High-throughput screening in droplets

Abstract

The present disclosure generally relates to compositions, methods, sorters, systems, devices, and uses for screening bioactive substances in emulsion droplets. In some embodiments, the composition is a continuous phase formulation for a stable emulsion. In some embodiments, the method is for preparing a monodisperse polyethylene glycol acrylamide (PEGA) copolymer resin or for preparing core-shell beads. In some embodiments, the system and device includes a sorter including an inlet channel, first and second outlet channels that merge with the inlet channel at a junction, and first and second electrodes proximate the first and second sides of the junction sorter, respectively. In some embodiments, the system and device includes a sorter including a microwell array plate configured to host one microdroplet per microwell, a fluorescent microscope, an imager configured for automated image assay of the droplets to identify desired droplets, and an automated microcapillary-based droplet sampling device configured for sequentially depositing the desired droplets into the hit wells.

Description

[Technical Field]

[0001] This disclosure generally relates to methods, compositions, sorters, systems, uses, and devices for screening bioactive substances in emulsion droplets. More specifically, this disclosure generally relates to methods, compositions, sorters, systems, uses, and devices for emulsion formulations, codeable compound beads, and high-throughput droplet selection, both as individual components and as aggregate platforms. [Background technology]

[0002] This disclosure generally relates to methods, compositions, sorters, systems, uses, and devices for screening bioactive substances in emulsion droplets. This disclosure also relates to microfluidic encapsulation, i.e., microencapsulated droplets containing cells and barcode compound beads. Such microencapsulated droplets have applications in a variety of research and analytical fields, including, but not limited to, high-throughput screening of compounds within microencapsulated droplets, carrying out a series of chemical reactions within microencapsulated droplets, and analyzing cell culture conditions.

[0003] In robot-based high-throughput screening (HTS) for discovering bioactive substances, screening compartments can be provided by microtiter well plates capable of containing reaction volumes up to tens of microliters and easily traceable via plate maps and plate barcodes. However, this process can be costly and resource-intensive (Inglese et al. 2007, MacArron et al. 2011, Ross 2017). HTS of large compound sets is typically performed in single doses due to the exorbitant cost, which can lead to high false-positive rates and failure to distinguish between weak but robust hit molecules and non-hits.

[0004] The significantly miniaturized picoliter compartments enabled by droplet microfluidics allow for scaling compound screening down to single-cell size, thus addressing many of the challenges of the robot-based high-throughput screening described above. Cell assays in HTS may require the development of biological assays in readily available artificially immortalized cell systems, which necessitate downstream hit triaging for more disease-related assay applications, such as using cells derived from induced pluripotent stem cells (iPSCs) or cells in primary disease states, which are difficult to scale. Reports of cell screens performed with picoliter-volume microdroplets exist. However, such methods have almost without exception been reported when the diversity source is an inherently hydrophilic biological molecule, such as proteins, peptides, oligonucleotides, or even organisms such as bacteria. Droplet-based cell screens using diversity sources such as libraries of more hydrophobic organic compounds are less common due to challenges such as compound retention over the duration of the assay, delivery of barcoded compound libraries such as fluorogenic silent barcoded compound libraries with high efficiency, and reliable selection of hit droplets at high frequency. Therefore, in relation to cell assays, it is important that the compound can be contained within the droplet and that the compound beads can be efficiently introduced into the droplet in the cell compatibility assay medium.

[0005] The precise delivery of individual library members into a single coded droplet can be challenging. Converting plate-based compound collections to coded compound droplets can present inherent challenges related to scalability and / or compound retention. Alternatively, a DNA-coded 1-bead 1-compound library loaded via a release linker allows for the delivery of individual library members into a single droplet (MacConnell, Price, and Paegel 2017; MacConnell et al. 2015). This method substantially reduces the requirement for compound retention from the in-situ release point to the assay readout point, while simultaneously leveraging the capabilities of combinatrices and achieving droplet and compound coding. However, these platforms present technical challenges in relation to pharmaceuticals. To date, library beads have not been demonstrated to simultaneously satisfy the following properties in cell compound screening within droplets: 1) Monodispersibility, 2) Biocompatibility, 3) Suspension and compressibility in aqueous media, 4) Low background autofluorescence, and 5) Compatibility with a wide range of library chemistry in organic phases.

[0006] TentaGel® (Rapp Polymere) is a PEG-grafted polystyrene backbone polymer used in previously reported DNA-coding 1-bead-1-compound libraries (MacConnell et al., 2015, 2017). It has also been widely used in solid-phase organic synthesis (Toy, 2004). However, it is also known for its autofluorescence (Townsend et al., 2010), and moreover, it can aggregate as a clamp in aqueous culture media, especially when loaded with relatively hydrophobic organic molecules.

[0007] PEGA resins are PEG-crosslinked polyacrylamide-backed hydrogel beads. PEGA may share the non-fluorescent hydrophilicity of polyacrylamide, while exhibiting excellent swelling in organic solvents and broader organic reaction compatibility than PEG-based polymer resins. The first compositions of PEGA resins were reported by Meldal et al. in the 1990s (Auzanneau et al., 1995; Meldal, 1992). At least one composition of PEGA resins with various functional handles is commercially available (Novabiochem / Merck-Millipore). However, these commercially available resins contain relatively large sizes (150-300 microns in diameter) and are polydispersible, thus failing to meet the criteria for compound delivery matrices for screening in droplets with a diameter of 10-200 microns.

[0008] Droplet-based microfluidics may include fluorescence-activated droplet sorting (FADS). FADS may offer advantages over traditional FACS approaches. For example, FADS may involve the integration of assays involving single-cell manipulation, real-time analysis of single cells, sequential cell treatment and final detection, or a combination thereof (Caen, O. et al, 2019). Furthermore, FADS can also enable the realization of complex cell assays, as well as reduce sample volume and waste. To date, a wide variety of FADS approaches exist, including pneumatic, acoustic, thermal, magnetic, and electric actuation (Xi, H. et al). However, to date, there is no off-the-shelf droplet sorter that can reliably perform cell compound screening in droplets under continuous operation, achieve robust high-throughput screens, and possess the flexibility of multiple wavelength readout and corresponding ratiometric sorting.

[0009] In addition to FADS, droplet arraying can provide further flexibility in assay readout. Further manipulation and selection of desired droplets remain untapped.

[0010] Therefore, there is a need in the art to provide an improved droplet platform that has sufficient compound holdings for cell assays in HTS, is in a library bead format suitable for efficient encapsulation into higher-throughput droplets, and offers a reliable and efficient sorting method for hit droplets.

[0011] This disclosure overcomes previous shortcomings in the art by providing novel methods, compositions, sorters, systems, uses, and devices for screening bioactive substances in emulsion droplets. [Overview of the Initiative]

[0012] The objective is to provide a droplet platform that enables efficient cell compound screening in droplets. This capability, coupled with established detection methods, provides a crucial tool for rigorous single-cell scale cell analysis that can reveal novel biological mechanisms and responses with unprecedented sensitivity and / or scale, making it available for testing, tuning, and use in the pharmaceutical industry. Another objective is to provide a droplet platform that enables efficient cell compound screening in droplets without excessive use of reagents or cells. This is particularly important for screening large compound libraries using rare cells.

[0013] This disclosure is based on the finding that significantly higher compound retention in droplets can be achieved by using the continuous phase formulations described herein. Specifically, the continuous phase formulation comprises at least one fluorescein dispersion oil (the fluorescein dispersion oil having an average fluorine content of about 70 wt% or more) and a droplet stabilizer comprising an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof.

[0014] This specification provides compositions, methods, sorters, devices, uses, and systems that can be used for screening compound libraries. In some embodiments, the methods, systems, and devices can be used for screening individual cells or groups of individual cells. In some embodiments, the methods, systems, and devices can be used for screening compound libraries. In some embodiments, the methods and devices provided herein can be used for screening compound libraries delivered as code beads. In some embodiments, the methods, systems, and devices provided herein can be used for screening compound libraries delivered as code beads and released into picoliter droplet compartments, enabling multi-order-of-magnitude scaling down of plate-based high-throughput screens (Figures 1A and 1B). In some examples, the systems and devices may include an entire platform and / or individual components.

[0015] In some embodiments, the assay compartment can be generated as either a water-in-oil (w / o) single emulsion or a water-in-oil (w / o / w) double emulsion. Figures 2A and 2B concretely depict a water-in-oil (w / o) single emulsion droplet (Figure 2A) and a water-in-oil (w / o / w) double emulsion droplet (Figure 2B). As illustrated in Figure 2A, the water-in-oil (w / o) single emulsion droplet (189) may comprise an aqueous phase (180), an oily continuous phase (181), and an aqueous-oil interface (182). The aqueous phase (180) may comprise an assay mixture (183) and barcoded compound beads (184). As illustrated in Figure 2B, a water-in-oil (w / o / w) double emulsion droplet (199) may contain an aqueous phase (190) in a core surrounded by an oily phase (192), subsequently surrounded by an outer aqueous continuous phase (191). The aqueous phase (190) may contain an assay mixture (193) and barcoded compound beads (194). In cell assays, the droplet size may be such that one or more encapsulated cells have sufficient nutrients available for the duration of the assay, for example, 10–200 microns in diameter for screening human cells in the range of 5–100 microns in diameter. In bacterial, cell-free, or biochemical assays, the droplet size can be selected from a wider range, for example, from 1–200 microns in diameter.

[0016] In some embodiments of the compositions, methods, uses, devices, sorters, and systems provided herein, the continuous phase may be any oil immiscible with water, such as mineral oil, hydrocarbon oil, silicone oil, or fluorescein oil. In some embodiments, the continuous phase may be a blend of fluorescein oils capable of achieving optimal compound retention while maintaining sufficient oxygen permeability, biocompatibility, and mechanical stability to a given assay buffer system. Examples of fluorescein oils, but not limited to, are perfluorocarbons, such as perfluorooctane, perfluoroheptane, perfluorohexane (FC-72), perfluoro-1,3-dimethylcyclohexane, octadecafluorodecahydronaphthalene (perfluorodecalin), perfluorinated oils, such as perfluoro-2-butyltetrahydrofuran, perfluoro-N-methylmorpholine (FC-3284), perfluorotripentylamine (FC-70), perfluorotributylamine (FC-43), perfluorotripropylamine (FC-3283), and perfluorotributylamine. Examples include mixtures with perfluoro(dibutylmethylamine) (FC-40), and hydrofluoroethers such as 2-(trifluoromethyl)-3-ethoxidedecafluorohexane (HFE-7500), ethyl perfluorobutyl ether (HFE-7200), 3-methoxyperfluoro(2-methylpentane) (HFE7300), methyl perfluoroisobutyl ether / methyl perfluorobutyl ether mixtures, mixtures of methoxynonafluorobutane and methoxynonafluoroisobutane (HFE7100), methoxynonafluorobutane, and methoxyheptafluoropropane (HFE7000).

[0017] Droplets can be stabilized by any emulsifier soluble in the continuous phase. In some embodiments, the alkyl-modified branched silicone emulsifier KF-6038 (lauryl PEG-9 polydimethylsiloxyethyl dimethicone) can be present in the continuous phase of silicone oil / mineral oil or squalene oil. In some embodiments, the emulsifier is composed of di- and / or tri-block copolymers, for example, those consisting of perfluorinated polyether (PFPE) and polyethylene glycol (PEG) and / or polypropylene glycol (PPG). Di- and tri-block copolymers can optionally be blended. In some embodiments, the emulsifier can be partially fluorinated silica nanoparticles. In some embodiments, the emulsifier can be any combination of the emulsifiers described herein. For example, the emulsifiers described herein can optionally be blended with partially fluorinated silica nanoparticles to enhance their properties.

[0018] In some embodiments, provided herein are compositions, methods, uses, sorters, systems, and devices for reducing droplet cross-contamination. In some embodiments, droplet cross-contamination can be reduced after droplet generation. In some embodiments, droplet cross-contamination can be reduced by replacing an emulsifier-loaded fluorinated oil continuous phase with an emulsifier-free fluorinated oil continuous phase, a Pickering emulsifier-loaded fluorinated oil continuous phase, or a combination thereof.

[0019] As described herein, it is understood that an "emulsifier" can be or can include a surfactant such as the surfactants described herein.

[0020] As used herein, “emulsion” is a stable mixture of at least two immiscible liquids. Generally, immiscible liquids tend to separate into two identifiable phases. Therefore, emulsions are stabilized by the addition of a “droplet stabilizer,” “surfactant,” or “emulsifier” that functions to reduce the surface tension between at least two immiscible liquids and / or to stabilize the interface. In some embodiments, the emulsions described herein include a discontinuous or dispersed phase formed of an aqueous substance (i.e., an isolated phase stabilized by a surfactant). The continuous phase may be formed of a fluorescein dispersion oil (e.g., a fluorocarbon). In some embodiments, this disclosure provides water in oil (w / o) single emulsions or water in oil in water (w / o / w) double emulsions having a dispersed aqueous phase and a fluorocarbon continuous phase. In some specific embodiments, the emulsions described herein are microemulsions. In some cases, microemulsions may contain droplets with an average diameter of approximately 10–200 micrometers, or in some cases, 50–100 micrometers.

[0021] As used herein, “droplet” means an isolated aqueous phase within a continuous phase having any shape, such as cylindrical, spherical, ellipsoidal, or irregular. Generally, in the emulsions of the present invention, the aqueous droplets are spherical or substantially spherical within the fluorocarbon continuous phase.

[0022] As used herein, “emulsifier” or “surfactant” defines a molecule that, when combined with a first component defining a first phase and a second component defining a second phase, facilitates and / or stabilizes the assembly of separate first and second phases. Emulsifiers may be triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, or mixtures thereof. Diblock and triblock copolymers typically comprise one or more fluorophilic main chains, one or both ends of which are soluble in the continuous phase of the emulsion, and one or more chains that are not soluble in the continuous phase of the emulsion (for example, these chains may be soluble in the aqueous phase). As an example, surfactants may be multiblock surfactants (for example, ABABABA...), in which case one component of the chain (for example, “A”) is soluble in the fluorescein phase, and another component of the chain (for example, “B”) is soluble in the aqueous phase. As used herein, a multiblock surfactant is a surfactant having an alternating copolymeric structure or (AB) structure (i.e., ABA, ABAB, ABABA, ABABABA, etc.). In some cases, one block may be soluble in the fluorescein phase of the emulsion, while another block may be soluble in the aqueous phase of the emulsion. In other cases, additional components may be present within the surfactant. For example, a multiblock surfactant may have other groups present in its polymeric structure, such as a linking moiety connecting A and B. For example, (AXB-) n , (AX 1 -BX 2 ) n In this case, "X" represents a covalent bond or connecting part as described below, and X 1 and X 2 If they exist, they may be the same or different.

[0023] As used herein, unless otherwise specified, a “fluorophilic” component or chain, for example in relation to the emulsifiers defined above, includes any fluorinated compound, e.g., linear, branched, cyclic, saturated, or unsaturated fluorinated hydrocarbon ethers or amines. A fluorophilic component may optionally contain at least one heteroatom (e.g., in the component's backbone, e.g., O). In some cases, a fluorophilic component may be highly fluorinated; that is, at least 30%, at least 50%, at least 70%, at least 90%, or at least 99% of the hydrogen atoms in the component are replaced with fluorine atoms. In some embodiments, 100% of the hydrogen atoms in the component are replaced with fluorine atoms; that is, it is perfluorinated; that is, the component contains fluorine atoms but no hydrogen atoms. A fluorophilic component may include, for example, fluorine-to-hydrogen ratios of at least 0.2:1, at least 0.5:1, at least 1:1, at least 2:1, at least 5:1, or at least 10:1. Fluorophilic components suitable for this disclosure may have low toxicity, low surface tension, and the ability to dissolve and transport gases. Examples of fluorophilic components are described herein.

[0024] As used herein, “stable emulsion” means that at least about 95% of the droplets of the emulsion do not, for example, coreless and form larger droplets over such a period. According to some embodiments, the compositions of the present disclosure are stable at a temperature of about 25 to 40 or 95 degrees Celsius and a pressure of 1 atm for at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 1 day, at least about 1 week, at least about 1 month, or at least about 2 months.

[0025] As used herein, the terms “thin-shell PEG” and “PEG coating” are interchangeable and mean that a long PEG polymer is grafted onto the surface of core beads supporting a compound and hard DNA. The long PEG may be, for example, 5 kDa or more, 10 kDa or more, or 40 kDa or more. The PEG group can be grafted onto the core beads using chemistry known in the art, for example, but not limited to strain-enhanced azide-alkyn click chemistry, copper-catalyzed azide-alkyn click chemistry, amide coupling, carbamoylation, urea or thiourea formation, sulfonamide formation, alkylation, reductive amination, etc.

[0026] Compounds and / or corresponding coding barcodes can be introduced as covalently barcoded compound beads. In some embodiments, compounds and / or corresponding coding barcodes can be introduced as reversibly covalently barcoded compound beads. In some embodiments, compounds and / or corresponding coding barcodes can be introduced via a code-1-bead-1-compound library technique. Compounds and / or barcodes can be directly attached to the beads or attached via a linker such as a cleavable linker (e.g., a photocleavable linker). See, for example, Figure 2C.

[0027] Dual barcoding for compounds and droplets can be independently achieved by, for example, oligonucleotide coding, color coding, fluorescent dye coding, RFID coding, or spatial arrangement of fluorescent beacons within beads (Meldal & Christensen, 2010), or any combination thereof. In some embodiments, barcoding for compounds and droplets can be achieved by DNA coding. In all coding schemes, each bead may have a bead-specific barcode that can act as both a droplet barcode for replica count and a compound-specific barcode for compound identity.

[0028] Aqueous droplets of a single aqueous droplet water-in-oil (w / o) emulsion co-encapsulating cells and coding compound beads can be generated with high frequency by adopting standard norms in the field of droplet microfluidics. Compressible hydrogel beads can allow for flexible quantitative adjustments of encapsulation efficiency at 1:1 or approximately 1:1 encapsulation in some embodiments, multiple droplet count per droplet in other embodiments, and efficiency at less than 1:1 encapsulation in yet other embodiments. In some embodiments, single cells can be co-encapsulated in addition to compound beads. In some other embodiments, multiple cells, for example, multiple cells of the same type, can be co-encapsulated. In other embodiments, two or more different cell types can be co-encapsulated.

[0029] In some embodiments, cells and coding compound beads can be co-encapsulated as described herein to generate a single (w / o) emulsion. In some embodiments, a double water-in-oil-in-water (w / o / w) emulsion can be generated. In some embodiments, a single (w / o) emulsion can be converted to a double (w / o / w) emulsion. In some embodiments, a single (w / o) emulsion can be converted to a double water-in-oil-in-water (w / o / w) emulsion before hit droplet selection. In some embodiments, cells and coding compound beads can be directly encapsulated in a double emulsion droplet.

[0030] The beads described herein can be made from any material that can be suspended in an aqueous buffer. In some examples, the beads may be hard beads, e.g., polystyrene, polystyrene-PEG hybrids, e.g., TentaGel® (Rapp Polymere), or hydrogels. Hydrogels can be selected from PEG (ChemMatrix), polyethylene glycol-acrylamide (PEGA) (Auzanneau et al., 1995, Meldal, 1992), agarose, alginate, collagen, and polyacrylamide (PA). In some examples, the beads may be compressible hydrogels. In some examples, the beads may be compressible hydrogels lacking autofluorescence. In some examples, the beads may be PEGA, which can exhibit excellent swelling properties in both aqueous and organic solvents, thus enabling versatile microfluidic handling while demonstrating good compatibility with a wide range of organic reactions.

[0031] Furthermore, as used herein, “beads” is understood to include capsules.

[0032] This specification provides monodisperse polyethylene glycol acrylamide (PEGA) resins and methods for preparing monodisperse polyethylene glycol acrylamide (PEGA) resins. In some embodiments, PEGA resins (e.g., polydisperse PEGA resins) can be generated in bulk. In some embodiments, the size distribution of the polydisperse PEGA resin can be adjusted by passing it through at least one filter. In some examples, the at least one filter is at least one cell strainer. In some examples, the size distribution of the polydisperse PEGA resin can be adjusted by passing it through a series of filters (e.g., a series of cell strainers). For example, to obtain beads in the range of 20 to 40 microns in diameter, beads larger than 40 microns can be filtered out using a 40-micron mesh strainer, and then the filtrate can be loaded onto a 20-micron mesh filter to remove beads smaller than 20 microns.

[0033] In some embodiments, a monodisperse PEGA resin can be generated in a microdroplet of a desired droplet size. The size of the PEGA resin can be adjusted to correspond to the size of the screening droplet and the required compound loading capacity. In some embodiments, the PEGA resin may have a diameter of 1 to 100 microns. In some embodiments, the screening droplet may have a diameter of 10 to 200 microns. The loading capacity of the PEGA resin may be such that the maximum concentration of the released ligand in the droplet is greater than 0.1 μM, preferably greater than 1 μM, and more preferably greater than 10 μM. The resin can carry compounds at a fixed dose or at a correspondingly decelerating dose from the maximum loading. In some examples, the resin can carry compounds screened by quantitative high-throughput screening (qHTS) (Inglese et al., 2006). In some examples, qHTS is achieved via controlled partial release of ligands from beads and / or via the preparation of dose-response beads having corresponding coding.

[0034] In some embodiments, hydrogel beads (e.g., PEGA resin) can be magnetic. Hydrogel resins, such as PEGA resin, can be made magnetic, for example, by co-encapsulation of appropriately coated magnetic microparticles or nanoparticles. In some examples, the coating of magnetic microparticles or nanoparticles can be selected based on biocompatibility, chemical resistance in organic synthesis, background fluorescence, mechanical stability, or any combination thereof. In some embodiments, examples of magnetic microparticles, but not limited to, include, for example, polystyrene-coated DynaBeads (InVitrogen, USA) or TurboBeads (TurboBeads LLC, Switzerland) or silica-coated BOCA beads (BOCA Scientific INC, USA). Magnetic microparticles can be spatially confined or covalently bonded via acrylamide surface modification. Magnetic hydrogels can be advantageous for bead handling during library preparation and post-screen bead processing, and / or enhance microfluidic handling, such as enabling the separation of bead-encapsulated droplets in bulk or on microfluidic chips.

[0035] In some embodiments, the codeable compound loading resins described herein can be encapsulated in a hydrogel matrix. In some examples, the encapsulating hydrogel matrix may be, for example, PEG, PEGA, polyacrylamide, alginate, collagen, or agarose, or any combination thereof. Encapsulation in a hydrogel matrix can enhance microfluidic handling properties, such as hydrophilicity, compressibility, or both. In some embodiments, encapsulation in a hydrogel matrix can increase droplet encapsulation efficiency. In some embodiments, the hydrogel matrix may include cavities that can act as, for example, cell carriers (Di Carlo et al., 2019). In some embodiments, hard beads can be encapsulated in a hydrogel matrix. The hard beads may be 1 to 50 microns in diameter, and the hydrogel shell may be sufficiently larger than the hard beads to allow sufficient compressibility. For example, encapsulation of 10-micron beads such as TentaGel® library beads is done with a 30 or 70-micron polyacrylamide gel. In some embodiments, the soft-shell beads described herein can be enriched by FACS. For example, soft-shell beads can be purified by FACS and gating, typically in a 1:1 ratio, but not limited to, to achieve suitable library bead loading for hydrogels. In the case of autofluorescent hard-shell beads, such as polystyrene or polystyrene-PEG hybrids, such as TentaGel®, the corresponding soft-shell beads can be enriched to have the desired bead loading amount.

