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] The present disclosure generally relates to methods, compositions, sorters, systems, uses, and devices for screening bioactive substances in emulsion droplets. In particular, the present disclosure generally relates to methods, compositions, sorters, systems, uses, and devices for emulsion formulations, codable compound beads, and high-throughput droplet selection, both as individual components and as aggregate platforms. [Background technology]

[0002] The present disclosure generally relates to methods, compositions, sorters, systems, uses, and devices for screening bioactive substances in emulsion droplets. The present disclosure also relates to microfluidic encapsulation, i.e., microencapsulated droplets containing cells and barcoded compound beads. Such microencapsulated droplets have applications in various areas of research and analysis, including, but not limited to, high-throughput screening of compounds in microencapsulated droplets, performing a series of chemical reactions in microencapsulated droplets, and analyzing cell culture conditions.

[0003] In robot-based high-throughput screening (HTS) for bioactive discovery, screening compartments can be provided by microtiter well plates that can contain reaction volumes up to tens of microliters and are easily tracked via plate maps and plate barcodes. However, this process can be expensive and resource- and time-consuming (Inglese et al. 2007, MacArron et al. 2011, Ross 2017). HTS of large compound sets is typically performed in single doses due to prohibitive costs, which can result in high false positive rates or an inability to distinguish non-hits from weak but robust hit molecules.

[0004] The greatly miniaturized picoliter compartments realized by droplet microfluidics can reduce the scale of compound screening to the size of a single cell, so many of the challenges can be addressed by the robot-based high-throughput screening specified above. Cellular assays in HTS may require the development of biological assays in artificial immortalized cell lines that provide sufficient quantities, requiring downstream hit triaging for more disease-related assay applications, such as using induced multipotent stem cell (iPSC)-derived or primary disease-state cells, which are difficult to scale. There are reports of cell screens performed in microdroplets of picoliter volume. However, such methods have been reported almost exclusively when the diversity source is an inherently hydrophilic biological molecule, e.g., 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 retention of compounds 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 frequencies. Therefore, in the context of cellular assays, it is important to be able to retain compounds in droplets and to efficiently introduce compound beads into droplets in a cell-compatible assay medium.

[0005] Precise delivery of individual library members into single coded droplets can be difficult to address. The conversion of plate-based compound collections to coded compound droplets can present inherent challenges with scalability and / or compound retention. Alternatively, DNA-coded one-bead one-compound libraries loaded via releasable linkers can deliver individual library members into single droplets (MacConnell, Price, and Paegel 2017; MacConnell et al. 2015). This method can substantially reduce the requirement for compound retention from the in-situ release point to the assay readout point, while at the same time harnessing the power of combinatrix and achieving droplet and compound coding. However, these platforms have technical challenges in the context of medicine. Thus far, no library beads have been demonstrated to simultaneously meet the following properties for cellular compound screening in droplets: 1) monodispersity, 2) biocompatibility, 3) suspendability and compressibility in aqueous media, 4) low background autofluorescence, and 5) compatibility with a wide range of library chemistries in the organic phase.

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

[0007] PEGA resins are PEG-crosslinked polyacrylamide-backbone hydrogel beads. PEGA may share the non-fluorescent hydrophilicity of polyacrylamide, while exhibiting excellent swelling in organic solvents and broader organic reaction compatibility of 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 resin with various functional handles is commercially available (Novabiochem / Merck-Millipore). However, this commercially available resin does not meet the criteria as a compound delivery matrix for screening in droplets with a diameter of 10 to 200 microns, as it contains relatively large sizes (diameters of 150 to 300 microns) and is polydisperse.

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

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

[0010] Therefore, there is a need in the art to provide improved droplet platforms with sufficient compound loading for cellular assays in HTS, yet in a library bead format suitable for efficient encapsulation into droplets for higher throughput, and with reliable and efficient methods for sorting of hit droplets.

[0011] The present disclosure overcomes previous shortcomings in the art by providing new methods, compositions, sorters, systems, uses, and devices for screening bioactive substances in emulsion droplets. Summary of the Invention

[0012] The objective is to provide a droplet platform that allows for efficient cellular compound screening in droplets. This capability, coupled with established detection methods, provides an important tool for rigorous cellular analysis at the single cell scale that can reveal new biological mechanisms and responses with unprecedented sensitivity and / or scale that can be tested, tuned, and exploited in the pharmaceutical industry. Another objective is to provide a droplet platform that allows for efficient cellular compound screening in droplets without excessive use of reagents or cells. This is particularly important for screens that use rare cells in the screening of large compound libraries.

