25°C to 40°C.

6. The microfluidic device (10) according to claim 3, wherein the critical solution temperature is a lower critical solution temperature above which the temperature-responsive hydrogel is in the contracted state and below which the temperature-responsive hydrogel is in the swollen state.

7. The microfluidic device (10) according to claim 3, wherein the critical solution temperature is an upper critical solution temperature above which the temperature-responsive hydrogel is in the swollen state and below which the temperature-responsive hydrogel is in the contracted state.

8. The microfluidic device (10) according to any one of claims 1 to 7, wherein the polymeric matrix of the hydrogel comprises a thermoresponsive polymer selected from homopolymers, copolymers of acrylic acid, alkyl (meth)acrylate, alkyl (meth)acrylamide, oligoethylene (meth)acrylate, sulfobetaine (meth)acrylate and N-acryloylglycine amide.

9. The microfluidic device (10) according to claim 8, the thermoresponsive polymer is selected from terpolymers of acrylic acid, alkyl (meth)acrylate, alkyl (meth)acrylamide, oligoethylene (meth)acrylate, sulfobetaine (meth)acrylate and N-acryloylglycine amide.

10. The microfluidic device (10) according to claim 8, the thermoresponsive polymer is selected from homopolymers of alkyl (meth)acrylamide.

11. The microfluidic device (10) according to any one of claims 1 to 7, the polymer matrix of the hydrogel comprising a thermoresponsive polymer, the thermoresponsive polymer being poly(N-isopropylacrylamide).

12. The microfluidic device (10) according to any one of claims 1 to 7, further comprising at least one inlet (24) and at least one outlet (26) allowing the introduction of reactants into the microfluidic device (10) or removal from the microfluidic device, respectively.

13. The microfluidic device (10) according to any one of claims 1 to 7, wherein in case the second substrate is not an actuatable hydrogel, the first substrate (14) and the second substrate (20) are independently made of a material selected from the group consisting of silicon, quartz, glass, polydimethylsiloxane, thermoplastic.

14. The microfluidic device (10) according to any one of claims 1 to 7, wherein in case the second substrate is not a hydrogel, the first substrate (14) and the second substrate (20) are independently selected from the group consisting of glass, polydimethylsiloxane.

15. The microfluidic device (10) according to any one of claims 1 to 7, wherein the plurality of enclosed patterns (16) are made of the actuatable swellable hydrogel and the second substrate (20) is made of a non-swellable material.

16. The microfluidic device (10) according to any one of claims 1 to 7, wherein the second substrate (20) is made of the actuatable swellable hydrogel and the enclosed patterns (16) are made of a non-swellable material.

17. The microfluidic device (10) according to any one of claims 1 to 7, wherein a plurality of ligands are grafted on the first substrate (14) and / or on the second substrate (20).

18. The microfluidic device (10) according to any one of claims 1 to 7, wherein a plurality of ligands conjugated to nucleic acids are associated with at least part of the grafted nucleic acids (22) by hybridization.

19. The microfluidic device (10) according to claim 17, wherein each ligand is independently selected from the group consisting of an antibody, a lectin, an aptamer.

20. The microfluidic device (10) according to any one of claims 1 to 7, wherein nucleic acids (22) sharing the same barcode have a plurality of sequences.

21. The microfluidic device (10) according to any one of claims 1 to 7, wherein nucleic acids (22) comprise one or any combination of the following sequences: 1) a restriction site or a photocleavable site for nucleic acid release, 2) a sequence complementary to amplification primers for further amplification, 3) a T7 RNA polymerase promoter sequence for in vitro transcription (IVT), 4) a hybridization site, a ligation site or a recombination site for nucleic acid labeling, and 5) a sequence of random nucleotide residues acting as unique molecular identifiers (UMIs).

22. A method of manufacturing the microfluidic device (10) according to claim 1, the method comprising: a) providing a first substrate (14), b) grafting a plurality of closed patterns (16) on the surface of the first substrate (14), c) providing a second substrate (20), d) grafting a plurality of nucleic acids (22) on the surface of the first substrate (14) or on the surface of the second substrate (20), wherein each nucleic acid (22) comprises a barcode encoding the position of the nucleic acid (22) on the first substrate (14) or on the second substrate (20); e) positioning the first substrate (14) and the second substrate (20) by placing the closed patterns (16) and the nucleic acids (22) between the first substrate (14) and the second substrate (20), f) bonding the first substrate (14) and the second substrate (20).

23. The method according to claim 22, further comprising: A) grafting a plurality of ligands directly on the surface of the first substrate (14) and / or on the surface of the second substrate (20); or B) associating a plurality of ligands with the plurality of grafted nucleic acids (22) by hybridization, the plurality of ligands being conjugated to nucleic acids complementary to at least a portion of the grafted nucleic acids (22); or C) coating at least a portion of the surface of the first substrate (14) and / or of the second substrate (20) with an adhesion coating and associating a plurality of ligands with the adhesion coating by non-covalent binding.