[0036] In some embodiments, the surface properties of the beads can be modified. For example, the beads can be coated with hydrophilic materials, such as, but not limited to, PEG, PPG, hyaluronic acid, polylactic acid, and other hydrophilic polymers, or with hydrogels, such as, but not limited to, polyacrylamide, PEG, alginate, agarose, or collagen, or any combination thereof. Modifying the surface properties can improve the properties of the beads. For example, modifications described herein, such as core-shell beads or hydrogel beads, can minimize bead aggregation in aqueous media and enhance microfluidic handling.

[0037] In some embodiments, compounds are releaseable from beads. For example, compounds are releaseable from beads within droplets. In some embodiments, compounds not linked by a linker ("linkerless compounds") are releaseable into droplets by at least one stimulus, for example, but not limited to, electromagnetic irradiation (e.g., light, UV, UVA, e.g., about 365 nm), enzymatic cleavage, pH change, reducing agent, or a combination thereof. In some embodiments, the stimulus may be electromagnetic irradiation. The stimulus for release (e.g., enzyme, pH trigger, reducing agent, or a combination thereof) may be contained in the assay medium, introduced via a separate channel, or introduced via injection (e.g., pico-injection). In some embodiments, the stimulus for release (e.g., UV irradiation) may be exposed on the tip in in-line procedures or in the bulk in offline procedures (or a combination thereof). The stimulus for release may include any combination of the stimuli described herein.

[0038] Assays described herein, such as bioassays, can be carried out by co-encapsulating barcoded compound beads and assay reagents in aqueous droplets of water in oil (w / o) or aqueous droplets of water in oil (w / o / w) to provide an assay compartment. Compounds, such as linkerless compounds, can be released by stimulation. The release can achieve a desired concentration of the compound, such as a linker-free compound, within the droplet. The droplets can be assayed, for example, by incubation over the duration of the assay. The results can be determined, for example, by the hit droplet selection method described herein, as described herein.

[0039] In some embodiments, assay droplets can be incubated inline in an integrated chip design or offline in an incubation chamber. In some embodiments, droplets can be reinjected into a device for hit droplet selection and / or decoding.

[0040] Assays may be biochemical assays, in-vitro transcription-translation (IVTT) assays, cell assays, organism assays, or combinations thereof. Biochemical assays may include, but are not limited to, fluorescence intensity (FLINT), fluorescence resonance energy transfer (FRET), time-resolved FRET (TR-FRET), fluorescence polarization, fluorescence lifetime, ELISA, and combinations thereof. IVTT assay formats may include, but are not limited to, two-hybrid systems, split GFP reporter assays, and combinations thereof. Cell assays may include, but are not limited to, intracellular reporter assays (e.g., gain or loss of fluorescent proteins or fluorescence-conjugating enzymes), secretion reporter and / or secretion enzyme binding assays, marker (e.g., cytokine marker) secretion assays, viability assays, cell-cell interaction assays, virus induction assays, and combinations thereof. In some examples, the assays described herein can be performed using detection beads. For example, marker (e.g., cytokine marker) secretion assays or ELISA assays can be performed using detection beads. Encoding compound beads may optionally act concurrently as detection beads. For example, in an ELISA assay, coding compound beads can act simultaneously as detection beads. Viral induction assays may include, but are not limited to, retroviral assays (e.g., lentiviruses, adeno-associated viruses (AAVs)).

[0041] In some preferred embodiments, the cell assay can be performed using a cell tracker and / or an internal reference fluorescent protein. The cell tracker and / or internal reference fluorescent protein can correct for the number of multiplets in a droplet, the relative expression levels of the reporter gene and / or protein, or a combination thereof.

[0042] In some preferred embodiments, the assay may be in a “mix-and-read” format, where the assay premix is ​​co-encapsulated with a barcoded compound, allowing the assay results to be determined without further manipulation. In other embodiments, the assay may include adding an assay reagent (e.g., a detection antibody) to determine the assay results. The assay reagent (e.g., a detection antibody) can be added sequentially, for example, by pico-injection, droplet merging, or a combination thereof.

[0043] In some preferred embodiments, hit droplets can be selected by fluorescence-activated droplet sorting (FADS). In some preferred embodiments, FADS may include sorting by a sorter as described herein, for example, a dual-electrode sorter as described herein. Sorting by a dual-electrode sorter, for example, a dual-electrode sorter as described herein, can enhance the speed and / or reliability of sorting.

[0044] In some embodiments, the droplet is selectable by FACS. In some embodiments, the droplet is selectable by FACS at a frequency of 12–14 kHz (Brower et al., 2019, 2020). In some embodiments, the droplet selected by FACS may be a w / o / w double emulsion droplet.

[0045] In some embodiments, assay droplets containing compound beads and bioassay products, generated using the preferred emulsion formulations described herein, can be converted to hydrogel beads by introducing biocompatible polymers and / or their precursors, for example, but not limited to, agarose, alginate, collagen, polyacrylamide, PEG and corresponding initiators, or any combination thereof, as required. The thus formed assay hydrogels can be isolated in aqueous buffer while preserving the compound beads and cells on the beads for off-droplet sorting using a conventional flow cytometer (Duarte et al., 2017; Yanakieva et al., 2020).

[0046] In some embodiments, core-shell beads containing members of a code library may be converted into water-in-oil droplets using preferred emulsion formulations described herein, comprising a cell-containing cavity, a compound released and incubated within a discrete droplet compartment, and a hydrogel supporting the beads and cells, which is extracted into an aqueous buffer for off-droplet sorting using a conventional flow cytometer (Di Carlo et al., 2019; Joseph de Rutte, Robert Dimatteo, Mark van Zee, Robert Damoiseaux, 2020).

[0047] In some embodiments, single or double emulsion droplets can be arrayed on a microwell plate, such as a 1536-well plate. The bottom of each well in the microwell plate may further contain an array of smaller wells. In this case, each well is of a size similar to the size range of the emulsion droplets, for example, a 100-micron grid. In some embodiments, each well in the array of smaller wells may contain one droplet. In some embodiments, the droplets can settle in the well. For example, in some further embodiments, w / o / w double emulsion droplets settle in the well. In some further embodiments, w / o / w double emulsion droplets settle in the well accompanied by a continuous phase formulation described herein as a continuous phase. In some embodiments, w / o / w double emulsion droplets settle in the well accompanied by a continuous phase formulation containing at least one fluorescein dispersion oil. In further embodiments, the fluorescein dispersion oil may have an average fluorine content of about 70 wt% or more. In other further embodiments, the continuous phase formulation may further include a droplet stabilizer comprising an emulsifier which is a triblock copolymer, a diblock copolymer, fluorinated silica nanoparticles, or a combination thereof. In some embodiments, the w / o single emulsion droplets settle in the well. In some further embodiments, the w / o single emulsion droplets settle in the well accompanied by a continuous phase oil having a density lower than that of the aqueous phase. In some even further embodiments, the continuous phase oil is mineral oil, squalene oil, or any other hydrocarbon-based oil.

[0048] In embodiments provided herein, where droplets settle (i.e., sink) in the wells of an array of smaller wells, the droplets are analyzable. For example, hit droplets can be isolated for hit deconvolution. For example, droplets can be analyzed by imaging techniques, such as fluorescence, luminescence, or a combination thereof. In some examples, hit droplets can be analyzed and automatically aspirated from the population for hit deconvolution. Picking can be fully automated. For example, picking can be performed by a robotic micromanipulator such as an automated cell picker.

[0049] In embodiments in which droplets are assayed by cell assays, the droplets may be w / o / w double emulsion droplets. In further embodiments, the w / o / w double emulsion droplets may be in a continuous phase formulation containing at least one fluorescein dispersion oil. In other further embodiments, the fluorescein dispersion oil has an average fluorine content of about 70 wt% or more. In other further embodiments, the continuous phase formulation further comprises a droplet stabilizer containing an emulsifier which is a triblock copolymer, a diblock copolymer, fluorinated silica nanoparticles, or a combination thereof. This droplet selection format, while not limiting, may enable versatile assay readouts, including phenotype imaging-based readouts, fluorescence-based readouts, bioluminescence-based readouts, and combinations thereof.

[0050] In some embodiments, hit droplets and optionally non-hit droplets can be analyzed. For example, droplets can be analyzed by decoding compound barcodes and / or droplet barcodes. In some embodiments, hit molecules can be deconvoluted. For example, deconvoluting hit molecules may include counting the number of positive droplets per compound. In some further embodiments, the number of positive droplets and the number of negative droplets per compound can be compared for the same compound. In some preferred embodiments, a DNA-coded-bead-compound library or compound beads having both beads and compound-specific barcodes can be employed. In further embodiments, decoding barcodes may include sequencing such as next-generation sequencing.

[0051] In the compositions, methods, assays, sorters, uses, and systems described herein, the droplets may be in the single emulsion format or the double emulsion format described herein.

[0052] Therefore, this technology provides a method, sorter, use, and device for screening substances such as bioactive materials using emulsion droplets.

[0053] According to several embodiments, continuous-phase formulations for stable emulsions are described. The continuous-phase formulation comprises at least one fluorescein dispersion oil (where the fluorescein dispersion oil has an average fluorine content of about 70 wt% or more (e.g., 75 wt% or more, 80 wt% or more)) and a droplet stabilizer comprising an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof.

[0054] According to several embodiments, continuous-phase formulations for stable emulsions are described. The continuous-phase formulation comprises a plurality of two or more fluorescein dispersion oils (where the plurality of fluorescein dispersion oils have an average fluorine content of about 70 wt% or more) and a droplet stabilizer comprising a plurality of two or more emulsifiers selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof.

[0055] According to several embodiments, a method for reducing cross-contamination between microdroplets (e.g., compound retention for more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, or more than 8 days) (e.g., water in oil (w / o) single emulsion) is described. The method comprises forming at least one aqueous microdroplet in a first continuous phase formulation (e.g., using a syringe pump), the continuous phase formulation comprising an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof. In some examples, the method comprises exchanging the first continuous phase formulation with fluorescein dispersion oil to provide aqueous microdroplets suspended in fluorescein dispersion oil (where the fluorescein dispersion oil has an average fluorine content of >70 wt% and does not contain an emulsifier) ​​(e.g., exchanging with fluorescein dispersion oil a total of 2, 3, 4, 5 times, or more).

[0056] In some embodiments, a method for reducing cross-contamination between microdroplets further includes replacing a first fluorescein dispersion oil with a second continuous phase formulation (for example, to increase droplet stability for further droplet operations) to provide aqueous microdroplets suspended in a second continuous phase formulation (where the second continuous phase formulation is the continuous phase formulation described herein).

[0057] According to several embodiments, methods for reducing cross-contamination between microdroplets are described. These methods include forming at least one aqueous microdroplet in a first continuous phase formulation described herein, replacing the first continuous phase formulation with a first fluorescein dispersion oil to provide an aqueous microdroplet suspended in the first fluorescein dispersion oil, and replacing the first fluorescein dispersion oil with a second fluorescein dispersion oil to provide an aqueous microdroplet suspended in the second fluorescein dispersion oil. In some examples, the first fluorescein dispersion oil has an average fluorine content of >70 wt% and contains a Pickering emulsifier (for example, an 8% wt formula dispersed in a mixture of perfluoro-2-butyltetrahydrofuran and 2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500)). [ka] Fluorinated silica nanoparticles (100 nm). In some examples, the second fluorine dispersion oil has an average fluorine content of >70 wt% and does not contain emulsifiers.

[0058] In some embodiments, the method further includes replacing the second fluorescein dispersion oil with the second continuous phase formulation to provide aqueous microdroplets suspended in the second continuous phase formulation (for example, to obtain increased droplet stability for further droplet operations). In such embodiments, the second continuous phase formulation is the continuous phase formulation described herein.

[0059] According to several embodiments, a method for preparing monodisperse polyethylene glycol acrylamide (PEGA) copolymer resins is described. The method involves dispersing multiple monomers in an aqueous buffer (e.g., TBSET (10 mM TBS (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 10 mM EDTA, 1% Tween)), combining the aqueous buffer and the multiple monomers with a continuous phase formulation containing oil and an emulsifier (e.g., emulsifiers in concentrations of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 0.5% to 1.5%, about 0.5% to about 2.0%, about 0.5% to about 2.5%), and forming at least one microdroplet (e.g., 1 to 200 microns, 10 to 200 microns, 1 to 100 microns) from the aqueous buffer and the multiple monomers. The process includes forming microdroplets (5-90 microns, 10-80 microns, 20-70 microns, 20-50 microns) and polymerizing monomers in at least one microdroplet to form a PEGA copolymer resin (using an initiator (e.g., tetramethylenediamine (TEMED) and ammonium persulfate), at a temperature of about 20°C, 30°C, 40°C, 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 60-70°C, or about 55-75°C, overnight polymerization). In some examples, the oil is selected from the group consisting of fluorescein oils, hydrocarbon oils, mineral oils, and silicone oils. In some examples, the monomers of the multiple monomers include acrylamide (e.g., acrylamide or N,N-dimethylacrylamide), bis-acrylamide PEG, and mono-acrylamide PEG containing a functionalized handle, and / or mono-acrylamide diamine containing a functionalized handle. In some examples, the monomers of the multiple monomers include acrylamide (e.g., acrylamide or N,N-dimethylacrylamide), bis-acrylamide PEG, mono-acrylamide PEG containing a functionalized handle, and mono-acrylamide diamine containing a functionalized handle.In some examples, the monomers of multiple monomers include acrylamide (e.g., acrylamide or N,N-dimethylacrylamide), bis-acrylamide PEG, and mono-acrylamide diamines containing functionalized handles.

[0060] In some embodiments, the PEGA resin is mechanically stable under reaction conditions typically used in solid-phase organic chemistry (e.g., heating and / or acidic or basic conditions), stable to handling in microfluidic devices such as pipetting, shaking, sonication, centrifugation, filtration, and handling protocols such as pipetting, centrifugation, filtration, chemical treatment, UV irradiation, droplet encapsulation, droplet rupture and recovery, and sorting in a flow cytometer, as measured by percentage bead recovery, for example, stable for at least 1 hour at 100°C, stable to acid (e.g., TFA) or base (e.g., DIP). EA) Stable for at least 1 hour against treatment, or at least 24 hours against shaking at 1500 rpm, or at least 3 minutes against centrifugation at 8000 rcf), and the PEGA resin is sufficiently biocompatible so that the integrity of the PEGA resin is not compromised over the duration of the assay by co-incubation and / or co-encapsulation in droplets with biological systems (e.g., cells, IVTT mix, and / or recombinant proteins, etc.) (e.g., co-incubation with cells for 24 hours results in >90% viability or greater viability compared to a beadless control). In some embodiments, the PEGA resin is sufficiently biocompatible (e.g., co-incubation with cells for 24 hours results in >90% viability or greater viability compared to a beadless control). In some cases, PEGA resins have the ability to swell in aqueous buffer solutions and organic solvents to enable efficient chemical reactions, effective solvent exchange, and suspension and handling on the entire resin (e.g., at least 5 ml / g, at least 6 ml / g, at least 7 ml / g, at least 8 ml / g, at least 9 ml / g, at least 10 ml / g, at least 11 ml / g, at least 12 ml / g, at least 13 ml / g, at least 14 ml / g, at least 15 ml / g, at least 16 ml / g).In some examples, PEGA resins are compressible enough to allow packing of hydrogel beads into microfluidic channels (e.g., achieving reversible diameter reduction without breakage when force is applied, such as diameter reduction >10%, >15%, >20%, >25%, or >30%), such that the beads are encapsulated in at least one microdroplet with superpoisson encapsulation efficiency (characterized, e.g., by analyzing droplets on a hemocytometer / microscope chamber slide (e.g., iBidi or Countess), or by comparing the number of encapsulated droplets to empty droplets (e.g., by observation under a microscope), such as droplets in a tightly packed monolayer).

[0061] In some embodiments, at least one microdroplet is formed in a microfluidic device (e.g., a microfluidic chip, a device optionally including a droplet splitter). In some examples, forming at least one microdroplet involves forming multiple monodisperse microdroplets in parallel.

[0062] According to several embodiments, a method for preparing monodisperse polyethylene glycol acrylamide (PEGA) copolymer resins is described. The method comprises dispersing a plurality of monomers in an aqueous buffer, polymerizing the monomers to form a polydisperse PEGA copolymer resin, and passing the polydisperse PEGA copolymer resin through a plurality of cell strainers to obtain a PEGA copolymer resin having a defined size distribution (e.g., 1-200 microns, 10-200 microns, 1-100 microns, 5-90 microns, 10-80 microns, 20-70 microns, 20-50 microns, 20-40 microns). In some examples, the monomers of the plurality of monomers include acrylamide (e.g., acrylamide or N,N-dimethylacrylamide), bis-acrylamide PEG, and mono-acrylamide PEG containing a functionalized handle, and / or mono-acrylamide diamine containing a functionalized handle.

[0063] According to several embodiments, a method for preparing core-shell beads is described. The method comprises: dispersing a plurality of monomers and at least one core bead in an aqueous buffer; forming at least one microdroplet from the aqueous buffer, the plurality of monomers, and the core bead, wherein the at least one microdroplet contains at least one core bead; and forming core-shell beads (for example, the thickness of the PEGA copolymer is greater than the radius of the bead) by polymerizing the monomers in the at least one microdroplet to form a hydrogel (e.g., PEGA, polyacrylamide, alginate, agarose, collagen, or a combination thereof) that encapsulates the beads.

[0064] According to several embodiments, a method for preparing thin-shell PEG-coated beads is described. The method involves grafting a hydrophilic coating onto compound load code beads. In some examples, the hydrophilic polymer coating consists of PEG. In some examples, the PEG is attached by acetylene-azidocric chemistry.

[0065] According to several embodiments, a sorter is described. The sorter includes an inlet channel, first and second outlet channels that merge into the inlet channel at a junction, and first and second electrodes adjacent to the first and second sides of the junction, respectively. In some embodiments, the first and second electrodes are configured to have a first state in which the first electrode receives a higher voltage than the second electrode, causing one or more target compositions to flow through the junction and into the first outlet channel, and a second state in which the second electrode receives a higher voltage than the first electrode, causing one or more target compositions to flow through the junction and into the second outlet channel. In some embodiments, the sorter includes a controller configured to switch the first and second electrodes between the first and second states.

[0066] According to several embodiments, systems (e.g., devices, devices in operation) for performing high-throughput screening are described. The system comprises a continuous phase formulation, a solid carrier, and a sorter. In some examples, the continuous phase formulation comprises a droplet stabilizer and at least one fluorescein dispersion oil. In some examples, the fluorescein dispersion oil has an average fluorine content of about 70 wt% or more. In some examples, the droplet stabilizer comprises an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof. In some examples, the solid carrier is selected from the group consisting of monodisperse polyethylene glycol acrylamide (PEGA) copolymer resins and core-shell beads. In some examples, monodispersible polyethylene glycol acrylamide (PEGA) copolymer resins are prepared by dispersing a first plurality of monomers in a first aqueous buffer; combining the first aqueous buffer and the first plurality of monomers with a continuous phase formulation containing oil and an emulsifier; forming at least one first microdroplet from the first aqueous buffer and the first plurality of monomers; and polymerizing the monomers in at least one first microdroplet to form a PEGA copolymer resin. In other examples, thin shell beads are prepared by grafting a hydrophilic coating onto a solid support. In some examples, the hydrophilic polymer coating consists of PEG. In some examples, the PEG is attached by acetylene-azidocric chemistry. In some examples, the oil is selected from the group consisting of fluorescein oils, hydrocarbon oils, mineral oils, and silicone oils. In some examples, the monomers of the first plurality of monomers include acrylamide, bis-acrylamide PEG, and mono-acrylamide PEG containing a functionalized handle, and / or mono-acrylamide diamine containing a functionalized handle.In some examples, core-shell beads are prepared by dispersing a second plurality of monomers and at least one core bead in a second aqueous buffer; forming at least one second microdroplet from the second aqueous buffer, the second plurality of monomers, and the core bead; and forming core-shell beads by polymerizing the monomers in at least one second microdroplet to form a hydrogel encapsulating the core bead. In some examples, at least one second microdroplet contains at least one core bead. In some examples, the sorter includes an inlet channel, first and second outlet channels that merge with the inlet channel at a junction, and first and second electrodes adjacent to the first and second sides of the junction, respectively. In some examples, the first and second electrodes are configured to have a first state in which the first electrode receives a higher voltage than the second electrode, causing one or more target compositions to flow through the junction and into the first exit channel, and a second state in which the second electrode receives a higher voltage than the first electrode, causing one or more target compositions to flow through the junction and into the second exit channel. In some examples, the sorter includes a controller configured to switch the first and second electrodes between the first and second states.

[0067] According to several embodiments, a system for performing high-throughput screening is described. The system comprises a continuous phase formulation, a solid carrier, and a sorter. In some embodiments, the solid carrier is in the form of code compound beads as described herein. In some examples, the continuous phase formulation comprises a droplet stabilizer and at least one fluorescein dispersion oil. In some examples, the fluorescein dispersion oil has an average fluorine content of about 70 wt% or more. In some examples, the droplet stabilizer comprises an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof. In some examples, the solid carrier is selected from the group consisting of monodisperse polyethylene glycol acrylamide (PEGA) copolymer resins and core-shell beads. In some examples, monodispersible polyethylene glycol acrylamide (PEGA) copolymer resins are prepared by dispersing a first plurality of monomers in a first aqueous buffer, combining the first aqueous buffer and the first plurality of monomers with a continuous phase formulation containing oil and an emulsifier to form at least one first microdroplet from the first aqueous buffer and the first plurality of monomers, and polymerizing the monomers in at least one first microdroplet to form a PEGA copolymer resin. In other examples, thin shell beads are prepared by grafting a hydrophilic coating onto a solid support. In some examples, the hydrophilic polymer coating consists of PEG. In some examples, the PEG is attached by acetylene-azidocric chemistry. In some examples, the oil is selected from the group consisting of fluorescein oils, hydrocarbon oils, mineral oils, and silicone oils. In some examples, the monomers of the first plurality of monomers include acrylamide, bis-acrylamide PEG, and mono-acrylamide PEG containing a functionalized handle, and / or mono-acrylamide diamine containing a functionalized handle.In some examples, core-shell beads are prepared by dispersing a second plurality of monomers and at least one core bead in a second aqueous buffer; forming at least one second microdroplet from the second aqueous buffer, the second plurality of monomers, and the core bead; and forming core-shell beads by polymerizing the monomers in at least one second microdroplet to form a hydrogel encapsulating the core bead. In some examples, at least one second microdroplet contains at least one core bead. In some examples, the sorter includes a microwell array plate configured to host one microdroplet per microwell, a fluorescence microscope, an imager configured to automate image assays of droplets to identify desired droplets, and an automated microcapillary-based droplet sampling device configured to sequentially select multiple desired droplets (e.g., sequential selection, bulk selection, high-throughput selection, e.g., about 1–3 cells / second or about 2 cells / second, selection by aspiration) and deposit them into hit wells (e.g., configured to allow access to droplets in the microwells or grid, with one or more capillary properties selected from a selection of sizes, angles, and heights adapted to the droplets and / or dividers).