[0013] The present disclosure is based on the discovery that significantly higher compound retention in the droplets is achieved by using the continuous phase formulations described herein. Specifically, the continuous phase formulations include at least one fluorous dispersion oil, where the fluorous dispersion oil has an average fluorine content of about 70 wt% or more, and a droplet stabilizing agent comprising an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof.

[0014] Provided herein are compositions, methods, sorters, devices, uses, and systems that can be used to screen compound libraries. In some embodiments, the methods, systems, and devices can be used to screen individual cells or multiple individual cells. In some embodiments, the methods, systems, and devices can be used to screen compound libraries. In some embodiments, the methods and devices provided herein can be used to screen compound libraries delivered as coded beads. In some embodiments, the methods, systems, and devices provided herein can be used to screen compound libraries delivered as coded beads and released into picoliter droplet compartments, which can scale down plate-based high-throughput screens by multiple orders of magnitude (FIGS. 1A and 1B). In some examples, the systems and devices can include entire platforms and / or individual components.

[0015] In some embodiments, the assay compartments can occur as either water-in-oil (w / o) single emulsions or water-in-oil-in-water (w / o / w) double emulsions. Figures 2A and 2B depict illustrative water-in-oil (w / o) single emulsion droplets (Figure 2A) and water-in-oil-in-water (w / o / w) double emulsion droplets (Figure 2B). As illustrated in Figure 2A, a water-in-oil (w / o) single emulsion droplet (189) can include an aqueous phase (180), an oil continuous phase (181), and an aqueous-oil interface (182). The aqueous phase (180) can include an assay mixture (183) and barcoded compound beads (184). As illustrated in FIG. 2B, a water-in-oil-in-water (w / o / w) double emulsion droplet (199) may comprise an aqueous phase (190) in a core surrounded by an oily phase (192) which is subsequently surrounded by an outer aqueous continuous phase (191). The aqueous phase (190) may comprise an assay mixture (193) and barcoded compound beads (194). For cellular assays, the droplet size may be such that sufficient nutrients are available to the encapsulated cell or cells for the duration of the assay, e.g., 10-200 microns in diameter for screening human cells which are in the range of 5-100 microns in diameter. For bacterial, cell-free, or biochemical assays, the droplet size may be selected from a broader range, e.g., 1-200 microns in diameter.

[0016] In some embodiments of the compositions, methods, uses, devices, sorters, and systems provided herein, the continuous phase can be any oil that is immiscible with water, such as mineral oil, hydrocarbon oil, silicone oil, or fluorous oil. In some embodiments, the continuous phase can be a blend of fluorous oils that can achieve optimal compound retention while maintaining sufficient oxygen permeability, biocompatibility, and mechanical stability for a given assay buffer system. Examples of fluorous oils include, but are not limited to, 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), perfluorotributylamine, and and mixtures with perfluoro(dibutylmethylamine) (FC-40), and hydrofluoroethers such as 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (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] The droplets can be stabilized by any emulsifier that is 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 a di- and / or triblock copolymer, such as one that consists of perfluorinated polyether (PFPE) and polyethylene glycol (PEG) and / or polypropylene glycol (PPG). The di- and triblock copolymers can be optionally blended. In some embodiments, the emulsifier can be subfluorinated silica nanoparticles. In some embodiments, the emulsifier can be any combination of the emulsifiers described herein. For example, the emulsifiers described herein can be optionally blended with subfluorinated silica nanoparticles to enhance their properties.

[0018] In some embodiments, compositions, methods, uses, sorters, systems, and devices are provided herein 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-based continuous phase with an emulsifier-free fluorinated oil-based continuous phase, a Pickering emulsifier-loaded fluorinated oil-based continuous phase, or a combination thereof.

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

[0020] An "emulsion" as used herein is a stable mixture of at least two immiscible liquids. In general, immiscible liquids tend to separate into two distinct phases. As such, emulsions are stabilized by the addition of "droplet stabilizers" or "surfactants" or "emulsifiers" that function to reduce the surface tension and / or stabilize the interface between the at least two immiscible liquids. In some embodiments, the emulsions described herein include a discontinuous or dispersed phase (i.e., an isolated phase stabilized by a surfactant) formed of an aqueous material. The continuous phase may be formed of a fluorous dispersed oil (e.g., a fluorocarbon). In some embodiments, the present 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 particular embodiments, the emulsions described herein are microemulsions. In some cases, microemulsions may include droplets having an average diameter of about 10 to 200 micrometers, or in some cases, 50 to 100 micrometers.