24. The method according to any one of claims 22 and 23, wherein the method further comprises: 1) hybridizing DNA comprising a sequence complementary to all or a portion of the constant sequence present in the grafted nucleic acids (22); or 2) extending the hybridized DNA by polymerization; or 3) ligating the grafted nucleic acids (22) to a DNA sequence; and 4) releasing all or a portion of the grafted nucleic acids (22) from the surface of the first substrate (14) or of the second substrate (20) by cleavage, the grafted nucleic acids having been previously modified by hybridization, extension or ligation according to 1), 2) or 3).

25. A method of performing a cellular analysis, the method comprising: a) providing a microfluidic device (10) according to any one of claims 1 to 7 and a preparation of cells; b) injecting the cells in suspension into the microfluidic device (10) under conditions in which the hydrogel is in a contracted state; c) changing conditions to actuate the hydrogel to a swollen state, thereby trapping cells in a cage formed by the first wall (12) and the second wall (18) of the microfluidic device (10) and in the closed patterns (16) of hydrogel in the swollen state; d) lysing the trapped cells, thereby releasing cellular nucleic acids in the cage; e) associating the barcodes of the grafted nucleic acids (22) with the released cellular nucleic acids, thereby forming barcoded nucleic acids; f) changing conditions to actuate the hydrogel to the contracted state; g) recovering and sequencing the barcoded nucleic acids; the method further comprising: after c) and before d): releasing the grafted nucleic acids (22) from the surface of the first substrate (14) or the second substrate (20) of the microfluidic device (10); or after f) and before g): releasing the grafted nucleic acids (22) from the surface of the first substrate (14) or the second substrate (20) of the microfluidic device (10).

26. The method of claim 25, further comprising: after a) and before b), all or part of the cells are associated with a common labeled nucleic acid sequence or with a plurality of different labeled nucleic acid sequences, and in step e), the barcodes of the grafted nucleic acids are also associated with the labeled nucleic acids, thereby forming barcoded nucleic acids.

27. The method of claim 25, further comprising, after c) and before d): cl) analyzing the captured cells and / or molecules they secrete using optical imaging.

28. The method of claim 27, further comprising, after g): h) mapping the barcoded sequencing data onto the data from optical imaging obtained in cl).

29. The method of claim 25, wherein the method further comprises: el) binding one or more analytes secreted or released by the captured cells to a ligand grafted directly or indirectly to the surface of the first substrate (14) and / or to the surface of the second substrate (20); e2) detecting the one or more analytes bound to the grafted ligand by binding to a labeled second ligand specific to the bound one or more analytes.

30. The method of claim 29, wherein in step e2), the detection is performed as follows: i) directly with one or more second ligands labeled with fluorescence; or ii) indirectly, with one or more second ligands labeled with a ligand identification nucleic acid specific to the one or more analytes bound to the grafted ligand, wherein the sequence of the ligand identification nucleic acid allows identification of the ligand and the one or more analytes bound to the grafted ligand and is associated with the barcode of the nucleic acid (22), thereby forming barcoded nucleic acids.

31. A method of performing organelle analysis, the method comprising: a) providing a microfluidic device (10) according to any one of claims 1 to 7 and a preparation of organelles; b) injecting the organelles in suspension into the microfluidic device (10) under conditions in which the hydrogel is in a contracted state; c) changing conditions to actuate the hydrogel to a swollen state, thereby trapping organelles in a cage formed by the first wall (12) and the second wall (18) of the microfluidic device (10) and in a closed pattern (16) of hydrogel in a swollen state; d) lysing the trapped organelles, thereby releasing organelle nucleic acids in the cage; e) associating barcodes of the grafted nucleic acids (22) with the released organelle nucleic acids, thereby forming barcoded nucleic acids; f) changing conditions to actuate the hydrogel to the contracted state; g) recovering and sequencing the barcoded nucleic acids; the method further comprising: after c) and before d): releasing the grafted nucleic acids (22) from the surface of the first substrate (14) or the second substrate (20) of the microfluidic device (10); or after f) and before g): releasing the grafted nucleic acids (22) from the first substrate (14) or the second substrate (20) of the microfluidic device (10).

32. The method of claim 31, further comprising: after a) and before b), all or part of the organelles are associated with a common marker nucleic acid sequence or with a plurality of different marker nucleic acid sequences, and in step e), the barcodes of the grafted nucleic acids are also associated with the marker nucleic acids, thereby forming barcoded nucleic acids.

33. The method of claim 31, further comprising after c) and before d): cl) analyzing the captured organelles and / or molecules secreted by them using optical imaging.

34. The method of claim 33, further comprising after g): h) mapping the barcoded sequencing data onto the data from optical imaging obtained in cl).

35. The method of claim 31, wherein the method further comprises: el) binding one or more analytes secreted or released by the captured organelles to a ligand grafted directly or indirectly to the surface of the first substrate (14) and / or to the surface of the second substrate (20); e2) detecting the one or more analytes bound to the grafted ligand by binding to a labeled second ligand specific to the bound one or more analytes.

36. The method of claim 35, wherein in step e2), the detection is performed: i) directly with one or more second ligands labeled with fluorescence; or ii) indirectly, with one or more second ligands labeled with a ligand identification nucleic acid specific to the one or more analytes bound to the grafted ligand, wherein the sequence of the ligand identification nucleic acid allows identification of the ligand and the one or more analytes bound to the grafted ligand and is associated with the barcode of the nucleic acid (22), thereby forming a barcoded nucleic acid.

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