[0068] Therefore, compositions, methods, sorters, systems, uses, and devices are provided for high-throughput screening in droplets. Such methods, compositions, sorters, systems, uses, and devices can complement or replace other methods, compositions, sorters, systems, uses, and devices for high-throughput screening in droplets. In some embodiments, the compositions, methods, sorters, systems, uses, and devices provided herein, as well as combinations thereof, can be used to perform one or more analyses selected from biochemical assays, cell-free assays, cell reporter assays, and cell phenotypic assays using fluorescent or bioluminescent and / or next-generation sequencing readouts and hit compounds deconvoluted by next-generation sequencing.

[0069] This patent or application file contains at least one drawing made in color. A copy of this patent or patent application publication with the color drawing will be provided by the Office upon request and payment of the necessary fees.

[0070] To better understand the various embodiments described, the following descriptions of embodiments should be referenced in relation to the drawings below, where the same reference numbers indicate corresponding parts throughout the figures. [Brief explanation of the drawing]

[0071] [Figure 1-1] Figure 1: Figures 1A and 1B illustrate a miniaturization embodiment from a 1536-well plate to approximately 500 picoliters of coded assay compartments. In a 1536-well plate (e.g., 651 plates for approximately 1 million wells), 5 μL of assay medium can be held in 1.7 mm diameter microwells (Figure 1A), while in droplets, approximately 500 picoliters of assay medium can be held in coded 100 micron diameter droplets (e.g., 1 million droplets in a single tube) (Figure 1B). [Figure 1-2] (As stated above.) [Figure 2-1] Figure 2: Figures 2A and 2B illustrate water-in-oil (w / o) single emulsion droplets (Figure 2A) and water-in-oil (w / o / w) double emulsion droplets (Figure 2B). Figure 2C illustrates compounds and barcodes mounted on compound loading beads. Ligands and barcodes can optionally be linked to common functionalization handles on the beads via split linkers (Figure 11A). Figures 2D and 2E illustrate compound loading beads 160 encapsulated in a hydrogel matrix 161 (Figure 2D) and a hydrophilic coating 162 (Figure 2E). Figure 2F illustrates core-shell beads in which compound loading beads 160 are encapsulated in a hydrogel shell 162 having a cell-adapted cavity 163. Figure 2G illustrates a dual-electrode droplet sorting device according to several embodiments. Figure 2H illustrates a microarray grid hosting 1 droplet / well and the isolation of hit droplets and / or beads according to several embodiments. [Figure 2-2] (As stated above.) [Figure 2-3] (As stated above.) [Figure 2-4] (As stated above.) [Figure 2-5] (As stated above.) [Figure 2-6] (As stated above.) [Figure 2-7] (As stated above.) [Figure 2-8] (As stated above.) [Figure 3] Figure 3 illustrates a microfluidic droplet generation device according to several embodiments. [Figure 4] Figure 4 shows monodisperse PEGA resin generated by microfluidic droplet generation devices according to several embodiments. [Figure 5-1]Figure 5: Figures 5A-5C illustrate coded compound loading beads with and without a hydrophilic polymer shell (Figure 5A) and with a hydrophilic polymer shell (Figures 5B-5C). Figures 5D-5E illustrate core-shell beads coated with a hydrophilic shell via acylation or azide-acetylene click chemistry or by grafting a hydrophilic polymer such as PEG (Figures 5D and 5D-1), and coated with a hydrophilic polymer mix such as polyacrylamide via surface polymerization (Figure 5E), according to several embodiments. Figure 5F illustrates the encapsulation of compound loading core beads in a hydrogel layer according to several embodiments. [Figure 5-2] (As stated above.) [Figure 5-3] (As stated above.) [Figure 5-4] (As stated above.) [Figure 5-5] (As stated above.) [Figure 5-6] (As stated above.) [Figure 5-7] (As stated above.) [Figure 6-1] Figure 6: Figures 6A-6B illustrate compound-loading hydrogel beads having magnetic microparticles mounted by non-covalent bonds (Figure 6A) and covalent bonds (Figure 6B) according to several embodiments. [Figure 6-2] (As stated above.) [Figure 7-1] Figure 7: Figures 7A-7H show a comparison of the molecular holdings of fluorescein (Figures 7A-7D) and resolphin (Figures 7E-7H) in w / o single emulsions generated using various commercially available continuous-phase formulations at the 1-hour time point. [Figure 7-2] (As stated above.) [Figure 7-3] (As stated above.) [Figure 7-4] (As stated above.) [Figure 7-5] (As stated above.) [Figure 7-6] (As stated above.) [Figure 7-7] (As stated above.) [Figure 7-8] (As stated above.) [Figure 8-1] Figure 8: Figures 8A-8D show the molecular retention of fluorescein (green) and resorphine (red) in droplets generated using continuous-phase oily compositions according to several embodiments. Figures 8E-8G show droplet stability and dye retention after 5 days of exchange with an emulsifier-free continuous-phase oily blend (containing 20% ​​HFE-7500, 20% FC-72, and 60% perfluorooctane, with emulsifier (Formula 1 (n is approximately 38 and m is approximately 9):Formula 2 (i and k are approximately 38 and j is approximately 9) 1:1, 2wt%)). Figure 8E shows bright-field imaging, Figure 8F shows 100 μM resorphine, and Figure 8G shows 100 μM fluorescein. Approximately 1 in 10 droplets contains the dye mixture to monitor cross-contamination across the droplets. Images are taken of droplets in ibidi imaging slides using a 100-micron height chamber. Figures 8H–8J show representative flow cytometry gating strategies for identifying viable cells observed by low red fluorescence (Figure 8H). Figure 8K shows the percentage of viable cells after co-encapsulation in 100-micron droplets containing specific di and triblock copolymers, respectively, and their subsequent retrieval. [Figure 8-2] (As stated above.) [Figure 8-3] (As stated above.) [Figure 8-4] (As stated above.) [Figure 8-5] (As stated above.) [Figure 8-6] (As stated above.) [Figure 8-7] (As stated above.) [Figure 8-8] (As stated above.) [Figure 8-9] (As stated above.) [Figure 8-10] (As stated above.) [Figure 8-11] (As stated above.) [Figure 9-1]Figure 9: Figures 9A-9F show fluorescein release in droplets from 10-micron TentaGel® beads (Figures 9A and 9B), 33-micron PEGA resin (Figures 9C and 9D), and 10-micron TentaGel® in a 33-micron polyacrylamide shell (Figures 9E and 9F) before (Figures 9A, 9C, and 9E) and after (Figures 9B, 9D, and 9F) cleavage of photocleavable linkers by UV irradiation. Figures 9G-9I show FACS enrichment of TentaGel® in polyacrylamide. The crude TentaGel® encapsulated population is 6% (Figure 9G). After FACS enrichment based on a subgroup of events with significantly higher fluorescence in FITC and PE channels, the encapsulated population increased to 75% (Figure 9H). Figure 9I shows the scatter plot and gate population. [Figure 9-2] (As stated above.) [Figure 9-3] (As stated above.) [Figure 9-4] (As stated above.) [Figure 9-5] (As stated above.) [Figure 9-6] (As stated above.) [Figure 9-7] (As stated above.) [Figure 9-8] (As stated above.) [Figure 9-9] (As stated above.) [Figure 10-1] Figure 10: Figure 10A shows the compressibility of polyacrylamide (PAA) versus PEGA 1× rod as measured by a texture analyzer. Figure 10B shows tight packing and quantitative 1:1 encapsulation into droplets of PEGA 1× resin in one channel according to several embodiments. Figures 10C and 10D show FACS results of biocompatibility of 33 micron PEGA resin as measured by cell viability after 24 hours in a 37° 5% CO2 incubator. [Figure 10-2] (As stated above.) [Figure 10-3] (As stated above.) [Figure 10-4] (As stated above.) [Figure 11-1]Figure 11: Figures 11A-11C illustrate exemplary DNA coding bead library linker and DNA barcode designs according to several embodiments. Figure 11A depicts azidrisine, which provides a click handle for DNA tag conjugation while using its N-terminus for further linker assimilation with a photocleavable motif such as an orthonitrobenzyl group. Figure 11B depicts an exemplary DNA barcode design in which a bead-specific barcode (BSB) follows the headpiece, as well as three positions for coding various building blocks, for example, followed by a library tag and an experiment-specific tag. Figure 11C depicts an example of a dose-response bead that can code DNA, allowing the split linker to be capped with an acetyl group to reduce the photocleavable linker functionalization handle, and enabling the pooling of dose-response beads for synthesis and screening. [Figure 11-2] (As stated above.) [Figure 11-3] (As stated above.) [Figure 12-1] Figure 12: Figures 12A-C illustrate embodiments in which a mix-and-read fluorescence reporter assay can be configured on a droplet-based screening platform to produce either signal gain or loss, illuminating the entire droplet (Figure 12A), a single or more cells and / or beads within the droplet (Figure 12B), or a secretion reporter from a single or more cells that illuminates the entire droplet (Figure 12C). [Figure 12-2] (As stated above.) [Figure 12-3] (As stated above.) [Figure 13]Figure 13 depicts a typical fluorescent reporter usable in cell-based assays. It depicts: i) a fluorescent protein detectable by a droplet sorter. For example, detection can be performed by rapid detection or sorting of fluorescence via a ratio (e.g., GFP / RFP ratio), where one fluorescent reporter reports on a biological response and the other is used to normalize expression and cell count. ii) a fluorescent reporter enzyme. For example, a β-lactamase usable as a reporter enzyme reporting on a cellular response using a FRET-based substrate. This can also be multiplexed with a toxic dye via a red fluorescent cell stain (ToxBlazer®, Thermo Fisher Scientific). Alternatively, the enzyme β-galactosidase can be used as a reporter in either its native or split form, enabling monitoring of a variety of cell-based events. The use of the β-galactosidase substrate DDAO galactoside (9H-(1,3-dichloro-9,9-dimethylacridine-2-on-7-yl)β-D-galactopyranoside) provides near-infrared fluorescence emission that is well separated from the typical background autofluorescence observed when the enzyme is expressed. iii) Secretory enzymes such as secretory alkaline phosphatase (SEAP) that can be used for detection of cellular responses by secretory form of FP or green fluorescent readout. An example shown is a fluorescence detection system for SEAP. [Figure 14-1] Figure 14: Figures 14A to 14E illustrate droplet sorting according to several embodiments. Figure 14A illustrates droplet flow without an applied electric field. Figure 14B illustrates an example of random droplet flow without an applied electric field. Figure 14C illustrates droplet sorting in which an electric field is applied to select non-hit stream paths. Figures 14D and 14E illustrate droplet sorting in which an electric field is applied to select hit streams. [Figure 14-2] (As stated above.) [Figure 14-3] (As stated above.) [Figure 14-4] (As stated above.) [Figure 14-5] (As stated above.) [Figure 15-1] Figure 15: Figure 15A shows the detection and sorting of fluorescent droplets by applying an electric field according to several embodiments. Figures 15B to 15E show the detection and sorting of droplets by AC voltage potential according to several embodiments. [Figure 15-2] (As stated above.) [Figure 15-3] (As stated above.) [Figure 15-4] (As stated above.) [Figure 15-5] (As stated above.) [Figure 16] Figure 16 shows double emulsion droplets in an imaging flow cytometer according to several embodiments. [Figure 17-1] Figure 17: Figures 17A-17F illustrate emulsion sorting by picking hit droplets from microwell plates according to several embodiments. Figure 17A illustrates a microwell plate in which the dimensions of the microwells are selected to be on the same size scale as the emulsion droplets for 1 droplet / well. Figure 17B illustrates emulsion droplets loaded onto a microwell plate so that each well contains 1 emulsion droplet. Figure 17C illustrates a microwell plate in which the locations of single and multiple hit groups are identified. Figure 17D illustrates a microwell plate in which single and multiple hit groups are mechanically removed from specific wells. Figure 17E illustrates single and multiple hit groups taken for analysis. Figure 17F is a microscopic image of double emulsion droplets with and without fluorescent dye arrayed in a microwell plate. [Figure 17-2] (As stated above.) [Figure 17-3] (As stated above.) [Figure 17-4] (As stated above.) [Figure 17-5] (As stated above.) [Figure 17-6] (As stated above.) [Figure 18-1]Figure 18: Figures 18A to 18D show microwell plates from which double emulsion is removed according to several embodiments. Figure 18A is a microwell plate containing double emulsion. Figure 18B is a microwell plate containing double emulsion and a capillary slightly larger than the double emulsion. Figures 18C and 18D show the defined volume aspirated to remove the double emulsion from the wells. [Figure 18-2] (As stated above.) [Figure 18-3] (As stated above.) [Figure 18-4] (As stated above.) [Figure 19-1] Figure 19: Figure 19A shows quantitative PCR of TentaGel® in PEGA resin, TentaGel® resin, and polyacrylamide resin. Three samples are analyzed: a blank prepared with MilliQ water (curve "C"), a control sample (beads mixed with DNA tags without DNA ligase - curve "B"), and sample beads (fully coded beads - curve "A"). Curve "D" corresponds to the calibration curve. Figure 19B shows PCR of TentaGel® resin with and without thin-shell PEG40K coating (PEG chains with an average molecular weight of 40kDa). Curve "A" is from null library beads without PEG40K coating. Curve "B" is from library beads with PEG40K coating. Curve "C" is MilliQ water (negative control). Sample beads with 40K PEG have the same amplification as control beads with the same DNA tags but without PEG. The overlapping curves clearly suggest that post-synthesis 40K PEG conjugation does not affect DNA tag amplification compared to the control sample without PEG. The procedure was performed five times for the two samples with beads, and triplicately for the MiliQ water samples. [Figure 19-2] (As stated above.) [Figure 20-1]Figure 20: Figures 20A and 20B illustrate the effect of a thin PEG coating on the dispersibility of compound-loaded TentaGel® beads in aqueous cell culture medium. A comparison of bead dispersion after 4 days of complete dispersion by probe sonication is shown; Figure 20A shows the beads without PEG coating, and Figure 20B shows the beads with 40K PEG coating. As described in Figure 11A, fluorescein is loaded onto the beads via a photocleavable linker. After 4 days of standing, large bead clamping is observed in the beads without PEG coating, while the beads with 40K PEG coating are mainly dispersed as a monodisperse. [Figure 20-2] (As stated above.) [Figure 21] Figure 21 illustrates the effect of magnetic beads on PEGA beads. The time courses of lateral traction of magnetic PEGA beads are shown from left to right at 1 second, 18 seconds, and 180 seconds. In 180 seconds, most of the magnetic PEGA beads are collected at the side of the tube. [Modes for carrying out the invention]

[0072] The following description provides exemplary methods, parameters, etc. However, it should be recognized that such description is not intended to limit the scope of this disclosure, but rather is provided as a description of exemplary embodiments.

[0073] There is a need for compositions, methods, devices, and systems to improve library screening in droplets. For example, there is a need for compositions that increase molecular retention in droplets for compounds within a range of physiological and chemical properties. There is also a need for droplets capable of achieving 1:1 or approximately 1:1 encapsulation efficiency. There is also a need to improve droplet imaging. There is also a need to improve monodispersity, biocompatibility, suspension, and compressibility in aqueous media, low background autofluorescence, compatibility with a wide range of library chemistry in organic phases, and any combination thereof.

[0074] In cell screening of biologics in droplets, the continuous phase may be a fluorinated oil, but this is due to its gas permeability, low cytotoxicity, and chemical inertness (C. Holtze et al. 2008). Di- and tri-block copolymers are used as emulsifiers to stabilize water in fluorinated oil emulsions (US Patent Application Publication No. 2010105112, Li et al. 2020, Christian Holtze et al. 2008). The retention of hydrophobic organic molecules associated with drug screening can be challenging (Etienne et al. 2018, Janiesch et al. 2015). Leakage into the carrier oil and cross-contamination across droplet compartments can obscure assay results, especially when longer incubation times are required for cell screening and / or when compound load bead encapsulation efficiency is high (see Lambda, MacConnell and Paegel 2017).

[0075] Figures 1-19 provide a description of exemplary compositions, devices, methods, sorters, uses, and systems for performing high-throughput screening in microdroplets.

[0076] The terms used in the descriptions of the various embodiments described herein are intended solely to describe specific embodiments and are not intended to limit them. Where used in the descriptions of the various embodiments and in the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless otherwise explicitly indicated in the context. The terms "and / or" as used herein will also be understood to mean and encompass all possible combinations of one or more of the related enumerated items. The terms "includes," "including," "comprises," and / or "comprising," when used herein, specifically indicate the presence of the specified features, integers, steps, operations, elements, and / or components, but will not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0077] As used herein and in the claims, the term "fluorescent dispersion oil" means a fluorinated oil capable of dispersing microdroplets.

[0078] As used herein and in the claims, the term “perfluorocarbon” means a compound composed entirely of fluorine and carbon.

[0079] As used herein and in the claims, the term “perfluorinated compound” means a compound containing carbon, fluorine, and preferably at least one heteroatom, and which does not contain CH bonds in its main chain.

[0080] As used herein and in the claims, the term “perfluorinated oil” means a perfluorocarbon, perfluoroether or partially fluorinated fluoroether, perfluoroamine or partially fluorinated perfluoroamine.

[0081] As used herein and in the claims, the term "hydrofluoroether" means an ether compound containing at least one hydrogen and at least one fluorine.

[0082] As used herein and in the claims, the terms “linked” or “linking” mean direct and / or indirect linking. Linking may include covalent or non-covalent linking, such as hydrogen bonding or ionic bonding. Linking may be reversible (e.g., photocleavable linker) or irreversible.

[0083] As used herein, the term “functionalized handle” means a mounting point that enables the linking of one or more molecules, such as barcodes and / or compounds (e.g., small molecules). The barcode may be a protein, peptide, enzyme, nucleic acid, or any other substance that can act as a barcode that enables the identification of the beads (bead-specific barcode, BSB) and a barcode that enables the identification of the compounds loaded on the beads (compound-specific barcode, CSB). In some embodiments, the nucleic acid is DNA.

[0084] This specification provides continuous-phase formulations for stabilizing and / or increasing molecular retention in droplet emulsions. In some examples, the continuous-phase formulation contains a droplet stabilizer comprising a fluorescein dispersion oil and an emulsifier. In some embodiments, the fluorescein dispersion oil may have an average fluorine content of about 70 wt% or more. In some embodiments, the emulsifier can be selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof.

[0085] Nanoparticles have been reported to stabilize droplets by coating the droplet interface and forming a Pickering emulsion (Gai et al., 2017, Tang et al., 2016). Such nanoparticles can be further modified to achieve compatibility with fluorinated oils with a higher fluorine content than 2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) to improve molecular retention while achieving sufficient droplet stability for microfluidic screening operations, and can be used alone or in combination with known emulsifiers such as FluoSurf (Emulseo), 008-FluoroSurfactant (RAN Biotechnologies), and PicoSurf (Sphere Fluidics, Ltd.).

[0086] In some embodiments, the triblock or diblock copolymers or combinations thereof described herein can be present in a continuous phase formulation at concentrations of, for example, 0.1% to 10% w / w, 0.2% to 8% w / w, 0.3% to 6% w / w, 0.4% to 5% w / w, or 0.5% to 3% w / w. In some embodiments, the triblock or diblock copolymers or combinations thereof can be present in a continuous phase formulation at concentrations of 0.3% to 4% w / w, for example, 2% w / w. In some embodiments, the copolymers described herein are polydispersible copolymers.

[0087] In some embodiments, the nanoparticles described herein can be present in a continuous phase formulation. In some further embodiments, the nanoparticles are partially derivatized silica nanoparticles. In some even further embodiments, the nanoparticles are present in the continuous phase formulation at concentrations of, for example, 0.1–15% w / w, 0.5–10% w / w, 1%–9% w / w, or 2%–8% w / w. In some embodiments, the nanoparticles are partially derivatized silica nanoparticles and are present in the continuous phase formulation at a concentration of 2–8% w / w.

[0088] In some embodiments, the fluorescein dispersion oil may contain perfluorocarbons, perfluorinated oils, hydrofluoroethers, or a combination thereof. In some embodiments, the fluorescein dispersion oil contains one or more oils selected from the group consisting of perfluorocarbons and perfluorinated oils and / or one or more hydrofluoroethers. In some embodiments, the fluorescein dispersion oil contains one or more oils selected from the group consisting of perfluorocarbons and perfluorinated oils and one or more hydrofluoroethers. In some embodiments, the fluorescein dispersion oil contains one or more oils selected from the group consisting of perfluorocarbons and perfluorinated oils or one or more hydrofluoroethers. In some embodiments, the fluorescein dispersion oil contains one or more oils selected from the group consisting of perfluorocarbons and perfluorinated oils. In some embodiments, the fluorescein dispersion oil contains one or more oils selected from one or more hydrofluoroethers. In some embodiments, the total concentration of perfluorocarbons and / or perfluorinated oils in the fluorescein dispersion oil is about 50% w / w or more. In some embodiments, the total concentration of perfluorocarbons and / or perfluorinated oils in the fluorous dispersion oil is about 50% w / w or more, and / or the concentration of one or more hydrofluoroethers in the fluorous dispersion oil is less than about 50% w / w.

[0089] In some embodiments, the total concentration of perfluorocarbons and perfluorinated oils in the fluorous dispersion oil is about 50% w / w or more, and the concentration of one or more hydrofluoroethers in the fluorous dispersion oil is less than about 50% w / w.

[0090] In some embodiments, the total concentration of perfluorocarbons or perfluorinated oils in the fluorous dispersion oil is about 50% w / w or more, and the concentration of one or more hydrofluoroethers in the fluorous dispersion oil is less than about 50% w / w.

[0091] In some embodiments, the total concentration of perfluorocarbons or perfluorinated oils in the fluorous dispersion oil is about 50% w / w or more, or the concentration of one or more hydrofluoroethers in the fluorous dispersion oil is less than about 50% w / w.

[0092] In some embodiments, the concentration of one or more hydrofluoroethers in the fluorescein dispersion oil is less than approximately 50% w / w. The selection of specific fluorescein dispersion oils, triblock and / or diblock copolymers, and fluorinated silica nanoparticles, and their respective concentrations, if present, can be adjusted to suit the specific buffer system in the screening biochemical / cellular assay.

[0093] The following are exemplary continuous-phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. A: Triblock and / or diblock copolymer (0.5% to 3% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oil (50% w / w or more) and hydrofluoroether (0 to 50% w / w), B: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbon and / or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% w / w), and C: Triblock and / or diblock copolymer (0.5-3% w / w) and Pickering emulsifier (2-8% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% w / w).

[0094] The following are exemplary continuous-phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. A: Triblock and diblock copolymers (0.5% to 3% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oil (50% w / w or more) and hydrofluoroether (0 to 50% w / w), B: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbon and / or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% w / w), and C: Triblock and diblock copolymers (0.5-3% w / w) and Pickering emulsifier (2-8% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% w / w).

[0095] The following are exemplary continuous-phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. D: Triblock and diblock copolymers (0.5% to 3% w / w) dispersed in a mixture of perfluorocarbons and perfluorinated oils (50% w / w or more) and hydrofluoroethers (0 to 50% w / w), E: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbons and perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w), and F: Triblock and diblock copolymers (0.5-3% w / w) and Pickering emulsifier (2-8% w / w) dispersed in a mixture of perfluorocarbons and perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w).

[0096] The following are exemplary continuous-phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. G: Triblock and diblock copolymers (0.5% to 3% w / w) dispersed in a mixture of perfluorocarbon or perfluorinated oil (50% w / w or more) and hydrofluoroether (0 to 50% w / w), H: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbons and perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w), and I: Triblock and diblock copolymers (0.5-3% w / w) and Pickering emulsifier (2-8% w / w) dispersed in a mixture of perfluorocarbon or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% w / w).