[0021] As used herein, "droplets" refers to isolated aqueous droplets within a continuous phase having any shape, e.g., cylindrical, spherical, ellipsoidal, irregular, etc. Generally, in emulsions of the present invention, the aqueous droplets are spherical or substantially spherical in the fluorocarbon continuous phase.

[0022] As used herein, "emulsifier" or "surfactant" defines a molecule that promotes and / or stabilizes the assembly of separate first and second phases when combined with a first component defining the first phase and a second component defining the second phase. The emulsifier can be a triblock copolymer, a diblock copolymer, a fluorinated silica nanoparticle, or a mixture thereof. Diblock and triblock copolymers typically contain one or more fluorophilic backbones, 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 (e.g., the chains may be soluble in the aqueous phase). As an example, the surfactant can be a multiblock surfactant (e.g., ABABABA...). In this case, one component of the chain (e.g., "A") is soluble in the fluorous phase and another component of the chain (e.g., "B") is soluble in the aqueous phase. As used herein, a multi-block surfactant is a surfactant having an alternating copolymeric or (AB) structure (i.e., ABA, ABAB, ABABA, ABABABA, etc.). In some cases, one block may be soluble in the fluorous phase of an emulsion and one block may be soluble in the aqueous phase of an emulsion. In other cases, additional components may be present within the surfactant. For example, a multi-block surfactant may have other groups present within its polymeric structure, such as a linking moiety connecting A and B. For example, (AXB-) n , (AX 1 -BX 2 ) n etc., in which "X" represents a covalent bond or a linking moiety as described below, and X 1 and X 2 When present, may be the same or different.

[0023] As used herein, unless otherwise specified, for example in the context of the emulsifiers defined above, a "fluorophilic" moiety or chain includes any fluorinated compound, for example linear, branched, cyclic, saturated or unsaturated fluorinated hydrocarbon ether or amine. The fluorophilic moiety may optionally include at least one heteroatom (e.g., in the backbone of the moiety, e.g., O). In some cases, the fluorophilic moiety 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 of the moiety are replaced by fluorine atoms. In some embodiments, 100% of the hydrogen atoms of the moiety are replaced by fluorine atoms. That is, it is perfluorinated. That is, the moiety contains fluorine atoms but no hydrogen atoms. The fluorophilic moiety may, for example, include a fluorine to hydrogen ratio 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 compatible with the present 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, a "stable emulsion" means that at least about 95% of the droplets of the emulsion, for example, do not coalesce to form larger droplets over such time periods. The compositions of the present disclosure, according to some embodiments, are stable at temperatures of about 25-40 or 95 degrees Celsius and at 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" can be used interchangeably and mean that long PEG polymers are grafted onto the surface of core beads carrying compounds and DNA. Long PEGs can be, for example, 5 KDa or more, 10 KDa or more, or 40 KDa or more. PEG groups can be grafted onto core beads using chemistries known in the art, such as, but not limited to, strain-promoted azide-alkyne click chemistry, copper-catalyzed azide-alkyne click chemistry, amide coupling, carbamoylation, urea or thiourea formation, sulfonamide formation, alkylation, reductive amination, and the like.

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

[0027] Dual barcoding for compounds and droplets can be achieved independently, for example, by oligonucleotide coding, color coding, fluorescent dye coding, RFID coding, or spatial placement 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 can have a bead-specific barcode that can act as a droplet barcode for replicate counts and a compound-specific barcode for compound identity.

[0028] Single aqueous droplets co-encapsulating cells and coded compound beads Aqueous droplets of water-in-oil (w / o) emulsions can be generated at high frequency by adopting standard practices in the droplet microfluidics field. Compressible hydrogel beads can allow flexible quantitative adjustment of encapsulation efficiency at 1:1 or about 1:1 encapsulation in some embodiments, multiple numbers of droplets per droplet in other embodiments, and less than 1:1 encapsulation in still other embodiments. In some embodiments, a single cell can be co-encapsulated in addition to the compound beads. In some other embodiments, multiple cells, e.g., 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 coded 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 prior to hit droplet selection. In some embodiments, cells and coded compound beads can be directly encapsulated in a double emulsion droplet.

[0030] The beads described herein can be made of any material that can be suspended in an aqueous buffer. In some examples, the beads can be hard beads, such as polystyrene, polystyrene-PEG hybrids, such as TentaGel® (Rapp Polymere), or hydrogels. The 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 can be compressible hydrogels. In some examples, the beads can be compressible hydrogels that lack autofluorescence. In some examples, the beads can be PEGA, which can have excellent swelling properties in both aqueous and organic solvents, allowing for versatile microfluidic handling while exhibiting sufficient compatibility with a wide range of organic reactions.