[0097] The following are exemplary continuous-phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. J: Triblock or diblock copolymer (0.5% to 3% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oil (50% w / w or more) and hydrofluoroether (0 to 50% w / w), K: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbon and / or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% w / w), and L: Triblock or diblock copolymer (0.5-3% w / w) and Pickering emulsifier (2-8% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% w / w).

[0098] The following are exemplary continuous-phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. M: Triblock or diblock copolymer (0.5% to 3% w / w) dispersed in a mixture of perfluorocarbons and perfluorinated oils (50% w / w or more) and hydrofluoroethers (0 to 50% w / w), N: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbons and perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w), and O: Triblock or diblock copolymer (0.5-3% w / w) and Pickering emulsifier (2-8% w / w) dispersed in a mixture of perfluorocarbons and perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w).

[0099] The following are exemplary continuous-phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. P: Triblock or diblock copolymer (0.5% to 3% w / w) dispersed in a mixture of perfluorocarbon or perfluorinated oil (50% w / w or more) and hydrofluoroether (0 to 50% w / w), Q: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbon or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% w / w), and R: Triblock or diblock copolymer (0.5-3% w / w) and Pickering emulsifier (2-8% w / w) dispersed in a mixture of perfluorocarbon or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% w / w).

[0100] Diblock copolymer In some embodiments, the diblock copolymer comprises a fluorophilic component. In other embodiments, the diblock copolymer comprises a fluorophilic component and a PEG group. In further embodiments, the diblock copolymer comprises a perfluorinated polyether (PFPE) and a PEG group. The fluorophilic component of the diblock copolymer described herein typically comprises a fluorophilic chain of at least C8 length (i.e., containing at least 8 carbon atoms). In some embodiments, the fluorophilic chain comprises at least C 10 Length, at least C 15length, at least C 20 length, at least C 25 length, or at least C 30 length. In other embodiments, the parent fluorophilic chain has at least C 50 length, at least C 75 length, at least C 100 length, or longer. As a non-limiting example, the structure -(C3F6O) 10 -containing parent fluorophilic component has 30 carbons equivalent to a C 30 chain. The parent fluorophilic component may be linear, branched, cyclic, saturated, unsaturated, etc.

[0101] In some embodiments, the fluorophilic component of the diblock copolymer includes non-carbon heteroatoms (e.g., oxygen (e.g., divalent oxygen), sulfur (e.g., divalent or hexavalent sulfur), nitrogen (e.g., trivalent nitrogen), etc.) in the structure of the component. Such heteroatoms may, for example, be bonded to carbon atoms in the backbone structure of the component. Additionally and / or alternatively, the fluorophilic component may include one or more branched branches extending from the main chain of the structure.

[0102] In some embodiments, diblock copolymers for use in the compositions, methods, sorters, uses, devices, and systems provided herein may include those described in U.S. Patent Application Publication No. 2010 / 0105112A1.

[0103] In some embodiments, the diblock copolymer is a copolymer of Formula 1 below or a combination thereof.

Chemical Formula

[0104] Triblock copolymer In some embodiments, the triblock copolymer includes a fluorophilic component. In some embodiments, the triblock copolymer includes at least one fluorophilic component, for example, two fluorophilic components. In other embodiments, the triblock copolymer includes at least one fluorophilic component, for example, two fluorophilic components and a PEG group. In further embodiments, the triblock copolymer includes at least one perfluorinated polyether (PFPE) chain, for example, two perfluorinated polyether (PFPE) chains and a PEG group. The fluorophilic components of the triblock copolymers described herein typically include a fluorophilic chain of at least C8 length (i.e., containing at least eight carbon atoms). In some embodiments, the fluorophilic chain includes at least C 10 Length, at least C 15 Length, at least C 20 Length, at least C 25 Length, or at least C 30 This is the length. In other embodiments, the fluorophilic chain is at least C 50 Length, at least C 75 Length, at least C 100 It is the length of or greater than [a certain length]. A non-restrictive example is the structure (C3F6O). 10 -Fluorophilic components having C 30 It has 30 carbon atoms equivalent to a chain. The fluorophilic component can be linear, branched, cyclic, saturated, or unsaturated.

[0105] In some embodiments, the fluorophilic component of the triblock copolymer contains heteroatoms (e.g., non-carbon atoms such as oxygen (e.g., divalent oxygen), sulfur (e.g., divalent or hexavalent sulfur), or nitrogen (e.g., trivalent nitrogen)) in the structure of the component. Such heteroatoms may be bonded to carbon atoms in the skeletal structure of the component, for example. Additionally and / or alternatively, the fluorophilic component may include one or more branched branches extending from the main chain of the structure.

[0106] In some embodiments, the triblock copolymers for use in the compositions, methods, sorters, uses, devices, and systems provided herein may include those described in U.S. Patent Application Publication No. 2010 / 0105112A1.

[0107] In some embodiments, the triblock copolymer is the copolymer of Formula 2, the copolymer of Formula 3, or a combination thereof. [ka] (In Formula 2, i, j, and k are precise or mean values ​​of polydispersible building blocks, and i and k are independently 35 to 45, and j is 1 to 23. In some embodiments, the precise or average molecular weight of the copolymer of Formula 2 is 2,000 to 20,000 Da. In some embodiments, i and k are independently about 38, and j is about 9, about 10, or about 11.), and [ka] (In Equation 3, p, q, r, s, and t are precise or mean values ​​of polydisperse building blocks, where p and t are independently 35 to 45, r is 1 to 23, q and s are each greater than 0, and the precise or mean value of the sum q+s is 3 to 6. In some embodiments, p and t are independently about 38, r is about 12.5, q and s are greater than 0, and the precise or mean value of the sum q+s is about 6. In some embodiments, p and t are independently about 38, r is about 39, q and s are each greater than 0, and the sum q+s is about 6. In some embodiments, the precise or average molecular weight of the copolymer of Equation 3 is 2,000 to 20,000 Da.)

[0108] Partially fluorinated silica nanoparticles By increasing the surface fluorophilicity of silica nanoparticles, it is possible to improve their dispersibility in perfluorocarbons. In some embodiments, the fluorinated silica nanoparticles are partially derivatized. In some embodiments, the fluorinated silica nanoparticles may have a size of about 100 nm, for example, 110 nm with a distribution of 80-130 nm. Examples of fluorinated silica nanoparticles for use in compositions, methods, uses, sorters, devices, and systems provided herein include partially fluorinated silica nanoparticles of formula 4 and partially fluorinated silica nanoparticles of formula 5. [ka] 1H,1H,2H,2H-Perfluorooctyltriethoxysilane [ka] 1H,1H,2H,2H-Perfluorodecyltriethoxysilane

[0109] Fluorescent Dispersion Oil In some embodiments, exemplary fluorescein dispersion oils for use in compositions, sorters, uses, methods, devices, and systems provided herein include the oils and combinations thereof shown in Table 1 below.

[0110] [Table 1]

[0111] In some embodiments, the continuous phase formulation comprises a fluorescein dispersion oil and a droplet stabilizer containing an emulsifier which is a diblock copolymer, a triblock copolymer, or a combination thereof. In some embodiments, the fluorescein dispersion oil is as follows: A mixture of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) in a ratio of 1:1:1 w / w or approximately 1:1:1 w / w. A mixture of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) in a ratio of 2:2:1 w / w or approximately 2:2:1 w / w, and A mixture of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) in a ratio of 1:3:1 w / w or approximately 1:3:1 w / w. The following combinations of diblock and triblock copolymers are available for selection: The diblock copolymer of Formula 1 described herein, and Triblock copolymers selected from the group consisting of compounds of formulas 2 and 3 described herein, and combinations thereof That is the case.

[0112] In some embodiments, the continuous phase formulations described herein may include a Pickering emulsifier such as fluorinated silica nanoparticles. In further embodiments, the Pickering emulsifier is 8% wt of the following formula 6: [ka] These are fluorinated silica nanoparticles (100 nm).

[0113] This specification provides methods for reducing cross-contamination between microdroplets. In some embodiments, the method may involve generating droplets in a continuous phase formulation, such as a continuous phase formulation containing the oil and emulsifier described herein. In some embodiments, the emulsifier can be present at concentrations of about 0.3% to about 4% w / w, or about 2% w / w. In some embodiments, the emulsifier can be present at concentrations of about 0.3%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 0.3% to about 2%, about 0.5% to about 2%, about 1% to about 2%, about 1% to about 1.5%, or about 1.5% to about 2%. In some embodiments, the oil may be a fluorescein oil, mineral oil, silicone oil, or any other water-immiscible oil. In some embodiments, the oil may be a hydrocarbon-based oil, such as mineral oil, hexadecane, silicone oil, sunflower oil, light mineral oil, kerosene, decane, undecane, dodecane, octane, cyclohexane, hexane, etc. In some embodiments, droplets can be generated by a syringe pump.

[0114] After droplet formation, the droplets can be collected (for example, in an Eppendorf tube). The continuous phase can be replaced with a fluorescein dispersion oil, such as a mixture of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) in a 1:3:1 ratio. In some examples, the volume of the second fluorescein dispersion oil can be approximately the same as the volume of the droplet (for example, 20 μL of fluorescein dispersion oil can be used for 20 μL of droplets). Replacement with fluorescein dispersion oil can be performed two or more times (for example, two, three, four, five or more times in total). Droplets can achieve long-term compound retention, e.g., compound retention for more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, or more than 8 days. In some embodiments, the droplets can be incubated for 30-60 minutes between one or more exchange steps. In some embodiments of the method for reducing cross-contamination between microdroplets, the continuous phase is replaceable with a first fluorescein dispersion oil to provide aqueous microdroplets suspended in a first fluorescein dispersion oil. In some embodiments, the first fluorescein dispersion oil is replaceable with a second fluorescein dispersion oil to provide aqueous microdroplets suspended in a second fluorescein dispersion oil. In some embodiments, the first fluorescein dispersion oil may have an average fluorine content of >70 wt% and may contain a Pickering emulsifier. In some further embodiments, the Pickering emulsifier is fluorinated silica nanoparticles dispersed in a mixture of perfluoro-2-butyltetrahydrofuran and 2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500), e.g., formula 6 [ka] Fluorinated silica nanoparticles, for example, 8% wt of formula 6 [ka] This may be fluorinated silica nanoparticles (100 nm). In some embodiments, the second fluorescein dispersion oil may have an average fluorine content of >70 wt% and may not contain emulsifiers. In some examples, the volume of the fluorescein dispersion oil may be approximately the same as the volume of the droplet (for example, 20 μL of fluorescein dispersion oil can be used for a 20 μL droplet). The fluorescein dispersion oil can be replaced two or more times (for example, two, three, four, five or more times in total). The droplets can achieve long-term compound retention, e.g., compound retention for more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, or more than 8 days. In some embodiments, the droplets can be incubated for 30 to 60 minutes between one or more replacement steps. In some embodiments, the second dispersion oil can be further replaced with a third dispersion oil having the emulsifier described above to provide stability for further droplet operations.

[0115] Compound library matrix In the compositions, methods, uses, sorters, devices, and systems provided herein, code library members are delivered in a single droplet. Matrices such as beads can be used as carriers for the compounds and corresponding barcodes. Examples of matrices include hydrogel beads, core-shell beads, hydrogel-shell beads, magnetic hydrogel beads, and capsules. In some embodiments, the beads are monodisperse.

[0116] Monodispersible hydrogel beads In some embodiments, the compound library matrix, such as beads, resins, or hydrogels, may be a composition of polyethylene glycol acrylamide copolymer (PEGA). This composition may exhibit excellent swelling in both aqueous and organic solvents, chemical compatibility and stability for a wide range of organic reactions, loading capacity to achieve target compound concentrations in droplets, dispersibility into aqueous media when loaded with hydrophobic library compounds, compressibility to achieve quantitative 1:1 encapsulation into droplets in microfluidic devices, and low background autofluorescence to minimize assay interference on droplet-based screens. Quantitative (1:1) encapsulation of compressible compound beads enhances screening throughput. While we do not wish to be bound by any theory, the enhancement of screening throughput is achieved by reducing the statistics from double Poisson to single Poisson, i.e., by using compressible beads so that every droplet contains assay-relevant compound beads, whereas using incompressible compound beads that cannot be packed into microfluidic channels results in many empty droplets during downstream sorter processing, reducing screening throughput. Furthermore, if a bioassay is used that allows for multiple cells per droplet in combination with compressible beads, more assay-related droplets will be generated, since most droplets will contain at least one cell.

[0117] This specification provides a method for preparing monodisperse PEGA resins via polymerization in droplets. In some embodiments, the method can be carried out using a microfluidic device capable of generating monodisperse droplets of a desired size. In some embodiments, monodisperse droplets can be generated with very high throughput. Monodispersity can improve the consistency of screening data against a droplet-based screen, and thus the compound loading capacity that allows for the determination of compound concentrations within the droplets. In the method provided herein, the size distribution of the droplet-generating PEGA resins can be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size distribution of the droplet-generating PEGA resins can be within 5%.

[0118] In some embodiments, it is possible to increase the throughput of polymerization in droplets by using one or more droplet splitters in the microfluidic chip design so that many droplets are generated in parallel.

[0119] Figure 3 concretely depicts an exemplary microfluidic device (i.e., a chip) for generating droplets. In some embodiments, the droplet generation chip 101 may include a continuous phase formulation input 102 and an aqueous buffer input 103 containing multiple monomers of a PEGA resin. In some embodiments, the monomers of the PEGA resin can be introduced into a microdroplet via the input 103 using the microfluidic device 101. The droplet can exit the device at a droplet output 104. In some embodiments, the monomers of the PEGA resin can undergo in-situ polymerization. In-situ polymerization can be initiated by a polymerization initiator such as TEMED in the continuous phase or ammonium persulfate (APS) in the aqueous phase. The PEGA resin may have three components: 1) acrylamide (e.g., an acrylamide skeleton), 2) bis-acrylamide PEG, and 3) mono-acrylamide PEG and / or mono-acrylamide diamine containing a functionalization handle. In some embodiments, the functionalized handle may be, but is not limited to, a free amine, a protected amine (e.g., a Boc-protected amine or an Fmoc-protected amine), or a protected or unprotected alcohol, acid or ester, azide, acetylene, or tetrazine. Figure 4 shows a monodisperse PEGA resin prepared using the droplet generating tip depicted in Figure 3.

[0120] In some embodiments, the monomer components of the PEGA resins exemplified herein are listed below. This specification describes exemplary compositions and methods for preparing amino-modified PEGA resins. Monomers containing other functional groups, such as those described herein, are understood to be usable. Furthermore, the methods described herein are understood to be applicable to the preparation of other PEGA resins, such as PEGA resins containing other functional groups.

[0121] In some embodiments, the acrylamide monomers for use in the compositions, methods, uses, sorters, devices, and systems provided herein may include acrylamide (i.e., CH2=CHC(O)NH2), N-methylacrylamide, N,N-dimethylacrylamide, and combinations thereof. In some embodiments, the acrylamide monomer is N,N-dimethylacrylamide. In some embodiments, the bis-acrylamide PEGs for use in the compositions, methods, uses, sorters, devices, and systems provided herein may include compounds of the following formula 7. [ka] (In Formula 7, R1 and R2 are independently H or -CH3, n1 is a precise value or mean value, and n1 is between 1 and 10⁸. In some embodiments, n1 is selected such that the bis-acrylamide PEG has an average molecular weight of 250 to 5000 daltons, and in further embodiments, n1 is selected such that the bis-acrylamide PEG has an average molecular weight of 800 to 2000 daltons.)

[0122] In some embodiments, the mono-acrylamide PEG for use in compositions, methods, uses, sorters, devices, and systems provided herein is the compound represented by Formula 8. [ka] (In formula 8, R3 to R6 are independently H or -CH3, R7 is hydrogen, Boc, or Fmoc, n2 is a precise value or mean value, and n2 is between 1 and 10⁹. In some embodiments, n2 is selected such that the monoacrylamide PEG has an average molecular weight of 158 to 5000 daltons. In some embodiments, n2 is 4.)

[0123] In some embodiments, the monoacrylamide ethylenediamine for use in compositions, methods, uses, sorters, devices, and systems provided herein is a compound of Formula 9. [ka] In Equation 9, R8 and R9 are independently H or -CH3, and R10 is selected from the group consisting of H, Boc, and Fmoc.

[0124] In some embodiments, the PEGA resin has the structure shown in formula 10. [ka] (In formula 10, each NH2 can independently be a functionalization handle for compound / DNA loading.)

[0125] In some embodiments, examples of monomer molar ratios may be N,N-dimethylacrylamide:bis-PEG-acrylamide(2000D):Boc-amino-PEG-acrylamide = 44:1:7 or about 44:1:7.

[0126] In some embodiments, PEGA resin beads can be prepared via a bulk emulsion method to prepare polydispersible hydrogel beads, which can then be passed through several cell strainers of different mesh sizes to obtain a resin with a defined size distribution.

[0127] Hydrogel-Shell Beads Figure 2D concretely depicts compound loading beads 160 encapsulated in a hydrogel matrix 161. Figure 2E concretely depicts compound loading beads 160 coated with a hydrophilic polymer 162.

[0128] Figures 5B-5C illustrate different embodiments in which compound loading beads, such as any hydrogel beads or polystyrene core beads described herein, can be modified to improve their physicochemical properties to facilitate bead handling in both library preparation and droplet microfluidics (Figure 5A). Such modifications can provide the same hydrophilic outer layer regardless of the type of linker and ligand loaded onto the beads, thus minimizing the compound dependence of bead behavior, such as aqueous suspension, which depends on the linker and ligand loaded onto the hydrogel beads or polystyrene core beads. Figure 5A depicts an embodiment in which compound loading beads 110 are linked to ligand 111 and barcode 112. Figure 5B depicts an embodiment in which core-shell beads 113 include a compound loading core 110 and a hydrophilic shell 114. The ligand 111 and barcode 112 are linked to the compound loading core. In some embodiments, the barcode is DNA. Figure 5C depicts an embodiment in which core-shell beads 113 include a compound loading core 110 and a hydrophilic shell 114. The ligand 111 is linked to the compound loading core 110, and the barcode 112 is linked to the hydrophilic shell 114. In some embodiments, the barcode is DNA.

[0129] Figure 5D illustrates an embodiment in which a compound loading core 110, for example, an amino-modified hydrogel as described herein, for example, PEGA or TentaGel® beads (Figure 5D-1), can be modified (e.g., partially modified) by installing a hydrophilic polymer coating 115 that provides a hydrophilic surface (e.g., containing a hydrophilic motif) capable of shielding hydrophobic ligands from exposure. In some embodiments, the modification may be acylation. In other embodiments, the modification may be azide-acetylene click chemistry. In some further embodiments, the modification may be acylation with a hydrophilic polymer. In further embodiments, the modification may be azide-acetylene click chemistry with an azide and DBCO (dibenzocyclooctin) functionalized hydrophilic polymer on a solid support. The hydrophilic motif may be, but is not limited to, PEG, PPG, polylactic acid, or a combination thereof.

[0130] In some embodiments, the compound library can be constructed on a compound loading core such as amino-modified PEGA resin 110 or TentaGel® beads before modification with a hydrophilic surface, for example, before the formation of dual-layer hydrogel beads or PEGylated TentaGel® beads. In such cases, the remaining azide handle on the split linker can be used as a handle, for example, via a click reaction with a PEG-conjugated alkyne, or via reduction of the azide followed by amide coupling with a PEG-conjugated acid or its activating acid.

[0131] Figure 5E illustrates an embodiment in which the compound loading core 110, for example, the amino-modified hydrogel described herein, for example, PEGA, can be modified (e.g., partially modified) with a monomer such as acrylamide to seed polymer shell formation, for example, acrylamide-based polymer shell formation. The polymer shell 116, for example, the acrylamide-based polymer, may be, but is not limited to, polyacrylamide and PEGA.

[0132] Figure 5F illustrates embodiments in which a hydrogel compound loading core bead 110, for example, an amino-modified hydrogel as described herein, such as PEGA, can be encapsulated in another layer of hydrogel, such as PEGA. In some embodiments, encapsulation can be performed in a microfluidic chip. In some embodiments, encapsulation can be performed with a 1:1 encapsulation efficiency. The library beads can be physically encapsulated by introducing the compound loading core bead 110, for example, an amino-modified PEGA resin, into a shell monomer 117, for example, a droplet of naive PEGA monomer to form a shell. A continuous phase formulation, for example, a continuous phase oil 118, is passed through to form the core-shell beads. Polymerization can be performed by an initiator, for example, APS in the aqueous phase and TEMED in the continuous phase. In some embodiments, polymerization can be started when droplets are generated. TEMED in the continuous phase can initiate the process in the aqueous droplets. In some embodiments, polymerization can be accelerated by heating.

[0133] In another embodiment, amino-functionalized core-naive PEGA shell dual-layer beads can be generated in a single microfluidic device, where a substrate for amino-modified PEGA resin can be introduced into the core of the droplet, and the naive PEGA resin without functionalized handles can be introduced into the periphery, and the layers can be rapidly polymerized in TEMED to give a core-shell structure in a single experiment.

[0134] Core shell beads This specification provides compositions, methods, uses, sorters, systems, and devices in which beads are encapsulated in a compressible hydrogel shell. In some embodiments, the beads are encapsulated by hydrogel formation in droplets. In some embodiments, hydrogel formation in droplets is carried out as described herein by a microfluidic device, such as a microfluidic device for preparing monodisperse PEGA resins. In some embodiments, the Poisson statistics of the bead encapsulation and / or clamping in aqueous medium are improved by encapsulation. In some embodiments, the Poisson statistics of the bead encapsulation and clamping of polystyrene-PEG hybrid beads, such as TentaGel® beads, in aqueous medium can be improved. In some further embodiments, the Poisson statistics of the bead encapsulation and clamping in aqueous medium can be improved by encapsulating polystyrene-PEG hybrid beads, such as TentaGel® beads, into a compressible hydrogel shell using hydrogel formation in droplets utilizing a microfluidic device described herein, such as a microfluidic device for preparing monodisperse PEGA resins. In some embodiments, the hydrogel may be, for example, PEGA, polyacrylamide, alginate, agarose, collagen, or a combination thereof. In some further embodiments, the hydrogel may be polyacrylamide. The beads can be suspended in a hydrogel monomer and encapsulated in the hydrogel using a microfluidic device. For example, 10-micron polystyrene-PEG hybrid beads, such as aminopolystyrene-PEG hybrid (e.g., TentaGel®) library beads, can be suspended in a polyacrylamide hydrogel monomer and encapsulated in polyacrylamide using a microfluidic device to give a hydrogel shell, such as a 30-micron hydrogel shell or approximately 30-micron hydrogel shell. Figures 20A-B illustrate the effect of the PEG-coated thin shell on the dispersibility of compound-loaded TentaGel® beads in aqueous cell culture medium.A comparison of bead dispersion after 4 days of complete dispersion by probe sonication is shown, with Figure 20A showing the bead without PEG coating and Figure 20B showing the bead with 40K PEG coating. As shown in Figure 11A, fluorescein is loaded onto the beads via a photocleavable linker. After 4 days of standing, large bead clamping is observed in the beads without PEG coating, while the beads with 40K PEG coating are dispersed mainly as a monodisperse.

[0135] Hydrogel-encapsulated library beads, such as hydrogel-encapsulated polystyrene-PEG hybrid library beads, can achieve flexibility in performing combinatorial chemistry and DNA ligation based on conventional methods (see, e.g., MacConnell et al., 2015, 2017) while retaining the flexibility of compressible hydrogel beads, allowing for encapsulation of hydrogel-encapsulated beads in droplets (e.g., microdroplets) in a 1:1 or approximately 1:1 ratio. Encapsulation can be measured, for example, by analyzing droplets with a hemocytometer / microscope chamber slide (e.g., iBidi or Countess) capable of producing tightly packed monolayers of droplets for observation under a comparable microscope, relative to the number of encapsulated droplets in an empty droplet.

[0136] In some embodiments, the Poisson distribution of bead (e.g., polystyrene-PEG hybrid beads, e.g., TentaGel®)) encapsulation in a hydrogel shell can be further improved by sorting the hydrogel-encapsulated beads, e.g., hydrogel-encapsulated polystyrene-PEG hybrid beads prepared using droplet microfluidics, based on specific scattering or fluorescence signatures, using standard FACS to enrich the hydrogel-encapsulated beads containing one or a desired number of core beads (e.g., one polystyrene-PEG hybrid bead, e.g., TentaGel®). Figures 9G-9H show an example of such enrichment process. In this case, crude TentaGel® in the polyacrylamide population is 6% (Figure 9G), while after FACS enrichment based on a subpopulation of events with significantly higher fluorescence in FITC and PE channels (Figure 9I), the TentaGel® encapsulated population increases to 75% (Figure 9H).

[0137] Poisson statistics and clamping of bead encapsulation can be improved with any of the beads described herein, for example, polystyrene-PEG hybrid beads, e.g., M30102 TentaGel® M NH2, Rapp Polymere GmbH. Hydrogels capable of encapsulating beads include the hydrogels described herein, e.g., PEGA, polyacrylamide, alginate, agarose, collagen, or combinations thereof. In some embodiments, the hydrogel is a polyacrylamide hydrogel composition formed by polymerization of acrylamide and bis-acrylamide (Zilionis et al., 2017). In some embodiments, the acrylamide and bis-acrylamide have the following structures. Acrylamide - [ka] and bis-acrylamide- [ka]

[0138] In some embodiments, the hydrogel has the following structure. [ka]

[0139] Magnetic monodispersible hydrogel beads Figures 6A and 6B illustrate embodiments in which the hydrogel beads described herein may contain magnetic microparticles, such as magnetic core beads. In some embodiments, the magnetic beads are suspendable in the hydrogel generating mixture, and droplets are generated as described herein. Figure 6A depicts an embodiment in which coated magnetic beads 120 are physically encapsulated within hydrogel beads 121. The coated magnetic beads are suspendable in the hydrogel generating mixture, and droplets are generated in such a way that the coated magnetic beads are encapsulated in the hydrogel but not covalently linked to provide magnetic hydrogel beads 121. Figure 6B depicts an embodiment in which coated magnetic beads 122 containing an amine group are functionalized with acrylamide to form acrylic magnetic core beads 123. The acrylic magnetic core beads 123 are suspendable in the hydrogel generating mixture, and droplets are generated in such a way that the coated magnetic beads are encapsulated in the hydrogel and covalently linked to provide magnetic core hydrogel beads 124.

[0140] In some embodiments, magnetic core beads can be coated, thereby preventing side reactions. In some embodiments, the coating encapsulates the magnetic core beads. Hydrogel beads containing magnetic core beads can be manipulated, isolated, and / or separated by magnetic force, thus increasing the efficiency of screening that can be performed, for example, by enriching bead-encapsulated droplets. For example, in bead washing steps in compound synthesis or phase coding, beads can be separated by magnetic force, thereby replacing more cumbersome separation steps such as filtration or centrifugation (Rana et al., 1999). In some embodiments, magnetic bead-encapsulated droplets can be separated from unencapsulated droplets, thereby reducing the number of droplets to be sorted (Ofner et al., 2017). In some embodiments, the magnetic core beads described herein may be fluorescent and capable of encoding their relative positions in a three-dimensional hydrogel grid (Meldal & Christensen, 2010).

[0141] In some embodiments, polystyrene-coated magnetic beads, such as acrylamide-functionalized amino-modified polystyrene-coated magnetic beads (e.g., DynaBeads (1 micron, amino-modified, Thermofisher)), can be suspended in a hydrogel-generating mixture. Droplets can be generated as described herein. In some embodiments, the beads (e.g., polystyrene-coated magnetic beads) may have a certain level of autofluorescence. In some further embodiments, beads having a certain level of autofluorescence may act as code tags.

[0142] In some embodiments, silica magnetic beads can be suspended in a hydrogel generating mixture. Droplets can be generated as described herein. In some embodiments, silica magnetic beads can be amino-modified. In some further embodiments, amino-modified silica magnetic beads can be functionalized with acrylamide. In some embodiments, the amino-modified silica magnetic beads are 1-micron amino-modified beads (BOCA Scientific). The beads can be suspended in a hydrogel generating mixture. Droplets can be generated as described herein. Silica magnetic beads may have low levels of autofluorescence.

[0143] Furthermore, in another embodiment, carbon-coated cobalt nanoparticles (e.g., TurboBeads) (Grass et al., 2007) (TurboBeads Llc, Zurich, Switzerland) can be embedded in a hydrogel as described herein. TurboBeads can offer advantages in handling, particularly because they may have high magnetic properties.

[0144] Furthermore, in another embodiment, iron oxide magnetic nanoparticles (e.g., TurboBeads) (Grass et al., 2007) (TurboBeads Llc, Zurich, Switzerland) can be embedded in a hydrogel as described herein.

[0145] Soluble monodisperse hydrogel beads In some embodiments, crosslinkers for compound loading and / or hydrogel-shell beads, such as, but not limited to, electromagnetic (e.g., light), enzyme, pH, temperature, and / or redox-sensitive linkers, are selectable in terms of degradability by a particular trigger. Such degradable polymers are well known in the field of controlled drug delivery (Gillies, 2020). In some further embodiments, the PEG-bis-acrylamide crosslinkers of monodisperse PEGA resins exemplified herein can replace the photo-unstable crosslinkers described in the literature (Kloxin et al., 2009, Raman et al., 2020) to produce monodisperse photo-unstable PEGA resins for the code bead library synthesis and screen systems described herein.

[0146] In some embodiments of the compositions, methods, uses, sorters, systems, and devices provided herein, the resin loading capacity can be determined by loading a known fluorophore (e.g., fluorescein) into the resin via a cleavage linker, such as a photocleavage linker. The beads can be individually encapsulated in a single droplet of a known volume. The fluorophore can be released from the beads within the droplet. The fluorescence intensity of the droplet can be determined by calibrating it against a reference droplet having a known fluorescein concentration.

[0147] Code 1 Beads 1 Compound Library In some embodiments, the compound library can be assembled on code beads. In some embodiments, the compound library can be combinatorially assembled on code beads. Methods for assembling the compound library are described in (MacConnell et al., 2015). In a code-1-bead-1-compound (eOBOC) library, each bead may have an atomolecular to femtomole of the same compound and a unique tag, preferably a DNA tag, loaded via a cleavable linker, preferably a photocleavable linker, to the bead itself (bead-specific barcode, BSB) and to the combinatorially generated compound itself (compound barcode). The cleavable linker may, alternatively, be an enzymatically cleavable, pH-sensitive, or reduction-sensitive (disulfide) linker. The tag may, alternatively, be an RFID, a fluorescent dye and / or bead, or a peptide mass tag, or a combination thereof.

[0148] Figure 2C concretely illustrates an embodiment of a compound and barcode mounted on a bead. The compound loading bead (110) is linked to the ligand (111) and the barcode (112). The compound is linked by (111) and a photocleavable linker (170). The ligand and barcode are optionally linked to a common functionalization handle on the bead via a split linker (Figure 11A).

[0149] Figures 11A–11C concretely illustrate embodiments of DNA coding bead library linkers and DNA barcode designs. Figure 11A illustrates an embodiment in which a split linker for compound loading and DNA coding via a photocleavable linker may contain 1) azidrisine for loading DNA headpieces via strain-enhancing click chemistry, and 2) an ortho-nitrobenzyl linker for photocleavage at a wavelength of 365 nm. Figure 11B illustrates an embodiment in which DNA coding can be achieved by clicking a DNA headpiece with a forward primer onto compound beads, then ligating first and second barcodes for bead-specific barcodes, followed by standard split-pool combinatorial chemistry in a microtiter well plate with ligation for building block code double-stranded oligonucleotides, further identifier sequences, and finally closing with a reverse primer (see, for example, Paegel et al. (MacConnell et al., 2015, 2017)).

[0150] The code bead libraries described herein can be optionally prepared with different compound loadings and / or different compound combinations to enable dose-response screens (qHTS) for the entire library. This method can add flexibility to photo-dose-response methods (see, for example, Paegel et al. (Price et al., 2016)) and may be particularly useful when partial cleavage of release linkers, such as enzymes or pH-unstable linkers, may be difficult.

[0151] Figure 11C illustrates an embodiment in which the linker supporting the compound loading site can be blocked at different doses to code for different dosage levels. This dose coding strategy can enable pooling of dose-response beads for library preparation and screening, thereby adding a vast amount of structure-activity resolution while maintaining the ease of pooled synthesis and screening.

[0152] Assay Figures 12A–C illustrate embodiments in which typical mix-and-read fluorescent reporter assays, resulting in either signal gain or loss using various reporters, can be configured on a droplet-based screening platform. In some embodiments, the assay is a biochemical or IVTT assay in a droplet (Figure 12A), a fluorescent cell or bead in a droplet (Figure 12B), a fluorescent reporter secreted from a cell in a droplet (Figure 12C), or a combination thereof. Assays include, but are not limited to, biochemical assays with fluorescent and / or next-generation sequencing readouts, cell-free assays, cell reporter assays, and cell phenotypic assays. The platform can support both compound screening and genetic screening assay methods. The signal may diffuse throughout the droplet (e.g., either a biochemical cell-free assay or a cell-based assay utilizing a secretion reporter system) or be contained within a cellular compartment detected via a secondary reporter bead (e.g., a cytokine secretion assay).

[0153] Figure 13 illustrates examples of reporters, such as fluorescent proteins (e.g., CFP, YFP, GFP, RFP, and related FP (Figure 13, Section i)), or fluorescent reporter enzymes, such as β-lactamases that provide ratiometric detection via a FRET substrate or β-galactosidases that provide near-infrared shift fluorescence to limit interference by autofluorescent components, which can be used to report on compounds whose expression is modulated via either transcription or post-transcriptional processes (Figure 13, Section ii), or in the form of secretion reporters (Figure 13, Section iii).

[0154] In some embodiments, compounds that degrade a target protein (POI) can be assayed on a platform by fusing the POI to GFP. In the assay, either suspension cells (e.g., K562, KG1, U937, Jarcut cells, etc.) or adherent cells (e.g., HEK293) can be engineered to express a GFP-POI-P2A-RFP reporter. This reporter expresses the GFP-POI fusion and the non-fusion RFP reporter as separate proteins at nearly equal levels within the cell using a ribosomal skipping sequence (P2A) incorporated between two genes. If the POI contains degron such as an IKZF3 tag (Sievers et al., 2018), the addition of a compound such as lenalidomide can induce degradation of the GFP fusion with signal loss.

[0155] In the case of secretion reporters, the reporter, such as GFP, can be constructed as a secretion protein, or the reporter can be a secretion enzyme such as secretion alkaline phosphatase (SEAP). The use of secretion reporters may have the advantage that rendering data processing and, consequently, sorting can be more direct and robust because the signal is diffused throughout the droplet rather than to a single focus (cell).

[0156] In another example, intracellular viral infectivity can be screened when investigating whether an agent enhances the effectiveness of antiviral drugs or virus-based therapies.

[0157] In some embodiments, the compositions, methods, uses, sorters, systems, and devices provided herein can be used, for example, to screen for agonists that modulate protein-protein interactions using a three-branched GFP system (Cabantous et al., 2013).

[0158] In some embodiments, the compositions, methods, uses, sorters, devices, and systems provided herein can be used to perform fluorescence-based biochemical displacement assays, such as biochemical displacement assays configured with probes for POIs containing a GFP fluorescence quencher. Similar types of assays can be constructed using fluorescence polarization as a readout (Hackler et al., 2020).

[0159] In some embodiments, the compositions, methods, uses, sorters, devices, and systems provided herein can be used to perform UV light cutting, and the imaging and / or methods provided herein may include UV light cutting and / or imaging. Example 20 describes UV light cutting and imaging.

[0160] The flexibility of this platform, including the IVTT system, can enable the implementation of assays that multiplex many compounds against many proteins and / or targets. An example of such an assay is selective translation inhibition, in which the sequence encoding the protein to be screened for translation inhibition is added next to the barcode sequence on the beads. In some embodiments, the assay involves selective stalling of human translation via small molecule engagement of nascent ribosomal chains (see, for example, Lintner et al. (2017) PLOS Biol. 15(3):e2001882). Protein expression is achievable via IVTT. When a positive result is achieved and protein translation is inhibited, the identity of the compound and protein can be determined via one-shot next-generation sequencing of the compound barcode and the protein-encoding gene.

[0161] Other possible assays include standard reporter gene assays, protein-protein interaction (PPI) assays using split reporter systems or TANGO systems (ThermoFisher Scientific) that probe GPCR function, or combinations thereof. Detection of secreted proteins such as cytokines from cells co-encapsulated with beads that capture the proteins for detection using fluorescently labeled antibodies is also possible.

[0162] Any of the droplet-based assays can be applied to screening a coding compound library, a genetic screen (e.g., CRISPER), or a combination thereof. Table 2 provides an overview of the assay types that can be performed on the droplet-based screening platform.

[0163] [Table 2]

[0164] Droplet sorting Fluorescence-activated droplet sorter (FADS) This specification provides methods, systems, and devices for sorting droplets. Figure 2G illustrates an exemplary embodiment of a sorter into which droplet 134 can flow into a path (200) between two electrode sets (130 and 131) to which sortable electric fields can be independently applied, allowing droplet 203 to be sorted into sorting channel 201 and droplet 204 to be sorted into sorting channel 202. Droplet 134 can be detected and identified as a hit droplet or a non-hit droplet before passing between the electrode sets (130 and 131).

[0165] The droplet sorters described herein are based on the remarkable finding that sorting by dual-electrode sorters, such as the dual-electrode sorters described herein, can enhance the speed and / or reliability of sorting.

[0166] Figures 14A–14E illustrate exemplary embodiments in which two electrode sets 130 and 131 exist. In some embodiments, the electrode sets may be present immediately before the droplet path splits into two sorting channels 132 and 133. In some further embodiments, at least two electrode sets may be on different sides of the droplet stream. The sorting may be high-frequency sorting. Figures 14A–14B illustrate embodiments in which droplet 134 can move to random channel 132 or 133 when there is no applied force, such as an electric field. When there is no applied force acting on the droplet, the droplet can move randomly with equal or nearly equal resistance (Figure 14B). Figure 14C illustrates embodiments in which an electric field is applied to a non-hit stream. In some embodiments, the electric field can be applied until the target droplet to be sorted reaches junction 135 where the droplet stream splits.

[0167] Figures 14D and 14E depict embodiments in which droplets 134 can be guided into a stream flowing through a sorting channel 133 by applying a force, for example, from electrode pair 131, such as an electric field (Figures 14D and 14E). When a target droplet arrives, the electric field for the hit stream can be activated at electrode pair 131. In some embodiments, the electric field for non-hit streams can be deactivated at electrode pair 130. As shown in Figure 14E, once a target droplet arrives, the activation of the electric field for the hit stream can redirect the target droplet 134 into its stream 133. This system can be implemented with the droplets, droplet-generating oil and emulsifier described herein, and combinations thereof. Droplet flow can be controlled, for example, by a pressure pump, a syringe pump, or a combination thereof.

[0168] Figures 15A–15E show examples of fluorescent droplets being detected and sorted. As shown in Figure 15A, droplet 144 is detected, for example, by fluorescence detection and sorted, for example, by the application of an electric field 145. Figures 15B–15E show examples of droplet 144 being detected and sorted. As shown in Figure 15B, droplet 144 flowing through the non-hit path 132 and the "hit" path 133 is detected by the sorter. Electrode pair 130 had an electric field applied (e.g., from 400V). Electrode pair 131 did not have an electric field applied. As shown in Figure 15C, a delay occurred after detection and before sorting. The delay (e.g., several milliseconds) can be introduced to match the time required for the detected droplet to reach the sorting junction. Sorting after the delay can minimize false positives in embodiments where bistandard droplets are directed to the hit path. In some embodiments, an evaluation algorithm can decide whether to apply an electric field within this time frame. As shown in Figure 15D, droplet 144 was identified as a hit, an electric field (e.g., arising from 800V) was applied to electrode pair 131, electrode pair (130) had no electric field, and sort droplet 144 was sorted in channel 133. As shown in Figure 15E, droplet 144 was not identified as a hit, and the droplet was sorted in non-hit stream 132.

[0169] In the methods, systems, and devices provided herein for sorting droplets, the droplets can be sorted by the application of an electric field, a magnetic field, a change in flow, or a combination thereof. In some embodiments, it is possible to position two or more electromagnets relative to electrodes at positions described herein. In some embodiments, droplets are sorted by changing the applied electric field. Dual sorting systems have been developed that allow for the rerouting of droplets by voltage. Non-hit droplets flow into the straight path at a lower voltage, while preventing them from rerouting into the side paths. In side path sorting, the voltage is turned on with the non-hit path voltage switched off.

[0170] Hydrogel bead sorting by FACS In some embodiments, assay droplets containing compound beads and bioassay products, generated using the preferred emulsion formulations described herein, can be converted to hydrogel beads by introducing biocompatible polymers and / or their precursors, for example, but not limited to, agarose, alginate, collagen, polyacrylamide, PEG and corresponding initiators, or any combination thereof, as required. The thus formed assay hydrogels can be isolated in aqueous buffer while preserving the compound beads and cells on the beads for off-droplet sorting using a conventional flow cytometer (Duarte et al., 2017; Yanakieva et al., 2020).

[0171] In some embodiments, core-shell beads containing members of a code library may be converted into water-in-oil droplets using preferred emulsion formulations described herein, comprising a cell-containing cavity, a compound released and incubated within a discrete droplet compartment, and a hydrogel supporting the beads and cells, which is extracted into an aqueous buffer for off-droplet sorting using a conventional flow cytometer (Di Carlo et al., 2019; Joseph de Rutte, Robert Dimatteo, Mark van Zee, Robert Damoiseaux, 2020).

[0172] Double emulsion sorting by FACS In some embodiments, water in oil (w / o / w) double emulsion droplets can be sorted. In some embodiments, sorting can be performed to screen droplets. For example, droplets can be sorted using fluorescence. Droplets such as double emulsion droplets can be sorted by FACS. This sorting method can be performed with the droplets described herein, the droplet-generating oil and emulsifier described herein, and combinations thereof. Commercially available FACS machines can be used to sort double emulsion droplets at frequencies of 10–12 kHz (Brower et al., 2019). Figure 16 shows water in oil (w / o / w) double emulsion droplets in an imaging flow cytometer (Amnis ImageStream X MkII, Luminex Corporation). In some embodiments, the imaged double emulsion droplets are monodisperse double emulsion droplets.

[0173] Sorting of arrayed droplets Figures 17A–17F depict embodiments for automated detection and / or sorting of emulsion droplets. Figure 17A depicts a grid 140 of partitions (e.g., microwell plate containing wells 141) where each partition (e.g., well) 141 is equal to or approximately equal to a size range of emulsion droplets. Figure 17B depicts a grid 140 in which emulsion droplets 142 are arrayed within the wells 141 of the grid. In some embodiments, one droplet is arrayed in each partition or well 141. Arraying emulsion droplets makes it possible to spatially trap the droplets. Compared to flow-based FADS, this spatial control can result in a fairly wide range of readout possibilities over a given time frame, e.g., bright-field, fluorescent or luminescent readout, or any combination thereof. In addition to standard live and dead cell staining readouts and fluorescent biosensors, spatial control enables high-resolution imaging of phenotypic readouts, e.g., cell or organelle morphology, localization of target proteins, etc. Figure 17C depicts a grid 140 containing droplets 141. After the application of one or more parameters (e.g., compound release from code beads via triggers such as electromagnetic irradiation (e.g., light, UV, UVA, e.g., about 365 nm) or enzymatic cleavage, incubation for peptide / protein expression using in-vitro transcription / translation, or simple incubation for a reporter, pH change, reducing agent, or a combination thereof), the assay can be analyzed and hit droplets 143 and 144 can be identified. After defining one or more desired groups, as illustrated in Figure 17D, the hit droplets can be removed from wells 145 of the microwell plate 140 (e.g., mechanically). Figure 17E depicts the analysis of single and multiple hit group droplets 143 and 144, respectively. In some embodiments, droplet removal can be automated. Droplets can be removed, for example, by a micromanipulator (e.g., an automated robotic micromanipulator), by irradiation, or a combination thereof.Figure 2H concretely illustrates an embodiment in which the hit droplet 143 is identified in the microwell plate 140.

[0174] In some embodiments, the defined emulsion droplets can be removed by an automated robotic micromanipulator. The removed droplets can be deposited at the defined location. In some embodiments, the droplets can be removed by irradiation (e.g., electromagnetic irradiation), thereby bursting the droplets and releasing their contents into the surrounding phase, allowing for analysis of the contents. In some further embodiments, the irradiation is by laser.

[0175] In some embodiments described herein, droplets for use in the sorting methods, devices, and systems described herein (e.g., arrayed droplets) can be denser than the continuous phase in which the droplets are suspended. In some embodiments, the droplets can be denser than the oily phase in which the droplets are suspended. When the droplets are denser than the liquid in the microplate, the droplets can settle to the bottom of the wells, thus facilitating imaging and / or analysis. In some embodiments, the droplets can be denser than the oily phase using fluorinated water-in-oil double emulsion droplets or water-in-oil droplets in which the oily phase is not denser than the aqueous phase. In an exemplary workflow, arrayed emulsion droplets can be arrayed and automatically imaged based on a defined hit criterion, and hit droplets can be automatically removed from the array for further analysis. Figure 17F shows a microscopic image of an array in which double emulsion droplets with and without a fluorescent dye (146 and 147) are arrayed in a microwell plate.

[0176] In some embodiments, barcodes associated with code beads can be identified. Barcode identification may include, for example, fluorescence microscopy / countering, RFID readers, mass spectrometry, DNA sequencing, or a combination thereof. In some embodiments, next-generation sequencing and data science can be used to deconvolute hits. In some embodiments, sequenced DNA barcodes from code beads can be sequenced. For example, in some embodiments, PCR can be performed directly on the DNA ligated to the beads.

[0177] In relation to droplet sorting and screening using NGS readouts, enrichment of unique bead-specific barcodes (BSBs) per compound-specific barcode (CSB) can be compared between assay hits sorted into "hit sort" (compounds active in the assay within the droplet) and "non-hit sort," and appropriate statistical models can be applied to assess the significance of enrichment. Using dataset randomization, it is possible to estimate the upper limit of enrichment background that calls a hit.