[0031] Also, as used herein, "beads" are understood to include capsules.

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

[0033] In some embodiments, monodisperse PEGA resin can be generated in microdroplets of 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 can be 1-100 microns in diameter. In some embodiments, the screening droplet can be 10-200 microns in diameter. The loading capacity of the PEGA resin can be such that the maximum concentration of released ligand in the droplet can be greater than 0.1 μM, preferably greater than 1 μM, more preferably greater than 10 μM. The resin can be loaded with compounds at fixed doses or correspondingly coded decreasing doses from a maximum loading. In some examples, the resin can be loaded with compounds to be screened by quantitative high throughput screening (qHTS) (Inglese et al., 2006). In some examples, qHTS is via controlled partial release of ligand from the beads and / or via preparation of dose-responsive beads with corresponding coding.

[0034] In some embodiments, the hydrogel beads (e.g., PEGA resin) can be magnetic. Hydrogel resins, e.g., PEGA resins, can be made magnetic, e.g., by co-encapsulation of appropriately coated magnetic microparticles or nanoparticles. In some examples, the coating of the 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 include, but are not limited to, microparticles of 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 attached via acrylamide surface modification. Magnetic hydrogels can be advantageous for bead handling during library preparation and post-screen bead processing, and / or can enhance microfluidic handling, such as enabling separation of bead-encapsulated droplets in bulk or on a microfluidic chip.

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

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

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

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

[0039] In some embodiments, the assay droplets can be incubated in-line in an integrated chip design or offline in an incubation chamber, hi some embodiments, the droplets can be re-injected into the device for hit droplet selection and / or decoding.

[0040] The assays may be biochemical assays, in-vitro transcription translation (IVTT) assays, cellular assays, organismal 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. Cellular assays may include, but are not limited to, intracellular reporter assays (e.g., gain or loss of fluorescent protein or fluorescent-conjugated enzyme), secreted reporter and / or secreted enzyme binding assays, marker (e.g., cytokine marker) secretion assays, live / dead 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, detection beads can be used to perform marker (e.g., cytokine marker) secretion assays or ELISA assays. The coded compound beads can optionally act as detection beads simultaneously. For example, in an ELISA assay, the coded compound beads can simultaneously act as detection beads. Virus-induced assays can include, but are not limited to, retroviral assays (e.g., lentivirus, adeno-associated virus (AAV)).

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

[0042] In some preferred embodiments, the assay may be in a "mix and read" format, where an assay premix is ​​co-encapsulated with the barcoded compounds to allow the assay result to be determined without further manipulation. In other embodiments, the assay may include adding an assay reagent (e.g., detection antibody) to determine the assay result. The assay reagent (e.g., detection antibody) may be added sequentially, for example, via 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, the FADS can include sorting by a sorter described herein, such as a dual electrode sorter described herein. Sorting by a dual electrode sorter, such as a dual electrode sorter described herein, can enhance the speed and / or reliability of sorting.

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

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

[0046] In some embodiments, core-shell beads containing members of the coding library can be converted into water-in-oil droplets using preferred emulsion formulations described herein to have cavities housing cells, compounds released and incubated in discrete droplet compartments, and hydrogels carrying beads and cells that are extracted into aqueous buffers for off-droplet sorting using traditional flow cytometers (Di Carlo et al., 2019; Joseph de Rutte, Robert Dimatteo, Mark van Zee, Robert Damoiseaux, 2020).

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

[0048] In embodiments provided herein where droplets settle (i.e., sink) into wells of an array of smaller wells, the droplets can be analyzed. For example, hit droplets can be isolated for hit deconvolution. For example, the 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 where the droplets are assayed by a cellular assay, 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 that includes at least one fluorous dispersion oil. In yet further embodiments, the fluorous dispersion oil has an average fluorine content of about 70 wt% or greater. In yet further embodiments, the continuous phase formulation further includes a droplet stabilizing agent that includes an emulsifier that is a triblock copolymer, a diblock copolymer, a fluorinated silica nanoparticle, or a combination thereof. This droplet selection format may enable versatile assay readouts, including, but not limited to, phenotypic imaging-based readouts, fluorescence-based readouts, bioluminescence-based readouts, and combinations thereof.