[0178] The droplet sorting methods, systems, uses, and devices described herein (e.g., double emulsion sorting by FADS or FACS, sorting of arrayed droplets) may comprise a black hole quencher assay, a secretion reporter assay, or a combination thereof. In some embodiments, droplet-based screening / sorting may comprise the use of an automatic cell picker. In some embodiments, the automatic cell picker may comprise a microwell array plate that hosts one microdroplet per microwell, a fluorescence microscope, an imaging setup for automatic imaging of assay droplets and identification of desired droplets, a microcapillary-based droplet sampling device for automatic picking of desired droplets and deposition into hit wells, or any combination of the foregoing. In some embodiments, the continuous phase formulation comprises a) one or more oils selected from the group consisting of perfluorocarbons and perfluorinated oils, and / or b) one or more hydrofluoroethers.

[0179] In some embodiments, the total concentration of perfluorocarbons and / or perfluorinated oils in the fluorous dispersion oil is about 50% w / w or more, and / or the concentration of one or more hydrofluoroethers in the fluorous dispersion oil is less than about 50% w / w.

[0180] In some embodiments, a) the perfluorocarbon is a perfluoroalkane selected from the group consisting of perfluorooctane, perfluoroheptane, perfluorohexane (FC-72), perfluoro-1,3-dimethylcyclohexane, and octadecafluorodecahydronaphthalene (perfluorodecalin), and / or b) the perfluorinated oil is perfluoro-2-butyltetrahydrofuran, perfluoro-N-methylmorpholine (FC-3284), perfluorotripentylamine (FC-70), perfluorotributylamine (FC-43), perfluorotripropylamine (FC-3283), perfluorotributylamine and perfluoro(dibutyl The hydrofluoroether is selected from the group consisting of (FC-40) a mixture with (Tylmethylamine), and / or c) the hydrofluoroether is selected from the group consisting of 2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500), ethyl perfluorobutyl ether (HFE-7200), 3-methoxyperfluoro(2-methylpentane) (HFE7300), methyl perfluoroisobutyl ether / methyl perfluorobutyl ether mixture, mixture of methoxynonafluorobutane and methoxynonafluoroisobutane (HFE7100), methoxynonafluorobutane, methoxyheptafluoropropane (HFE7000), HFE712, HFE649, HFE7100DL, HFE71DE, HFE71IPA, HFE7200DL, HFE72DA, HFA72DE, HFE72FL, HFE73DE, HFE7700, and HFE8200.

[0181] In some embodiments, the perfluorocarbon is a perfluoroalkane selected from the group consisting of perfluorooctane, perfluoroheptane, perfluorohexane (FC-72), perfluoro-1,3-dimethyl-cyclohexane, and octadecafluorodecahydronaphthalene (perfluorodecalin), and / or the perfluorinated oil is selected from the group consisting of perfluoro 2-butyltetrahydrofuran, perfluoro-N-methylmorpholine (FC-3284), perfluorotripentylamine (FC-70), perfluorotributylamine (FC-43), perfluorotripropylamine (FC-3283), and a mixture of perfluorotributylamine and perfluoro(dibutylmethylamine) (FC-40), and / or the hydrofluoroether is selected from the group consisting of 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500), ethyl perfluorobutyl ether (HFE-7200), 3-methoxyperfluoro(2-methylpentane) (HFE7300), methyl perfluoroisobutyl ether / methyl perfluorobutyl ether mixture (HFE7100), and methoxynonafluorobutane (HFE7000).

[0182] In some embodiments, the perfluorocarbon is a perfluoroalkane selected from the group consisting of perfluorooctane, perfluoroheptane, perfluorohexane (FC-72), perfluoro-1,3-dimethylcyclohexane, and octadecafluorodecahydronaphthalene (perfluorodecalin), and the perfluorinated oil is perfluoro-2-butyltetrahydrofuran, perfluoro-N-methylmorpholine (FC-3284), perfluorotripentylamine (FC-70), perfluorotributylamine (FC-43), and perfluorotripropylamine (FC-70). -3283) is selected from the group consisting of a mixture of perfluorotributylamine and perfluoro(dibutylmethylamine) (FC-40), and the hydrofluoroether is selected from the group consisting of 2-(trifluoromethyl)-3-ethoxidedecafluorohexane (HFE-7500), ethyl perfluorobutyl ether (HFE-7200), 3-methoxyperfluoro(2-methylpentane) (HFE7300), methyl perfluoroisobutyl ether / methyl perfluorobutyl ether mixture (HFE7100), and methoxynonafluorobutane (HFE7000).

[0183] In some embodiments, the perfluorocarbon is a perfluoroalkane selected from the group consisting of perfluorooctane, perfluoroheptane, perfluorohexane (FC-72), perfluoro-1,3-dimethylcyclohexane, and octadecafluorodecahydronaphthalene (perfluorodecalin).

[0184] In some embodiments, the perfluorinated oil is selected from the group consisting of perfluoro-2-butyltetrahydrofuran, perfluoro-N-methylmorpholine (FC-3284), perfluorotripentylamine (FC-70), perfluorotributylamine (FC-43), perfluorotripropylamine (FC-3283), and a mixture of perfluorotributylamine and perfluoro(dibutylmethylamine) (FC-40).

[0185] In some embodiments, the hydrofluoroether is selected from the group consisting of 2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500), ethyl perfluorobutyl ether (HFE-7200), 3-methoxyperfluoro(2-methylpentane) (HFE7300), methyl perfluoroisobutyl ether / methyl perfluorobutyl ether mixture (HFE7100), and methoxynonafluorobutane (HFE7000).

[0186] In some embodiments, the fluorescein dispersion oil consists of perfluorohexane (FC-72), perfluorooctane, and 2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500), each present in amounts ranging from 10 to 90% w / w.

[0187] In some embodiments, the fluorescein dispersion oil is selected from the group consisting of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxidedecafluorohexane (HFE-7500) in a 1:1:1 w / w ratio or about 1:1:1 w / w ratio, perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxidedecafluorohexane (HFE-7500) in a 2:2:1 w / w ratio or about 2:2:1 w / w ratio, and perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxidedecafluorohexane (HFE-7500) in a 1:3:1 w / w ratio or about 1:3:1 w / w ratio.

[0188] In some embodiments, the triblock and / or diblock copolymer is fluorinated. In some embodiments, the triblock or diblock copolymer comprises perfluoropolyether (PFPE) and polyethylene glycol (PEG) (for example, perfluoropolyether (PFPE), polypropylene glycol (PPG), and polyethylene glycol (PEG), two perfluoropolyethers (PFPE) and polyethylene glycol (PEG), or two perfluoropolyethers (PFPE), two polypropylene glycols (PPG), and polyethylene glycol (PEG).

[0189] In some embodiments, the diblock copolymer is derived from formula 1 [ka] (In the formula, n and m are precise or average values ​​of polydispersible building blocks, the average molecular weight of the diblock copolymer is 1,000 to 10,000 Da, n is 35 to 45, and m is 2 to 24.) It has. In some embodiments, the triblock copolymer is [ka] (wherein i, j, and k are precise or average values ​​of polydispersible building blocks, the average molecular weight of the triblock copolymer is 2,000 to 20,000 Da, i and k are independently 35 to 45, and j is 1 to 23.), and [ka] (In the formula, p, q, r, s, and t are the precise or mean values ​​of the polydisperse building blocks, the average molecular weight of the triblock copolymer is 2,000 to 20,000 Da, p and t are independently 35 to 45, q and s are each greater than 0, the precise or mean value of the sum q + s is 3 to 6, and r is 1 to 23.) has a formula selected from the group consisting of:

[0190] In some embodiments, the emulsifier comprises a diblock copolymer and a triblock copolymer in a ratio of about 1:1 to 1:9 (w / w), respectively. In such embodiments, the diblock copolymer and the triblock copolymer are present in the continuous phase formulation at a combined concentration of about 0.3 to 4 wt% (e.g., about 2% w / w).

[0191] In some embodiments, the droplet stabilizer comprises fluorinated silica nanoparticles (e.g., partially fluorinated silica nanoparticles comprising fluorinated alkyl side chains).

[0192] In some embodiments, the fluorinated silica nanoparticles may include those described in U.S. Patent Application Publication No. 2016 / 0114325A1.

[0193] In some embodiments, the fluorinated silica nanoparticles have the formula Chemical formula comprising one or more substituents selected from the group consisting of the substituents of.

[0194] In some embodiments, the fluorinated silica nanoparticles are present in the continuous phase formulation at a concentration of about 2 to 8% w / w.

[0195] In some embodiments, the fluorinated silica nanoparticles are Pickering emulsifiers.

[0196] In some embodiments, the Pickering emulsifier is 8% wt of Formula 6 dispersed in perfluoro-2-butyltetrahydrofuran:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) at a 1:1 (w / w) ratio Chemical formula These are fluorinated silica nanoparticles (100 nm).

[0197] In some embodiments, the method for preparing a monodispersible PEGA copolymer resin further includes coating the PEGA copolymer resin with a hydrophilic material (for example, a hydrophilic polymer or hydrogel, such as polyethylene glycol (PEG), polypropylene glycol (PPG), hyaluronic acid, polylactic acid, polyacrylamide, alginate, agarose, or collagen).

[0198] In some embodiments, a method for preparing a monodisperse PEGA copolymer resin further includes linking a ligand and / or barcode (e.g., nucleic acid, DNA) to the PEGA copolymer resin either reversibly (e.g., by a photocleavable linker) or irreversibly (e.g., by mounting to a functionalized handle). In some embodiments, a method for preparing a monodisperse PEGA copolymer resin further includes linking a ligand and / or barcode (e.g., nucleic acid, DNA) to the PEGA copolymer resin either reversibly (e.g., by a photocleavable linker) or irreversibly (e.g., by mounting to a functionalized handle). In some embodiments, a method for preparing a monodisperse PEGA copolymer resin further includes linking a ligand or barcode (e.g., nucleic acid, DNA) to the PEGA copolymer resin either reversibly (e.g., by a photocleavable linker) or irreversibly (e.g., by mounting to a functionalized handle).

[0199] In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further includes reversibly linking ligands to the PEGA copolymer resin and irreversibly linking barcodes (e.g., nucleic acids, DNA) (e.g., mounting onto a functionalized handle). In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further includes reversibly linking ligands to the PEGA copolymer resin and irreversibly linking barcodes (e.g., nucleic acids, DNA) (e.g., mounting onto a functionalized handle). In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further includes reversibly linking ligands to the PEGA copolymer resin and reversibly linking barcodes (e.g., nucleic acids, DNA) (e.g., mounting onto a functionalized handle). In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further includes reversibly linking ligands to the PEGA copolymer resin and reversibly linking barcodes (e.g., nucleic acids, DNA) (e.g., mounting onto a functionalized handle).

[0200] In some embodiments, the ligand is reversibly linked to the PEGA copolymer resin, the barcode is irreversibly linked to the PEGA copolymer resin, and the barcode is DNA.

[0201] In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further includes embedding magnetic particles in the PEGA copolymer resin by covalent linkage or physical encapsulation. In such embodiments, the magnetic particles may have a size of 1 nanometer to 10 microns in diameter.

[0202] In some embodiments, the monodisperse PEGA copolymer resin has a diameter of 1 to 100 microns, or at least one microdroplet is a plurality of monodisperse microdroplets, and the size distribution of the PEGA copolymer resin (e.g., within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) is within 5%.

[0203] In some embodiments of the method for preparing monodispersible PEGA copolymer resins, bis-acrylamide PEG has a precise or average molecular weight of 250 to 5,000 daltons (e.g., 800 to 2,000), and / or mono-acrylamide PEG has a precise or average molecular weight of 150 to 5,000 daltons.

[0204] In some embodiments of the method for preparing monodispersible PEGA copolymer resin, bis-acrylamide PEG is expressed as follows: Formula 7: [ka] (In Equation 7, R1 is H or -CH3, R2 is H or -CH3, and the precise or average n1 is between 1 and 10⁸.) It has the following structure. In such embodiments, mono-acrylamide PEG is the following formula 8: [ka] The structure is, or mono-acrylamide diamine is, formula 9: [ka] It has the structure of [the object]. (In Equation 8, R3 to R6 are independently H or -CH3, R7 is hydrogen, Boc, or Fmoc, and precise or average n2 is 1 to 10⁹, and In Equation 9, R8 and R9 are independently H or -CH3, and R 10 (This is selected from the group consisting of H, Boc, and Fmoc.)

[0205] In some embodiments of the method for preparing a monodispersible PEGA copolymer resin, the monomers dispersed in an aqueous buffer include N,N-dimethylacrylamide, bis-PEG-acrylamide (2000 daltons), and Boc-amino-PEG-acrylamide.

[0206] In some embodiments of the method for preparing monodispersible PEGA copolymer resins, the functionalization handle is selected from the group consisting of protective amines (e.g., Boc protective amines or Fmoc protective amines), unprotected amines (e.g., free amines), alcohols, acids, esters, azides, acetylenes, and tetrazines.

[0207] In some embodiments of the method for preparing core-shell beads, core beads are linked to monomers corresponding to multiple monomers. In such embodiments, monomer polymerization includes covalently linking core beads to a hydrogel that encapsulates the beads.

[0208] In other embodiments, thin-shell (e.g., PEG-coated) core-shell beads are prepared by grafting a hydrophilic polymer coating onto a solid carrier. In some embodiments, the solid carrier is a polystyrene matrix, for example, a low-crosslinked polystyrene matrix grafted with polyethylene glycol. In some examples, the hydrophilic polymer coating includes PEG. In some examples, the PEG is mounted by acetylene-azidocric chemistry.

[0209] The swelling capacity (degree of swelling) of the resulting network is known to be affected by the amount of crosslinking (crosslinking density). The swelling capacity of the resin in both organic and aqueous media is particularly important when performing DNA ligation in an aqueous buffer while carrying out chemistry in an organic solvent. In photocrosslinking, a sufficient amount of crosslinkable monomer is used to give the polymer beads dimensional stability so that they swell rather than dissolve in aqueous and / or organic media. Lower levels of crosslinking usually provide a larger surface area and adsorption capacity in the final product, but optimal performance will also depend on the type of monomer and other conditions, such as the degree of swelling and even other process conditions. For this reason, the slightly (or lightly) crosslinked polystyrene matrix of this disclosure with PEG grafting is selected so that the swelling capacity of the polymer is sufficient in both aqueous and organic media.

[0210] In some embodiments, the formation of core-shell beads includes forming a mixture of core-shell beads and empty beads. In such embodiments, the method further includes enriching the mixture of core-shell beads and empty beads (e.g., by flow cytometry) so that core-shell beads are obtained in which one core bead is encapsulated per core-shell bead.

[0211] In some embodiments, forming core-shell beads involves forming a plurality of core-shell beads that are sufficiently stable and compressible so that the core-shell beads can be packed in a microfluidic channel and encapsulated in a microdroplet with super Poisson encapsulation efficiency.

[0212] In some embodiments of the method for preparing core-shell beads, the hydrogel is selected from the group consisting of PEGA, polyacrylamide, alginate, agarose, and collagen.

[0213] In some embodiments of the method for preparing core-shell beads, at least one core bead includes a ligand and / or a barcode, or the method further includes attaching a ligand and / or a barcode to at least one core bead of the core-shell beads. In some embodiments of the method for preparing core-shell beads, at least one core bead includes a ligand and / or a barcode, or the method further includes attaching a ligand and / or a barcode to at least one core bead of the core-shell beads. In some embodiments of the method for preparing core-shell beads, at least one core bead includes a ligand and a barcode, or the method further includes attaching a ligand and a barcode to at least one core bead of the core-shell beads. In some embodiments of the method for preparing core-shell beads, at least one core bead includes a ligand or a barcode, or the method further includes attaching a ligand or a barcode to at least one core bead of the core-shell beads.

[0214] In some embodiments of the method for preparing core-shell beads, the method further includes ligating a ligand and / or a barcode to a hydrogel encapsulating the core beads.

[0215] In some embodiments of the method for preparing core-shell beads, the method further includes ligating a ligand and / or a barcode to the hydrogel encapsulating the core beads. In some embodiments of the method for preparing core-shell beads, the method further includes ligating a ligand or a barcode to the hydrogel encapsulating the core beads. In some embodiments of the method for preparing core-shell beads, the method further includes ligating a ligand and a barcode to the core beads. In some embodiments of the method for preparing core-shell beads, the method further includes ligating a ligand to the core beads and ligating a barcode to the hydrogel encapsulating the core beads. In some embodiments of the method for preparing core-shell beads, the method further includes ligating a ligand to the hydrogel encapsulating the core beads and ligating a barcode to the core beads.

[0216] In some embodiments, at least one core bead is selected from the group consisting of hydrogel beads, magnetic hydrogel beads, divinylbenzene crosslinked polystyrene beads, low crosslinked polystyrene matrices grafted with polyethylene glycol, magnetic beads, silica beads, glass beads, and ceramic beads.

[0217] In some embodiments, the diameter of the core-shell beads is approximately 1 to 70 microns (for example, approximately 10 microns, approximately 15 microns, approximately 20 microns, approximately 30 microns, approximately 33 microns, approximately 40 microns, approximately 50 microns, approximately 60 microns, approximately 70 microns, approximately 20 to 50 microns, approximately 5 to 40 microns, approximately 10 to 35 microns, or approximately 30 to 40 microns), or the diameter of the core-shell beads is 70% or less of the diameter of the droplet.

[0218] In some embodiments, the core-shell beads include core beads encapsulated with polyacrylamide. In such embodiments, the core beads are a low-crosslinked polystyrene matrix grafted with polyethylene glycol.

[0219] In some embodiments of the method for preparing core-shell beads, at least one microdroplet is a water-in-oil (w / o) single emulsion microdroplet.

[0220] In some embodiments, the sorter further includes a sensor configured to detect a target composition having selective properties that flows through an inlet channel upstream of the junction. In such embodiments, the controller is configured to switch the states of first and second electrodes from a first state to a second state in response to the sensor detecting the target composition.

[0221] In some embodiments, the sensor is a fluorescence intensity sensor that includes at least one photomultiplier tube.

[0222] In some embodiments, the selection characteristics are selected from the group consisting of a threshold amount of fluorescence intensity, emission of fluorescence intensity within a threshold range, emission of a specific combination of a fluorescence signal and its threshold intensity, and emission of a specific combination of fluorescence signal intensity within a threshold ratiometric range.

[0223] In some embodiments, the controller is configured to return the first and second electrodes to a first state after the target compound has passed through the junction.

[0224] In some embodiments, the junction has a symmetric geometry, and the first and second electrodes have a third state in which multiple target compounds flow through the junction and into both the first and second outlet channels without either the first or second electrode receiving a voltage.

[0225] In some embodiments, the diameter of the inlet channel is 1 to 200 microns.

[0226] In some embodiments, the second electrode does not receive a voltage in the first state, and the first electrode does not receive a voltage in the second state.

[0227] In some embodiments, both the inlet channel and the first outlet channel have straight geometry and are aligned with each other. In such embodiments, the second outlet channel has curved geometry.

[0228] In some embodiments, in a first state, the first electrode receives a first voltage. In such embodiments, in a second state, the second electrode receives a second voltage that is greater than the first voltage.

[0229] In some embodiments, the first side of the junction is on the opposite side of the second side of the junction. [Examples]

[0230] Example 1: Perfluor polypropylene-polyethylene glycol (PFPE-PEG) [ka] 50 mL of Krytox157-FSH (carboxylic acid-terminated perfluoropolyether) (51.07 g, 7.60 mmol) in HFE-7100 was charged into an oven-dried 100 mL two-neck round-bottom flask equipped with a stirring bar and a findenser. The reaction mixture was cooled to 0°C under an N2 line. Oxalyl chloride (9.98 mL, 114 mmol) was added dropwise at 0°C, the ice bath was removed, and the mixture was heated to 70°C for 5 hours. The solvent was evaporated and the mixture was placed under high vacuum for 18 hours to yield acid chloride 11 (average molecular weight = approximately 6739) as an opaque oil, which was used in the next step.

[0231] In a 100 mL oven-dried two-necked round-bottom flask equipped with a stirring bar and a findenser, 10 mL of THF and DIPEA (672 μl, 3.85 mmol) containing poly(ethylene glycol) methyl ether 500 (1812 mg, 3.85 mmol) was charged under an N2 atmosphere. 10 mL of HFE-7100 containing 11 (7628 mg, 1.132 mmol) was added dropwise, and the mixture was heated at 60°C for 3 hours. The reaction mixture was cooled to room temperature and stirred overnight under an N2 atmosphere.

[0232] To remove unreacted amines, 6 mL of DCM, followed by 6 mL of FC-72, was added to the reaction mixture, and the contents were transferred to a separatory funnel. The separatory funnel was shaken and left for several hours until two clear layers were obtained. The top layer containing organic matter was discarded, and the bottom layer was evaporated to yield an opaque oil.

[0233] To remove the PEGamine salt, the latter oil was washed with 1N HCl, DI water, and then MeOH. The resulting oil was dissolved in 7 mL of HFE-7100, and 10 mL of THF and then 4 mL of MeOH were added. The resulting mixture was heated at 75°C for 10 minutes to remove the top organic layer. The bottom fluorescein layer was evaporated to give the desired diblock copolymer 12 as an opaque oil (74% yield). FT-IR(cm-1): 2871(CH), 1720(CO). 19 F NMR (376 MHz, benzene-d6) δ -76.32~-84.77 (m), -131.29 (s), -133.19~-133.79 (m), -145.16~-146.81 (m).

[0234] Example 2: Perfluor polypropylene-polyethylene glycol-perfluor polypropylene (PFPE-PEG-PFPE) [ka] 50 mL of Krytox157-FSH (carboxylic acid-terminated perfluoropolyether) (51.07 g, 7.60 mmol) in HFE-7100 was charged into an oven-dried 100 mL two-neck round-bottom flask equipped with a stirring bar and a findenser. The reaction mixture was cooled to 0°C under an N2 line. Oxalyl chloride (9.98 mL, 114 mmol) was added dropwise at 0°C. The ice bath was removed and the reaction was heated to 70°C for 5 hours. The solvent was evaporated and the reaction was placed under high vacuum for 18 hours to give an opaque oil of 11 (average molecular weight = approximately 6739), which was used in the next step.

[0235] In an oven-dried 100 mL two-necked round-bottom flask equipped with a stirring bar and a findenser, 10 mL / 5 mL of THF / DMF and poly(ethylene glycol)diamine 400 (156 mg, 0.389 mmol) in DIPEA (0.340 mL, 1.945 mmol) were charged under an N2 atmosphere. 10 mL of HFE-7100 containing 11 (6553 mg, 0.972 mmol) was added dropwise, and the mixture was heated at 60°C for 30 min. 1 mL of poly(ethylene glycol)methyl etheramine 500 (694 mg, 1.389 mmol) in THF was added. The mixture was heated at 60°C for 2.5 hours, cooled to room temperature, and stirred overnight under an N2 atmosphere.

[0236] To remove unreacted amines, 10 mL of DCM, followed by 10 mL of FC-72 (perfluorohexane), was added to the reaction mixture, and the contents were transferred to a separatory funnel. The separatory funnel was shaken and allowed to stand until two clear layers were obtained. The top layer, containing organic matter, was discarded. The bottom layer was evaporated to yield an opaque oil.

[0237] To remove the PEGamine salt, the latter oil was washed with 1N HCl, DI water, and then MeOH. The resulting oil was dissolved in 5 mL of HFE-7100, and 8 mL of THF was added. The resulting mixture was heated at 75°C for 10 minutes to remove the top organic layer. 3 mL of MeOH was added, and the resulting mixture was heated at 75°C for 10 minutes. The top layer was removed, and the bottom fluorescein layer was evaporated to give the desired triblock copolymer 13 as a pale yellow oil (79% yield). FT-IR(cm-1): 2870(CH), 1721(CO). 19 F NMR (376 MHz, benzene-d6) δ -80.08~-86.48 (m), -131.41 (s), -133.18~-133.84 (m), -144.83~-147.14 (m).