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

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

[0052] The present technology therefore provides methods, sorters, uses and devices that allow for screening of substances, such as bioactive substances, in emulsion droplets.

[0053] According to some embodiments, a continuous phase formulation for a stable emulsion is described, the continuous phase formulation comprising at least one fluorous dispersion oil, the fluorous dispersion oil having an average fluorine content of about 70 wt% or more (e.g., 75 wt% or more, 80 wt% or more), and a droplet stabilizing agent comprising an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof.

[0054] According to some embodiments, a continuous phase formulation for a stable emulsion is described, the continuous phase formulation comprising a plurality of two or more fluorous dispersion oils, where the plurality of fluorous dispersion oils have an average fluorine content of about 70 wt% or more, and a droplet stabilizing agent 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 some embodiments, a method of 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., a single water-in-oil (w / o) emulsion) is described. The method includes forming (e.g., using a syringe pump) at least one aqueous microdroplet in a first continuous phase formulation, the continuous phase formulation including an emulsifier selected from the group consisting of a triblock copolymer, a diblock copolymer, a fluorinated silica nanoparticle, and combinations thereof. In some examples, the method includes exchanging the first continuous phase formulation with a fluorous dispersion oil (e.g., exchanging and then re-exchanging with a fluorous dispersion oil a total of 2, 3, 4, 5, or more times) to provide aqueous microdroplets suspended in the fluorous dispersion oil, where the fluorous dispersion oil has an average fluorine content of >70 wt% and does not include an emulsifier.

[0056] In some embodiments, the method of reducing cross-contamination between microdroplets further comprises exchanging the first fluorous dispersion oil with a second continuous phase formulation (e.g., increasing droplet stability for further droplet manipulations) to provide aqueous microdroplets suspended in a second continuous phase formulation (wherein the second continuous phase formulation is a continuous phase formulation described herein).

[0057] According to some embodiments, a method for reducing cross-contamination between microdroplets is described. The method includes forming at least one aqueous microdroplet in a first continuous phase formulation described herein, exchanging the first continuous phase formulation with a first fluorous dispersion oil to provide aqueous microdroplets suspended in the first fluorous dispersion oil, and exchanging the first fluorous dispersion oil with a second fluorous dispersion oil to provide aqueous microdroplets suspended in the second fluorous dispersion oil. In some examples, the first fluorous dispersion oil has an average fluorine content of >70 wt% and contains a Pickering emulsifier (e.g., 8% wt of formula 10000 dispersed in a mixture of perfluoro 2-butyltetrahydrofuran and 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500). [ka] fluorinated silica nanoparticles (100 nm). In some examples, the second fluorous dispersion oil has an average fluorine content of >70 wt% and does not contain an emulsifier.

[0058] In some embodiments, the method further comprises exchanging the second fluorous dispersion oil with the second continuous phase formulation to provide aqueous microdroplets suspended in the second continuous phase formulation (e.g., to provide increased droplet stability for further droplet manipulations). In such embodiments, the second continuous phase formulation is a continuous phase formulation described herein.

[0059] According to some embodiments, a method for preparing a monodisperse polyethylene glycol acrylamide (PEGA) copolymer resin is described. The method includes dispersing a plurality of 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 plurality of monomers with a continuous phase formulation including oil and an emulsifier (e.g., about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 0.5%-1.5%, about 0.5%-about 2.0%, about 0.5%-about 2.5% emulsifier), and forming at least one microdroplet (e.g., 1-200 microns, 10-200 microns, 1-100 microns) from the aqueous buffer and the plurality of monomers. , 5-90 microns, 10-80 microns, 20-70 microns, 20-50 microns) and polymerizing the monomers in the at least one microdroplet (e.g., using an initiator (e.g., tetramethylenediamine (TEMED) and ammonium persulfate), 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) to form a PEGA copolymer resin. In some examples, the oil is selected from the group consisting of fluorous oil, hydrocarbon oil, mineral oil, and silicone oil. 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 with functionalized handle, and / or mono-acrylamide diamine with functionalized handle. In some examples, the monomers of the plurality of monomers include acrylamide (e.g., acrylamide or N,N-dimethylacrylamide), bis-acrylamide PEG, mono-acrylamide PEG with a functionalized handle, and mono-acrylamide diamine with a functionalized handle. In some examples, the monomers of the plurality of monomers include acrylamide (e.g., acrylamide or N,N-dimethylacrylamide), and bis-acrylamide PEG, and mono-acrylamide PEG with a functionalized handle.In some examples, a monomer of the plurality of monomers includes an acrylamide (eg, acrylamide or N,N-dimethylacrylamide), a bis-acrylamide PEG, and a mono-acrylamide diamine that includes a functionalized handle.