[0238] Example 3: Dye retention in droplets To enable screening in picoliter droplets, each screening vessel is a stable and sealed entity. The droplets can be stabilized for the duration of bioassays and screenings, which can range from minutes to weeks, by adding emulsifiers and / or nanoparticles to the continuous phase. The emulsion formulation is prepared by dissolving any or all of the required amount of emulsifier, such as di and triblock copolymers and / or partially fluorinated silica nanoparticles, in a fluorinated oil. The water-in-oil droplets are then generated by a microfluidic device, such as the one shown in Figure 3. In this case, the aqueous phase contains the bioassay material and its culture medium, and the continuous phase contains the oil and emulsifier.

[0239] Figures 7A-7H show the droplet retention of 100 μM fluorescein and 100 μM resorphine (in 1 M HEPES buffer) at time zero (top) and 1 hour later (bottom) for four formulations: FluoSurf (Emulseo) (Figures 7A and 7E), PicoSurf (Sphere Fluidics, Ltd.) (Figures 7B and 7F), 008-FluoroSurfactant (RAN Biotechnologies) (Figures 7C and 7G), and droplet-generating oil 1864006 (Bio-Rad) (Figures 7D and 7H). All showed good retention of the hydrophilic and loading electrodyne fluorescein (Figures 7A-7D), but none were able to retain neutral and more hydrophobic dyes such as resorphine (Figures 7E-7H).

[0240] By reducing the net dipole moment of the oily composition in the continuous phase, the distribution of organic molecules across the droplet boundary can be restricted, while simultaneously achieving biocompatibility, including cell viability, and mechanical stability for droplet manipulation. The fluorophilicity of 2-(trifluoromethyl)-3-ethoxidedecafluorohexane (HFE-7500) (fluorine content 66%) was increased by wetting with the cosolvent perfluorocarbon / perfluorinated oil (perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxidedecafluorohexane (HFE-7500) in a 1:1:1 w / w ratio, perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxidedecafluorohexane (HFE-7500) in a 2:2:1 w / w ratio, and perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxidedecafluorohexane (HFE-7500) in a 1:3:1 w / w ratio, resulting in a fluorine content >75%), and consequently, increased compound retention. The formulation contained 2% wt of copolymers of formula 1 (average n is approximately 38, and average m is approximately 9) and formula 2 (average i and k are approximately 38, and average j is approximately 9-11) in a 1:1 ratio. No leakage of resolphin (100 μM) or fluorescein (100 μM) in the HEPES buffer was observed. Figures 8A-8D show the molecular holdings of fluorescein (green, Figure 8D) and resolphin (red, Figure 8C) in droplets in compositions containing perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) in a 1:3:1 w / w ratio and 2% wt of the block copolymers described above.

[0241] When conventional di- or tri-block copolymer emulsifiers, including the commercially available formulations described above (C. Holtze et al., 2008, Christian Holtze et al., 2008), were used with these highly nonpolar hydrofluoroether / perfluorocarbon carrier oil systems, adequate droplet stability was not provided (IFT γ > 10 mN / m). However, when the formulations described above, which contain a mixture of 2% wt of block copolymers, were used, excellent droplet stability (> 1 week) was achieved.

[0242] The effectiveness of a 1:1:1 w / w formulation perfluorohexane (FC-72):perfluorooctane:HFE-7500 containing 2% wt of the copolymer of formula 1 (mean n is approximately 38, and mean m is approximately 9) was determined for the retention of commonly used water-soluble dyes (fluorescein derivatives, rhodamine derivatives, and resolphins) shown below. The physiological and chemical properties ranged from 200 to >500 Da (mol wt. 200 to >500 Da) to 50 to 150 Å (TPSA). 2 Excellent molecular retention in droplets was achieved for compounds having hydrophobicity (logD -2 to +4, and net charge -1 to +1).

[0243] [Table 3]

[0244] [Table 4]

[0245] [Table 5]

[0246] In addition, the retention of resolphin (100 μM) and fluorescein (100 μM) was evaluated in droplets containing different buffer systems with various salt concentrations (PBS, 1× to 10×, Tris 100 mM to 1 M, and HEPES 100 mM to 1 M) and assay media (RPMI cell culture medium). In a 1:1:1 w / w ratio formulation of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) containing the copolymer of formula 1 (mean value n is approximately 38 and mean value m is approximately 9) (as described above) at 2% wt, no detectable leaks were observed, and retention was independent of the buffer system used. Furthermore, the properties of the buffer system had little effect on droplet stability.

[0247] Example 4: Cell viability in the presence of diblock copolymer emulsifiers and / or triblock copolymers The effects of di and triblock copolymers on cell viability were determined by encapsulating jarcut cells in 100-micron droplets and incubating them on a benchtop for 24 hours. Prior to encapsulation, the cell concentration was adjusted to 2.0 × 10^6 / mL in fresh medium (RPMI 10%, FBS 1%, Pen Strep 2.5%, Ficoll type 400 (Sigma-Aldrich F5415-50ML)), and the droplets were collected in Eppendorf tubes. The droplets were then ruptured using 10% final concentration 1H,1H,2H,2H-perfluoro-1-octanol (PFO). The aqueous layer containing the cells was collected, washed, stained with SYTOX RED (ThermoFisher S348969) at a 1:1000 dilution, and analyzed using a BD Fortessa (BD Bioscience 647645) flow cytometer (excitation 633 nm, emission filter 670 ± 15 nm).

[0248] Example 5: Mechanical stability and surface tension of emulsion formulations Mechanical stability was assessed by microscopic examination of residual coreless droplets after repeated pipetting and centrifugation of emulsion droplets at 16900 rcf. Surface tension was determined by the pendant droplet method (DSA 30, Kruss, Germany).

[0249] [Table 6]

[0250] Example 6: Replacement with emulsifier-free continuous phase to minimize compound cross-contamination A 2 μL fluorescently labeled droplet with a diameter of 100 microns (containing 100 μM fluorescein and 100 μM resolphin in RPMI buffer) was mixed with an 18 μL unlabeled droplet with a diameter of 100 microns (in RPMI buffer). An oily layer containing an emulsifier (Formula 1 (n is approximately 38 and m is approximately 9):Formula 2 (i and k are approximately 38 and j is approximately 9-11) 1:1, 2 wt%), 20% HFE-7500, 20% FC-72, and 60% perfluorooctane) was removed from the emulsion. The droplets were washed with 20 μL of emulsifier-free oily blend (20% HFE-7500, 20% FC-72, and 60% perfluorooctane). The oil removal and washing process was repeated 3 times. The droplets were incubated in eppi tubes for >5 days, and leakage was monitored by fluorescence spectroscopy (CY3 10 ms and FITC 10 ms exposure). No leakage of resolphin or fluorescein to unlabeled droplets was observed (Figure 8E-8G).

[0251] Example 7: Synthesis of Boc-amino-PEG functionalized monodisperse PEGA resin PEGA hydrogel microbeads were synthesized via microfluidic encapsulation. A 1 mL aqueous phase was prepared containing 100 μL of TBSET buffer, 30 μL of 10% (w / v) APS (Sigma-Aldrich, A9164), 37.6 mg of dimethylacrylamide (Sigma-Aldrich, 274135), 25.1 mg of acrylamide-PEG-acrylamide 2KD (Biochempeg, 10060), 19.7 mg of Boc-NH-PEG4-acrylamide (Biochempeg, 12310), and 787.6 mL of water. The solution was filtered using a 0.2 micron syringe filter before loading it into a 1 mL syringe (Becton Dickinson, 309628). 2.5 mL of carrier oil (RAN Biotechnologies, 008-FluoroSurfactant-2wtH-50G) and 10 μL of TEMED (Sigma-Aldrich, T9281-25ML) were mixed and loaded into a 3 mL syringe (Becton Dickinson, 309657). These two syringes were connected to the inlet of a droplet generation device using PE2 tubing (Scientific Commodities, BB31695-PE / 2). The aqueous solution was pumped at 900 μL / h and the oil at 1800 μL / h. Emulsion droplets were collected at the outlet of the microfluidics tip into an Eppendorf tube containing 50 μL of mineral oil (Sigma-Aldrich, M5310-1L) and incubated overnight at 65°C. The Eppendorf tube was centrifuged and the oily phase was discarded. The beads were washed twice with 500 μL of 20% (vol / vol) PFO (Alfa Aesar, B20156) in HFE7500 (Novec7500), and the droplets were ruptured. The beads in the aqueous phase were washed twice with 1% Span80 (Sigma-Aldrich, S6760-250ML) in hexane (Sigma-Aldrich, 227064-1L), and then washed three times with TBSET buffer. The beads were filtered through a 70 μm cell strainer (Corning, 352350) and stored in TET buffer for up to 6 months in four stages.

[0252] Example 8: Preparation of 10 μm TentaGel® and monodispersible 33 micron PEGA resin supporting fluorescein via a photocleavable linker. To determine the loading capacity and photo-emission compatibility of TentaGel® and PEGA resin, fluorescein probes were loaded using a standard solid-phase organic synthesis protocol with some modifications. Briefly, Boc protecting groups on newly prepared 33-micron diameter monodisperse PEGA resin were removed by HCl treatment in methanol for 2-3 hours. The resin was washed by centrifugation and decanting the supernatant, then acylated with an acid linker supporting fluorescein via a photocleavable linker using standard coupling conditions with DIC / HOAt, and the resin was washed again by decanting the supernatant, with repeated solvent changes. The fluorescein-loaded 33-micron aminoPEGA resin thus prepared was kept in the dark and refrigerated until further use. [ka]

[0253] Example 9: Loading capacity of aminopolystyrene-PEG hybrid (TentaGel®) and amino-PEGA resin The loading capacities of 10-micron TentaGel® NH2 (Rapp Polymere) (aminopolystyrene-PEG hybrid) and 33-micron amino-PEGA resins were determined by loading fluorescein into the resin via a photocleavable linker, individually encapsulating the beads in a single droplet of known volume via a microfluidic device, photo-emitting fluorescein from the beads within the droplet, and determining the fluorescence intensity of the droplet. For a 100-micron droplet, the loading capacity of 10-micron TentaGel® NH2 (Rapp Polymere) was determined to be at least 5 femtomoles per bead, achieving a concentration of at least 10 micromoles. The loading capacity of the 33-micron amino-PEGA resin prepared as described in Example 7 was determined to be approximately 20 femtomoles per bead, achieving a concentration of approximately 35 micromoles in a 100-micron diameter droplet. PEGA resin achieved complete release of fluorescein from the beads into the droplets (Figures 9C-9D (before release and approximately 10 seconds after release, respectively)), while aminopolystyrene-PEG hybrid (TentaGel®) resin did not completely release fluorescein (Figures 9A-9B (before release and approximately 10 seconds after release, respectively)).

[0254] Example 10: Quantitative determination of PEGA resin in droplets using 1:1 encapsulation. 33 micron PEGA 1× hydrogel beads were washed multiple times with TET buffer and HBW buffer by vortexing, centrifugation at 1000 g for 3 minutes, and careful removal of supernatant between washes. The resulting washed beads were mixed with an equal volume of 2× bead concentrate mix, vortexed, centrifuged, and all supernatant was removed to prepare tightly packed PEGA 1× beads. A PE-2 tube with a 25 gauge needle was mounted on a 1 mL syringe. The beads were loaded into the tube by inserting the open end of the tube into the beads and gently pulling the plunger. After bead loading, the open end of the tube was mounted on a syringe with a 25 gauge needle filled with HF-7500 carrier oil. These beads were encapsulated in droplets using a microfluidic device containing three inlets (two for aqueous solutions, one for oil-based solutions) and one outlet. Two aqueous inlets were used to connect the beads and cell culture medium, and one organic inlet was used to connect the carrier oil (RAN Biotechnologies, 008-FluoroSurfactant-2wtH-50G). The flow rates to the three inlets were optimized so that one bead / one droplet encapsulation occurred, as shown in Figure 10B.

[0255] reagent 1.Tris-EDTA-Tween buffer (TET) a. 480 mL nuclease-free water b. 5 mL 1 ml Tris-HCl (pH 8.0) c. 10 mL 0.5 M EDTA d. 5 mL of nuclease-free water containing 10% (vol / vol) Tween-20 e. Filter the solution through a 0.2 μm membrane. 2. Hydrogel bead washing buffer (HBW) a. 980 mL nuclease-free water b. 10 mL 1 M Tris-HCl (pH 8.0) c. 200 μL 0.5 M EDTA d. 10 mL of nuclease-free water containing 10% (vol / vol) Tween-20 e. Filter the solution through a 0.2 μm membrane. 3.2x Beads Concentrated Mix a. 6.1 mL nuclease-free water b. 4.2 mL 5 × First Strand Buffer i.Thermo Fisher Scientific cat.No.18080-044 1.250 mM Tris-HCl (pH 8.3 at room temperature) 2.375 mM KCl 3.5 mM MgCl2 c.210μL 10%(vol / vol)Igepal CA-60

[0256] Figure 10A shows the compressibility of the reference polyacrylamide resin against DropSeq (PAA) versus PEGA 1× rod as measured by a texture analyzer. The compressibility of PEGA 1× roughly matches that of the polyacrylamide reference gel (PAA).

[0257] Figure 10B shows tight packing of PEGA 1× resin in a single channel and quantitative 1:1 encapsulation into droplets.

[0258] Example 11: Cell viability in the presence of PEGA resin 1 x 10 6 Jarcut cells suspended in RPMI 20% FBS 1% Pen Strep and resin suspended in water were mixed 1:1 in a 96-well microtiter plate to a final volume of 200 μL, and then incubated at 37°C and 5% CO2 for 24 hours. Cells were also mixed with water in the same manner. Appropriate samples were stained with SYTOX RED (ThermoFisher Scientific S348959) at a 1:1,000 dilution and read using a BD Fortessa flow cytometer. Figures 10C and 10D show FACS results regarding the biocompatibility of 33 micron PEGA resin. PEGA resin had minimal impact on cell viability as determined by FACS after 24 hours of co-incubation in a microtiter well plate.

[0259] Cells cultured in the presence of PEGA 32μM 2000 PEG 1× survived similarly to cells cultured with 50% water, but cells cultured with PEGA 32μM 3700 PEG 2× showed approximately 70% reduced viability.

[0260] Example 12: Compound and DNA tag loading onto 10-micron TentaGel® and 33-micron PEGA resin Reference compound beads were prepared on 10-micron TentaGel® NH2 and 33-micron PEGA resins by adopting the protocol described by Paegel et al. (Paegel et al., 2020) with some modifications. Briefly, the native resin was functionalized with glycine, pbf-protected arginine, glycine, azidrisine, and glycine by sequential loading of each Fmoc-protected amino acid using DIC / Oxima / DIPEA in DMF. Each loading step was carried out by a capping step with a 20% acetic anhydride solution in DMF, followed by Fmoc deprotection with a 20% 4-methylpiperidine solution in DMF. The resin was then reacted with the reference compound derivatized with a photocleavable linker using DIC in a DCM / DMF 1 / 1 mixture. The DNA headpiece was then loaded by click chemistry between the azide handle on the resin and the DBCO-derivative headpiece. Finally, the beads were fully encoded by performing multiple simultaneous enzymatic ligations using T4 ligase in Bis-Tris buffer modified with NaCl, ATP, and MgCl2.

[0261] Example 13: Encapsulation of fluorescein loading TentaGel® into polyacrylamide hydrogel beads, droplet encapsulation, and determination of photoemission and loading capacity within droplets. 0.15 mg of fluorescein-labeled TentaGel® (TG) beads (mounted via a photo-unstable linker) were dispersed in 1.5 mL of hydrogel precursor (PAA / APS and water). The fluorescein-labeled TG beads were encapsulated in polyacrylamide hydrogel using a dolomite microfluidic device. The aqueous line contained the hydrogel precursor, and TEMED was dispersed in oil (2 wt% 008-fluorsurfactant (RAN Biotechnologies) in HFE). A 60 micron hydrogel was generated by incubating the droplets at 65°C for 24 hours.

[0262] The hydrogel was encapsulated in a 100-micron droplet (Figure 9E) and exposed to 365 nm UV light. Uniform diffusion of fluorescein from TG to the hydrogel and surrounding droplet was observed (Figure 9F). A concentration of 10 μM was achieved after 100 ms of exposure.

[0263] Example 14: FACS enrichment of polyacrylamide hydrogel beads with TentaGel® Ten μl of TentaGel® in polyacrylamide beads was loaded into a countess slide (Countess Cell Counting Chamber Slide C10228) and imaged under bright-field conditions at 4× magnification using an EVOS FLoid Imaging System (ThermoFisher Scientific 4471136). The beads in the images were manually counted, and the co-encapsulation rate was calculated. Crude TentaGel® in polyacrylamide beads was enriched using a SONY SH800 FACS sorter configured with a 9PSI 130μM sorting tip. The beads were sorted at a moderate flow rate (6 / 10). All events passing through the machine were analyzed for FITC (e.g., 488nM em. 525 / 50nm) and PE (e.g., 561nm em. 600 / 60nm) fluorescence. Beads with relatively higher FITC and PE fluorescence than other events analyzed were enriched. Figure 9I shows the scatter plot and gated population. Subpopulations of beads were sorted at 10K beads / well in 96-well microtiter plates containing 100 µl of water. Samples were taken, pooled, imaged, and quantified as described above. Images show enrichment of co-encapsulated beads from 6% to 75% (Figures 9G and 9H, respectively).

[0264] Example 15: Quantitative PCR of TentaGel® in PEGA resin, TentaGel® resin, and polyacrylamide resin Figure 19 shows the amplification curves of different samples relative to the standard curve (green). Three samples are analyzed: a blank prepared with MilliQ water (cyan curve), a control sample (beads mixed with DNA tags without DNA ligase - pink curve), and sample beads (fully coded beads - purple curve). The control beads simply show low amplification compared to the blank sample, while the sample beads show unambiguous amplification compared to both the blank and the control, confirming the success of enzymatic ligation. All samples were prepared in at least triplicates. Based on the number of beads in the sample, the amount per bead can be estimated using the standard curve.

[0265] Example 16: UV light sectioning and image acquisition. A 25 μL aliquot of a 100 μm water-in-oil droplet without mineral oil overlay (to avoid cracking / degradation of the ibidi polymeric slide bottom) was loaded into one channel of an uncoated ibidi 100 μm channel slide (ibidi cat.#80661) and sealed with Kwik-Cast sealant (World Precision Instruments cat.#KWIK-CAST). The sample was positioned in a Zeiss Axio Observer Z1 microscope with the hydrogel beads in focus, and a snapshot of the sample was taken with a wide-field (WF) detector before UV exposure. UV light cutting was performed with 10 msec intervals of UV exposure (385 nm Colibri LED at 10% producing 20.7 mW at 365 nm), and the total number of cycles was adjusted to produce a total exposure time of up to 10 sec (e.g., 100 cycles = 1 sec). To capture video of fluorescein emission from the beads into the droplet, we also used 10 msec 2% 488 nm Colibri LED excitation. The total image acquisition time was considerably longer than the sum of all cycles of the 10 msec burst due to the switching time for the optics.

[0266] Example 17: Verification and encapsulation of TentaGel® beads by UV light cutting and image acquisition of polyacrylamide hydrogel. A 25 μL aliquot of a 100 μm water-in-oil droplet without mineral oil overlay (to avoid cracking / degradation of the ibidi polymeric slide bottom) was loaded into one channel of an uncoated ibidi 100 μm channel slide (ibidi cat.#80661) and sealed with Kwik-Cast sealant (World Precision Instruments cat.#KWIK-CAST). The sample was positioned in a Zeiss Axio Observer Z1 microscope with focus on the bead, and a snapshot of the sample was taken with a wide-field (WF) detector before UV exposure. A WF UV section acquisition template was loaded, and UV sectioning was performed with a 10 msec interval of UV exposure (385 nm Colibri LED at 10% producing 20.7 mW at 365 nm), and the total number of cycles was modified to produce a total exposure time of up to 10 sec (e.g., 100 cycles = 1 sec). To capture video of fluorescein emission from the beads into the droplet, we also used 10 msec 2% 488 nm Colibri LED excitation. The total image acquisition time was considerably longer than the sum of all cycles of the 10 msec burst due to the switching time for the optics.

[0267] Example 17a: PEG coating of fluorescein-loaded TentaGel® beads for the production of thin shell beads 20 million fluorescein-labeled TentaGel® (TG) beads (mounted via a photo-unstable linker) were suspended in a 7:3 mixture of acetonitrile containing 1% Pluronic F127 (a block polymer of PEG and PPG, also known as Poloxamer or Poloxamer 407) and 30 mM TEAA, pH 7.4. 20.8 mg of PEG 40K DBCO (Creative PEGworks, #PSB-707) was added. The suspension was mixed on a shaker at room temperature for 5 days. The suspension was then centrifuged at 6000 rcf for 2 minutes, the supernatant was discarded, and the mixture was resuspended in cell culture medium. The process was repeated three times to ensure complete buffer exchange and removal of excess PEG-DBCO. The suspension was kept in the ridge until further use.

[0268] Thin-shell beads with a PEG10K coating were prepared from 20 million fluorescein-labeled TentaGel® (TG) beads (mounted via a photo-unstable linker), similar to the above, except that 26 mg of PEG 10K DBCO (BroadPharm #BP-22462) and a 1:1 mixture of DMSO and 30 mM TEAA, pH 7.4 were used while shaking overnight.

[0269] Example 17b: UV light sectioning and image acquisition of PEGylated thin-shell fluorescein load TentaGel® beads Using a similar protocol to that described in Example 17, UV light sectioning and image acquisition of fluorescein probes using 10K or 40K PEG-loaded fluorescein-loaded TentaGel beads were confirmed. Approximate loading was determined to be up to 25 fmole per bead for 10K PEG-coated beads and up to 36 fmole per bead for 40K PEG-coated beads. Example 17c: PCR of PEGylated (thin-shell) TentaGel® beads (Figure 19B) The amplification of DNA tags using 40K PEG-coated null library beads was confirmed using the same protocol as described in Example 15. Although the amplification of control beads without PEG was low, a clear amplification was present when compared to the negative control MilliQ water. Sample beads with 40K PEG showed the same amplification as control beads with the same DNA tag but without PEG. The overlapping curves clearly suggest that post-synthesis 40K PEG conjugation does not affect the amplification of DNA tags when compared to the control sample without PEG. The two samples with beads were run five times, and the MilliQ water was run tripletically. Based on the number of beads per sample, the amount of DNA per bead can be estimated using a standard curve.

[0270] Example 18: Emulsion Array Figures 18A–18D show microwell plates containing aqueous solutions loaded with water-fluorinated oil-in-water double emulsion droplets. The double emulsions sank to the bottom, occupying a maximum of one double emulsion per well. Figure 18A shows a microwell plate containing double emulsion 150 to be removed. Figure 18B shows a microwell plate into which a capillary 151, slightly larger than the double emulsion (150), is dropped. The volume of the solution is aspirated to remove the double emulsion from the wells (Figures 18C and 18D). Single detection emulsions were aspirated from the microwell plates using a CELLector tool from Molecular Machines & Industries (Eching, Germany), with a modified aspirator capillary tube designed for droplet aspiration from a grid, without disturbing nearby droplets. The CELLector tool included a modified aspirator capillary tube designed for droplet aspiration from a grid, in contrast to the aspiration of cells from flat-bottom microwell plates.