[0060] In some embodiments, the PEGA resin is mechanically stable under reaction conditions typically utilized in solid phase organic chemistry (e.g., stable to elevated temperatures and / or acidic or basic conditions, e.g., stable to pipetting, shaking, sonication, centrifugation, filtration, and handling in a microfluidic device, e.g., as measured by percentage bead recovery following handling protocols such as pipetting, centrifugation, filtration, chemical treatment, uv irradiation, droplet encapsulation, droplet rupture and recovery, sorting in a flow cytometer, e.g., stable at 100° C. for at least 1 hour, stable to acids (e.g., TFA) or bases (e.g., DIP EA) treatment for at least 1 hr, or for at least 24 hours with shaking at 1500 rpm, or for at least 3 min with centrifugation at 8000 rcf), and the PEGA resin is sufficiently biocompatible such that co-incubation and / or co-encapsulation in droplets with biological systems (e.g., cells, IVTT mixes, and / or recombinant proteins, etc.) does not compromise the integrity of the PEGA resin for the duration of the assay (e.g., co-incubation with cells for 24 hr results in >90% viability or greater viability when compared to a no-beads control). In some embodiments, the PEGA resin is sufficiently biocompatible (e.g., co-incubation with cells for 24 hr results in >90% viability or greater viability when compared to a no-beads control). In some examples, the PEGA resin is capable of swelling in aqueous buffer solutions and organic solvents to allow for efficient chemical reactions on the entire resin, effective solvent exchange and suspensibility and handling (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, the PEGA resin is sufficiently compressible to allow packing of hydrogel beads into microfluidic channels (e.g., achieving a reversible reduction in diameter without rupture upon application of force such as a diameter reduction of >10%, >15%, >20%, >25%, or >30%) such that the beads are encapsulated into at least one microdroplet with super-Poisson encapsulation efficiency (e.g., characterized by analyzing droplets in a hemacytometer / microscope chamber slide (e.g., iBidi or Countess), e.g., by comparing the number of encapsulated droplets to empty droplets, e.g., droplets in a tightly packed monolayer (e.g., by observation under a microscope)).

[0061] In some embodiments, the at least one microdroplet is formed in a microfluidic device (e.g., a microfluidic chip, a device that optionally includes a droplet splitter). In some examples, forming the at least one microdroplet includes forming a plurality of monodisperse microdroplets in parallel.

[0062] According to some embodiments, a method for preparing a monodisperse polyethylene glycol acrylamide (PEGA) copolymer resin is described. The method includes 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 with functionalized handle, and / or mono-acrylamide diamine with functionalized handle.

[0063] According to some embodiments, a method for preparing core-shell beads is described, comprising 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, the at least one microdroplet comprising at least one core bead, and forming a core-shell bead (e.g., 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 combinations thereof) that encapsulates the beads.

[0064] According to some embodiments, a method for preparing thin-shell PEG-coated beads is described. The method includes grafting a hydrophilic coating onto compound-loaded beads. In some examples, the hydrophilic polymer coating is comprised of PEG. In some examples, the PEG is attached by acetylene-azide click chemistry.