[0271] Example 19: Next-generation sequencing (NGS) and deconvolution of a DNA-coded, one-bead, one-compound library. NGS and informatics for bead barcode counting and hit deconvolution were performed using a mock code library screen set consisting of a 1% positive and negative control mixture and approximately 99% 10K null libraries. Next-generation sequencing and data science were used to deconvolute hits. DNA barcodes from the code beads were sequenced. This specification describes a method of performing PCR directly on DNA ligated to beads, among several approaches.

[0272] Barcodes were reconstructed from the bead library via PCR and NGS workflows. PCR was performed using the KAPA HiFi HotStart ReadyMix PCR kit (KAPA Biosystems KR0370). The bead library was combined in a 96:1 ratio, and PCR reactions were performed on 2,500 beads in 50 μl reactions. PCR products were purified using AMpure XP beads (Beckman Coulter Life Sciences, A63882) and analyzed using the Agilent D1000 ScreenTape system. Library preparations for NGS were prepared using the Illumina EB#e7545S / L Version 5. New England BioLabs Inc. NEBNext Ultra II DNA Library Prep Kit, in accordance with the "Miseq System Denature and Dilute Libraries guide" Document #15039740 version 10 Illumina.

[0273] NGS Data Preprocessing Following a sequencing run on a Miseq system (where paired-end 150-base reads are sequenced), NGS data in the form of pooled raw sequencing BCL files was demultiplexed for each sample using Bcl2Fastq (Illumina) into separate paired-end read (R1 and R2) fastq files (e.g., sample1.Read1.fastq and sample1.Read2.fastq), with the Illumina adapter sequence trimmed during processing. A sequencing quality report was generated for each R1 / R2 fastq file pair using FASTQC (www.bioinformatics.babraham.ac.uk / projects / fastqc / ).

[0274] Tools from the BBTools suite (jgi.doe.gov / data-and-tools / bbtools / ) were used for subsequent processing steps. The remnant Illumina adapter sequence was trimmed using BBTools / bbduk. Read pairs with identical sequences containing the same UMI (Unique Molecular Index) were combined using BBTools / dedupe to remove sequencing bias that may have been introduced by the PCR step, and the deduplated read pairs were combined and reported as a fastq file. Then, using the default quality filter, reads R1 and R2 were merged into a unique sequence using BBTools / bbmerge, which requires at least 145 base pair overlap. At this point, the majority (75%) of the reads were preserved.

[0275] Before counting the bead-specific and compound-specific barcodes, we further trimmed the common sequences at the 5' and 3' ends of all reads using BBTools / bbduk.

[0276] Decoding / Hit Calling The pre-processed barcode sequences were decoded into a compound-specific barcode (CSB) portion (approximately 39 bp) and a related bead-specific barcode portion (BSB, approximately 45 bp), allowing up to two mismatches across an approximately 85 bp trimmed sequence. The majority (81%) of the pre-processed sequences mapped to the expected barcodes at this mismatch level.

[0277] CSB+BSB pairs with a read count less than 2 (which can vary depending on the sequencing read depth) were not considered.

[0278] For each compound, the number of unique bead barcodes with two or more read counts was counted, and the compounds (CSBs) were ranked according to the number of unique beads.

[0279] Example 20: Acrylation of amino-functionalized polystyrene-coated iron oxide magnetic nanoparticles 15 mL Falcon tubes were filled with amino-functionalized polystyrene-coated iron oxide magnetic nanoparticles (TurboBeads, Zurich, Switzerland) (200 mg, 0.086 mmol amine equivalent). The nanoparticles were suspended in 5 mL of 20% N-methylmorpholine in DMF, vortexed, and sonicated in an ultrasonic bath for 1 hour. The nanoparticles were centrifuged (1000 rcf, 5 min), the supernatant was removed, 3 mL of DMF was added, and the tube was vortexed. This washing process was repeated two more times with DMF (3 mL each).

[0280] The washed magnetic nanoparticles were suspended in 3 mL of DMF and treated with NHS ester-functionalized PEG acrylate (Laysan Bio, ACRL-PEG-SVA-2000, 123 mg) in 3 mL of DMF, followed by treatment with DIPEA (50 μL). The suspension was sonicated for 5 minutes and then mixed overnight on a shaker. The beads were washed 3 times with 3 mL of DMF each, followed by 3 times with 3 mL of water each, and the final suspension was adjusted to a concentration of 150 mg / mL in water.

[0281] Example 21: Preparation of Magnetic PEGA Hydrogel Beads A 100 μL aliquot (150 mg / mL in water) of the acrylic magnetic beads prepared in Example 20 was added to 900 μL of the PEGA hydrogel mix described in Example 7. The suspension was cooled on ice and bath and probe sonication was performed at maximum amplitude (55 W) using a QSonica Q55 Sonicator Ultrasonic Processor (Qsonica, Vernon Hills, IL, USA) with 4 cycles of 15-second pulses. The suspension was then passed through a 20 μm cell strainer (CellTrics 20 μm, Sysmex, Kobe, Japan), and the filtrate was loaded into a 1 mL syringe (Beckton Dickinson, Vaud, Switzerland). Droplets were generated using the custom PDMS device described in Example 7. The continuous phase was 2% RAN in HFE-7500 (RAN biotechnologies, Beverly, MA). The flow rates were 500–600 μL / hr for the dispersed phase and 1000 μL / hr for the continuous phase. The generated droplets were collected in a total of two 1.5 mL Eppendorf tubes, and the top end of each was covered with 50 μL of light mineral oil. The tubes were then incubated at 60°C for 18 hours.

[0282] The fluorescein oil was removed from each Eppendorf tube using a syringe, and the emulsion was treated with 20% perfluorooctanol in HFE7500 and vortexed. The tubes were centrifuged (1000 rcf, 3 min), and the fluorescein oil was removed using a syringe. The beads were washed again with 20% PFO, followed by two more washes in Span80 in heptane. The beads were then transferred to two 15 mL Falcon tubes with 1% Pluronic F127 in water, vortexed, and then centrifuged at 1000 rcf for 5 min. The supernatant was removed, and the beads were suspended in 1% Pluronic F127 in 1 × PBS (approximately 3 mL each).

[0283] Larger hydrogel particles were removed by vortexing a Falcon tube, and then the larger, heavier particles were allowed to settle for approximately 30 seconds. The supernatant was then transferred to a single 15 mL Falcon tube. The supernatant thus collected, containing smaller suspended magnetic hydrogel beads, was then collected onto a 50 μm mesh cell strainer (CellTrics, Sysmex, Kobe, Japan) by transferring the suspension with a syringe. The beads were washed 3 × with 1% Pluronic F127 in 1 × PBS (3 mL each). The beads were then resuspended in 1% Pluronic F127 in 1 × PBS, centrifuged, and the supernatant was removed as needed to collect the beads in a 1.5 mL Eppendorf tube. The average size and %CV were determined by observing the magnetic beads under a microscope using 5 fields at 20 × magnification. A total of 46 beads were analyzed, resulting in an average diameter of 120 μm with a 16% CV.

[0284] Example 22: Assessment of Magnetic PEGA Beads Next, the magnetic PEGA beads were tested for their magnetic properties by leaving them on a magnetic rack (MagJET Separation Rack, Thermo Scientific, Waltham, MA). Visual inspection revealed that most of the hydrogel was collected from the side of the tube after approximately 3 minutes.

[0285] The above description is written with reference to specific embodiments for illustrative purposes. However, the above illustrative considerations are not intended to be exhaustive or to limit the invention to the precise forms of this disclosure. With regard to the above teachings, many modifications and variations are possible. These embodiments have been selected and written to best illustrate the principles of the art and their practical applications. Thereafter, those skilled in the art can make the best use of the art and its various embodiments by making various modifications to suit their intended specific use.

[0286] While the present disclosure and examples are adequately described with reference to the accompanying drawings, it should be noted that various modifications and alterations will be apparent to those skilled in the art. Such modifications and alterations should be understood to fall within the scope of the present disclosure and examples as defined by the claims. The present invention includes the following embodiments. <1> below: A fluorescein dispersion oil comprising at least one fluorescein dispersion oil having an average fluorine content of approximately 70 wt% or more, A droplet stabilizer comprising an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof, Continuous phase formulations for stable emulsions, including those mentioned above. <2> below: A plurality of two or more fluorescein dispersion oils, wherein the plurality of fluorescein dispersion oils have an average fluorine content of approximately 70 wt% or more, A droplet stabilizer comprising two or more emulsifiers selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof, Continuous phase formulations for stable emulsions, including those mentioned above. <3> The fluorescein dispersion oil is as follows: One or more oils selected from the group consisting of perfluorocarbons and perfluorinated oils, and / or One or more hydrofluoroethers, including, <1> or <2> The continuous phase formulation described above. <4> The total concentration of the perfluorocarbon and / or the perfluorinated oil in the fluorus dispersion oil is approximately 50% w / w or more, and / or The concentration of one or more hydrofluoroethers in the fluorescein dispersion oil is less than approximately 50% w / w. <3> The continuous phase formulation described above. <5> The perfluorocarbon is a perfluoroalkane selected from the group consisting of perfluorooctane, perfluoroheptane, perfluorohexane (FC-72), perfluoro-1,3-dimethylcyclohexane, and octadecafluorodecahydronaphthalene (perfluorodecalin), and / or The perfluorinated oil is selected from the group consisting of perfluoro-2-butyltetrahydrofuran, perfluoro-N-methylmorpholine (FC-3284), perfluorotripentylamine (FC-70), perfluorotributylamine (FC-43), perfluorotripropylamine (FC-3283), and / or a mixture of perfluorotributylamine and perfluoro(dibutylmethylamine) (FC-40), and / or The hydrofluoroether is selected from the group consisting of 2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500), ethyl perfluorobutyl ether (HFE-7200), 3-methoxyperfluoro(2-methylpentane) (HFE7300), methyl perfluoroisobutyl ether / methyl perfluorobutyl ether mixture (HFE7100), and methoxynonafluorobutane (HFE7000). <3> or <4> The continuous phase formulation described above. <6> The fluorescein dispersion oil consists of perfluorohexane (FC-72), perfluorooctane, and 2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500), each present in an amount ranging from 10 to 90% w / w. <1> ~ <5> A continuous phase formulation as described in any of the following. <7> The fluorescein dispersion oil is as follows: Each is a 1:1:1 w / w ratio or approximately 1:1:1 w / w ratio of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500), Each of these is perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) in a 2:2:1 w / w ratio or approximately 2:2:1 w / w ratio, and Each is a 1:3:1 w / w ratio or approximately 1:3:1 w / w ratio of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500), Selected from the group consisting of, <1> ~ <6> A continuous phase formulation as described in any of the following. <8> The aforementioned triblock or diblock copolymer is as follows: Perfluoropolyethers (PFPE) and polyethylene glycols (PEG), Two perfluoropolyethers (PFPE) and polyethylene glycol (PEG), or Two perfluoropolyethers (PFPE), two polypropylene glycols (PPG), and polyethylene glycol (PEG), including, <1> ~ <7> A continuous phase formulation as described in any of the following. <9> a) The diblock copolymer is of formula

change

change

change

change

change

change

change

change

[0287] References Auzanneau,F.-I,Meldal,M.,& Bock, K.(1995).Synthesis,characterization and biocompatibility of PEGA resins.In Journal of Peptide Science.https: / / doi.org / 10.1002 / psc.310010106 Brower,K.K.,Brower,K.K.,Carswell-Crumpton,C.,Klemm,S.,Cruz,B.,Kim,G.,Calhoun,S.G.K.,Nichols,L.,Fordyce,P.M.,Fordyce,P.M.,Fordyce,P.M.,& Fordyce,P.M.(2020).Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery.Lab on a Chip.https: / / doi.org / 10.1039 / d0lc00261e Brower,K.K.,Carswell-Crumpton,C.,Klemm,S.,Cruz,B.,Kim,G.,Calhoun,S.G.K.,Nichols,L.,& Fordyce,P.M.(2019).Optimized double emulsion flow cytometry with high-throughput single droplet isolation.In bioRxiv (p.803460).Cold Spring Harbor Laboratory.https: / / doi.org / 10.1101 / 803460 Cabantous,S.,Nguyen,H.B.,Pedelacq,J.D.,Koraichi,F.,Chaudhary,A.,Ganguly,K.,Lockard,M.A.,Favre,G.,Terwilliger,T.C.,& Waldo,G.S.(2013).A new protein-protein interaction sensor based on tripartite split-GFP association.Scientific Reports.https: / / doi.org / 10.1038 / srep02854 Di Carlo, D., Wu, C., Destgeer, G., & Ouyang, M. (2019). Particle-drop structures and methods for making and using the same (Specification of US Patent Application Publication No. 20190381497A1). Duarte, J.M., Barbier, I., & Schaerli, Y. (2017). Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology. ACS Synthetic Biology. https: / / doi.org / 10.1021 / acssynbio.7b00111 Gai, Y., Kim, M., Pan, M., & Tang, S.K.Y. (2017). Amphiphilic nanoparticles suppress droplet break-up in a concentrated emulsion flowing through a narrow constriction. Biomicrofluidics. https: / / doi.org / 10.1063 / 1.4985158 Gillies, E.R. (2020). Reflections on the Evolution of Smart Polymers. In Israel Journal of Chemistry. https: / / doi.org / 10.1002 / ijch.201900075 Grass, R.N., Athanassiou, E.K., & Stark, W.J. (2007). Covalently functionalized cobalt nanoparticles as a platform for magnetic separations in organic synthesis. Angewandte Chemie - International Edition. https: / / doi.org / 10.1002 / anie.200700613 Hackler,AL,Fitzgerald,FG,Dang,VQ,Satz,AL,& Paegel,BM(2020).Off-DNA DNA-Encoded Library Affinity Screening. ACS Combinatorial Science Holtze,C.,Rowat,AC,Agresti,JJ,Hutchison,JB,Angile,FE,Schmitz,CHJ,Koester,S,Duan,H,Humphry,KJ,Scanga,RA,Johnson,JS,Pisignano,D.,& Weitz,DA(2008).Biocompatible surfactants for water-in-fluorocarbon emulsions.Lab on a Chip.https: / / doi.org / 10.1039 / b806706f Holtze, Christian, Guerra, RE, Agresti, J., Weitz, DA, Ahn, K., HUTCHISON, JB, Griffiths, A., HARRAK, A. EL, MILLER, OJ, BARET, J.-C., TALY, V., RYCHELLYNCK, M., & MERTEN, C. (2008).Fluorocarbon emulsion stabilizing surfactants(specific 9012390B2 surfactants). Inglese,J.,Auld,D.S.,Jadhav,A.,Johnson,R.L.,Simeonov,A.,Yasgar,A.,Zheng,W.,& Austin,C.P.(2006).Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries.Proceedings of the National Academy of Sciences of the United States of America.https: / / doi.org / 10.1073 / pnas.0604348103 Joseph de Rutte,Robert Dimatteo,Mark van Zee,Robert Damoiseaux,D.D.C.(2020).Massively parallel encapsulation of single cells with structured microparticles and secretion-based flow sorting.BioRxiv.https: / / doi.org / 10.1101 / 2020.03.09.984245;t Kloxin,A.M.,Kasko,A.M.,Salinas,C.N.,& Anseth,K.S.(2009).Photodegradable hydrogels for dynamic tuning of physical and chemical properties.Science.https: / / doi.org / 10.1126 / science.1169494 MacConnell,A.B.,McEnaney,P.J.,Cavett,V.J.,& Paegel,B.M.(2015).DNA-Encoded Solid-Phase Synthesis:Encoding Language Design and Complex Oligomer Library Synthesis.ACS Combinatorial Science.https: / / doi.org / 10.1021 / acscombsci.5b00106 MacConnell,A.B.,Price,A.K.,& Paegel,B.M.(2017).An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.ACS Combinatorial Science.https: / / doi.org / 10.1021 / acscombsci.6b00192 Meldal,M.(1992).Pega:a flow stable polyethylene glycol dimethyl acrylamide copolymer for solid phase synthesis.Tetrahedron Letters.https: / / doi.org / 10.1016 / S0040-4039(00)79604-3 Meldal,M.,& Christensen,S.F.(2010).Microparticle matrix encoding of beads.Angewandte Chemie - International Edition.https: / / doi.org / 10.1002 / anie.200906563 Ofner,A.,Moore,D.G., Ruehs,P.A.,Schwendimann,P.,Eggersdorfer,M.,Amstad, E., Weitz,D.A.,& Studart,A.R.(2017).High-Throughput Step Emulsification for the Production of Functional Materials Using a Glass Microfluidic Device.Macromolecular Chemistry and Physics.https: / / doi.org / 10.1002 / macp.201600472 Paegel,B.M.,MacConnell,A.B.,Kodadek,T.,& McEnaney,P.J.(2020).Encoded Solid Phase Compound Library with Polynucleotide Based Barcoding (US Patent Application Publication No. 20200190507A1 Specification). Price,A.K.,MacConnell,A.B.,& Paegel,B.M.(2016).HνSABR:Photochemical Dose-Response Bead Screening in Droplets. Analytical Chemistry.https: / / doi.org / 10.1021 / acs.analchem.5b04811 Raman,R.,Hua,T.,Gwynne,D.,Collins,J.,Tamang,S.,Zhou,J.,Esfandiary,T.,Soares,V.,Pajovic,S.,Hayward,A.,Langer,R.,& Traverso,G.(2020).Light-degradable hydrogels as dynamic triggers for gastrointestinal applications.Science Advances.https: / / doi.org / 10.1126 / sciadv.aay0065 Rana,S.,White,P.,& Bradley,M.(1999).Synthesis of magnetic beads for solid phase synthesis and reaction scavenging. Tetrahedron Letters.https: / / doi.org / 10.1016 / S0040-4039(99)01572-5 Sievers,Q.L.,Petzold,G.,Bunker,R.D.,Renneville,A.,Slabicki,M.,Liddicoat,B.J.,Abdulrahman,W.,Mikkelsen,T.,Ebert,B.L.,& Thomae,N.H.(2018).Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN.Science.https: / / doi.org / 10.1126 / science.aat0572 Tang,S.,Pan,M.,Lyu,F.,& Derda,R.(2016).Fluorinated pickering emulsion(Patent No.US20160114325A1).USPTO. Townsend,J.,Do,A.,Lehman,A.,Dixon,S.,Sanii,B.,& Lam,K.S.(2010).3-nitro-tyrosine as an internal quencher of autofluorescence enhances the compatibility of fluorescence based screening of OBOC combinatorial libraries.Combinatorial Chemistry & High Throughput Screening. Toy,P.H.(2004).TentagelTM.Encyclopedia of Reagents for Organic Synthesis.https: / / doi.org / 10.1002 / 047084289X.RN00470 Yanakieva,D.,Elter,A.,Bratsch,J.,Friedrich,K.,Becker,S.,& Kolmar,H.(2020).FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells.Scientific Reports.https: / / doi.org / 10.1038 / s41598-020-66927-5 Zilionis,R.,Nainys,J.,Veres,A.,Savova,V.,Zemmour,D.,Klein,A.M.,& Mazutis,L.(2017).Single-cell barcoding and sequencing using droplet microfluidics.Nature Protocols.https: / / doi.org / 10.1038 / nprot.2016.154

Claims

1. At least one fluorescein dispersion oil, A droplet stabilizer comprising an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof, A continuous phase formulation for a stable emulsion, including, The fluorescein dispersion oil is as follows: i. Perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) in a 1:1:1 w / w ratio, ii. Perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) in a 2:2:1 w / w ratio, and iii. Perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) in a 1:3:1 w / w ratio, respectively. Selected from the group consisting of, Perfluorohexane (FC-72), perfluorooctane, and 2-(trifluoromethyl)-3-ethoxide decafluorohexane (HFE-7500) are each present in amounts ranging from 10% to 90% w / w. The emulsifier mentioned above i. Each comprising a diblock copolymer and a triblock copolymer in a ratio of 1:1 to 1:9 (w / w), and ii. The diblock copolymer and triblock copolymer are present in the continuous phase formulation at a combined concentration of 0.3 to 4 wt%, a) The diblock copolymer is of the formula 【Chemistry 1】 (In the formula, n and m are precise or average values ​​of polydisperse building blocks, the average molecular weight of the diblock copolymer is 1,000 to 10,000 Da, n is 35 to 45, and m is 2 to 24.) It has and b) The triblock copolymer is 【Chemistry 2-1】 (wherein i, j, and k are precise or average values ​​of polydispersible building blocks, the average molecular weight of the triblock copolymer is 2,000 to 20,000 Da, i and k are independently 35 to 45, and j is 1 to 23), or 【Transformation 3】 (In the formula, p, q, r, s, and t are precise or mean values ​​of polydispersible building blocks, the average molecular weight of the triblock copolymer is 2,000 to 20,000 Da, p and t are independently 35 to 45, q and s are each greater than 0, the precise or mean value of the sum q + s is 3 to 6, and r is 1 to 23.) Having an expression represented by, Continuous phase formulations for stable emulsions.

2. A water-in-oil (w / o) single emulsion comprising an aqueous dispersed phase and the continuous phase described in claim 1, or A water-in-oil (w / o / w) double emulsion comprising an aqueous dispersed phase and the continuous phase described in claim 1.

3. The water-in-oil single emulsion or the water-in-oil double emulsion according to claim 2, wherein the aqueous phase comprises an assay mixture and barcoded compound beads.

4. The beads are selected from the group consisting of hydrogel beads, magnetic hydrogel beads, divinylbenzene crosslinked polystyrene beads, low crosslinked polystyrene matrix grafted with polyethylene glycol, magnetic beads, silica beads, glass beads, and ceramic beads, or The beads are core-shell beads containing beads encapsulated with polyacrylamide, and the core is a low-crosslinked polystyrene matrix grafted with polyethylene glycol, or The beads are thin shell beads containing beads coated with a hydrophilic polymer, and the core is a low-crosslinked polystyrene matrix grafted with polyethylene glycol. The water-in-oil single emulsion or water-in-oil double emulsion according to claim 3.

5. The water-in-oil single emulsion or water-in-oil double emulsion according to claim 4, wherein the diameter of the beads is 1 to 70 microns, or the diameter of the beads is 70% or less of the diameter of the droplet.

6. A high-throughput screening system comprising a continuous phase formulation, a solid carrier, and a sorter, The continuous phase compound is defined as described in claim 1, The solid carrier is selected from the group consisting of monodispersible polyethylene glycol acrylamide (PEGA) copolymer resin, core-shell beads, and thin shell beads. High-throughput screening system.

7. A high-throughput screening system according to claim 6, The aforementioned sorter is as follows: Inlet channel and A first exit channel and a second exit channel merge with the aforementioned inlet channel at a junction, A first electrode and a second electrode adjacent to the first and second sides of the junction, respectively, wherein the first electrode and the second electrode are A first state in which the first electrode receives a first amount of voltage and the second electrode receives no voltage at all, A second state in which the second electrode receives a second amount of voltage and the first electrode receives no voltage at all, A first electrode and a second electrode are configured to have, A controller configured to switch between the first and second electrodes between the first and second states, A high-throughput screening system, including...

8. A high-throughput screening system according to claim 6, The aforementioned sorter is as follows: A microwell array plate comprising an array of microwells, wherein each well contains one microdroplet per microwell, Fluorescence microscope and, Imager and, Automated microcapillary-based droplet sampling device, including, High-throughput screening system.

Images