[0065] According to some embodiments, a sorter is described. 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 proximate 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 voltage greater than the second electrode, thereby causing one or more target compositions to flow through the junction into the first outlet channel, and a second state in which the second electrode receives a voltage greater than the first electrode, thereby causing one or more target compositions to flow through the junction 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 some embodiments, a system (e.g., a device, a device in operation) for performing high throughput screening is described. The system includes a continuous phase formulation, a solid support, and a sorter. In some examples, the continuous phase formulation includes a droplet stabilizer and at least one fluorous dispersion oil. In some examples, the fluorous dispersion oil has an average fluorine content of about 70 wt% or more. In some examples, the droplet stabilizer includes an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof. In some examples, the solid support is selected from the group consisting of monodisperse polyethylene glycol acrylamide (PEGA) copolymer resins and core-shell beads. In some examples, a monodisperse polyethylene glycol acrylamide (PEGA) copolymer resin is 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 comprising an 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 the at least one first microdroplet to form a PEGA copolymer resin. In other examples, the thin shell beads are prepared by grafting a hydrophilic coating on a solid support. In some examples, the hydrophilic polymer coating comprises PEG. In some examples, the PEG is attached by acetylene-azide click chemistry. In some examples, the oil is selected from the group consisting of fluorous oils, hydrocarbon oils, mineral oils, and silicone oils. In some examples, the monomers of the first plurality of monomers comprise acrylamide, bis-acrylamide PEG, and mono-acrylamide PEG with functionalized handles, and / or mono-acrylamide diamine with functionalized handles.In some examples, the 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 polymerizing the monomers in the at least one second microdroplet to form a hydrogel encapsulating the core bead to form the core-shell bead. In some examples, the at least one second microdroplet includes 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 proximate 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 greater voltage than the second electrode, thereby causing one or more target compositions to flow through the junction into the first outlet channel, and a second state in which the second electrode receives a greater voltage than the first electrode, thereby causing one or more target compositions to flow through the junction into the second outlet 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 some embodiments, a system for performing high throughput screening is described. The system includes a continuous phase formulation, a solid support, and a sorter. In some embodiments, the solid support is in the form of a coded compound bead as described herein. In some examples, the continuous phase formulation includes a droplet stabilizer and at least one fluorous dispersion oil. In some examples, the fluorous dispersion oil has an average fluorine content of about 70 wt% or more. In some examples, the droplet stabilizer includes an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof. In some examples, the solid support is selected from the group consisting of monodisperse polyethylene glycol acrylamide (PEGA) copolymer resins and core-shell beads. In some examples, a monodisperse polyethylene glycol acrylamide (PEGA) copolymer resin is 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 comprising an 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 the at least one first microdroplet to form a PEGA copolymer resin. In other examples, the thin shell beads are prepared by grafting a hydrophilic coating onto a solid support. In some examples, the hydrophilic polymer coating comprises PEG. In some examples, the PEG is attached by acetylene-azide click chemistry. In some examples, the oil is selected from the group consisting of fluorous oils, hydrocarbon oils, mineral oils, and silicone oils. In some examples, the monomers of the first plurality of monomers comprise acrylamide, bis-acrylamide PEG, and mono-acrylamide PEG with functionalized handles, and / or mono-acrylamide diamine with functionalized handles.In some examples, the 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 polymerizing the monomers in the at least one second microdroplet to form a hydrogel encapsulating the core bead to form the core-shell bead. In some examples, the at least one second microdroplet comprises 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 for automated image assay of the droplets to identify desired droplets, and an automated microcapillary-based droplet sampling device configured to sequentially select (e.g., sequential selection, bulk selection, high throughput selection, e.g., at about 1-3 cells / sec or about 2 cells / sec, selection by aspiration) a plurality of desired droplets and deposit them into hit wells (e.g., configured with one or more capillary properties selected from among sizes, angles, and heights selected to accommodate the droplets and / or dividers to allow access to the droplets in the microwells or grids).

[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, and 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] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0070] For a better understanding of the various described embodiments, reference should be made to the following description of the embodiments in connection with the following drawings, in which like reference numerals refer to corresponding parts throughout the drawings, in which: [Brief description of the drawings]

[0071] [Figure 1-1] Figure 1: Figures 1A and 1B illustrate miniaturization embodiments from a 1536-well plate to approximately 500 picoliters coded assay compartments. In a 1536-well plate (e.g., a 651 plate for approximately 1 million wells), 5 μL of assay medium can be held in a 1.7 mm diameter microwell (Figure 1A), while in a droplet, approximately 500 picoliters of assay medium can be held in a coded 100 micron diameter droplet (e.g., 1 million droplets in a single tube) (Figure 1B). [Figure 1-2] (As stated above.) [Figure 2-1] FIG. 2: FIGS. 2A and 2B illustrate water-in-oil (w / o) single emulsion droplets (FIG. 2A) and water-in-oil-in-water (w / o / w) double emulsion droplets (FIG. 2B). FIG. 2C illustrates compounds and barcodes attached to compound-loaded beads. Ligands and barcodes can be optionally linked to a common functionalized handle on the beads via a split linker (FIG. 11A). FIGS. 2D and 2E illustrate compound-loaded beads 160 encapsulated in a hydrogel matrix 161 (FIG. 2D) and a hydrophilic coating 162 (FIG. 2E). FIG. 2F illustrates a core-shell bead in which compound-loaded beads 160 are encapsulated in a hydrogel shell 162 with a cavity 163 to accommodate cells. FIG. 2G illustrates a dual-electrode droplet sorting device according to some embodiments. FIG. 2H illustrates a microarray grid hosting one droplet / well and isolation of hit droplets and / or beads according to some 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.) [Diagram 3] FIG. 3 depicts a microfluidic droplet generation device according to some embodiments. [Figure 4] FIG. 4 shows monodisperse PEGA resin generated with a microfluidic droplet generation device according to some embodiments. [Figure 5-1]Figure 5: Figures 5A-5C illustrate coded compound-loaded beads without (Figure 5A) and with (Figures 5B-5C) a hydrophilic polymer shell. 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 on-surface polymerization (Figure 5E) according to some embodiments. Figure 5F illustrates encapsulation of compound-loaded core beads in a hydrogel layer according to some 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] FIG. 6: FIGS. 6A-6B illustrate compound-loaded hydrogel beads with non-covalently attached (FIG. 6A) and covalently attached (FIG. 6B) magnetic microparticles according to some embodiments. [Figure 6-2] (As stated above.) [Figure 7-1] FIG. 7: FIGS. 7A-7H show a comparison of molecular retention of fluorescein (FIGS. 7A-7D) and resorufin (FIGS. 7E-7H) in w / o single emulsions generated using various commercially available continuous phase formulations at the 1 hr 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] FIG. 8: FIGS. 8A-8D show molecular retention of fluorescein (green) and resorufin (red) in droplets generated using a continuous phase oil composition according to some embodiments. FIGS. 8E-8G show droplet stability and dye retention after 5 days of replacement with an emulsifier-free continuous phase oil blend (20% HFE-7500, 20% FC-72, and 60% perfluorooctane containing emulsifier (Formula 1 (n is about 38 and m is about 9):Formula 2 (i and k are about 38 and j is about 9) 1:1, 2 wt%)). FIG. 8E shows bright field, FIG. 8F shows 100 μM resorufin, and FIG. 8G shows 100 μM fluorescein. Approximately 1 in 10 droplets contains the dye mixture to monitor cross contamination across droplets. Images are taken with droplets in an ibidi imaging slide using a 100 micron high chamber. Figures 8H-8J show representative flow cytometry gating strategies to identify viable cells as observed by low red fluorescence (Figure 8H), and Figure 8K shows the percent viable cells after coencapsulation into 100 micron droplets with certain di- and triblock copolymers, respectively, and their subsequent recovery. [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 the photocleavable linker 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 the subpopulation of events with significantly higher fluorescence in the FITC and PE channels, the encapsulated population increased to 75% (Figure 9H). Figure 9I shows the scatter plot and gated populations. [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 polyacrylamide (PAA) vs. PEGA 1x rod compressibility as measured by texture analyzer. Figure 10B shows dense packing of PEGA 1x resin in one channel and quantified 1:1 encapsulation into droplets according to some 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 depict exemplary DNA-encoded bead library linker and DNA barcode designs according to some embodiments. Figure 11A depicts an azidolysine that provides a click handle for DNA tag conjugation while using the N-terminus for further linker assimilation with a photocleavable motif such as an ortho-nitrobenzyl group. Figure 11B depicts an exemplary DNA barcode design with a headpiece followed by a bead-specific barcode (BSB) and three positions for encoding various building blocks, for example, followed by a library tag and an experiment-specific tag. Figure 11C depicts an example of a dose-responsive bead where the split linker can be capped with an acetyl group to reduce the photocleavable linker functionalized handle and DNA can be encoded to allow pooling of dose-responsive beads for synthesis and screening. [Figure 11-2] (As stated above.) [Figure 11-3] (As stated above.) [Figure 12-1] FIG. 12: FIGS. 12A-C depict embodiments in which mix-and-read fluorescent reporter assays can be configured on a droplet-based screening platform that result in either gain or loss of signal to illuminate the entire droplet (FIG. 12A), a single or multiple cells and / or beads within a droplet (FIG. 12B), or a secreted reporter from a single or multiple cells that illuminates the entire droplet (FIG. 12C). [Figure 12-2] (As stated above.) [Figure 12-3] (As stated above.) [Figure 13]FIG. 13 depicts exemplary fluorescent reporters that can be used in cell-based assays. They depict: i) fluorescent proteins detectable by droplet sorters. For example, detection can be by rapid detection or sorting of fluorescence via ratios (e.g., GFP / RFP ratios), where one fluorescent reporter reports on a biological response and the other is used to normalize expression and cell number. ii) fluorescent reporter enzymes. For example, β-lactamase can be used as a reporter enzyme to report on cellular responses using a FRET-based substrate. This can also be multiplexed with toxic dyes via a red fluorescent cell stain (ToxBlazer™, Thermo Fisher Scientific). Alternatively, the enzyme β-galactosidase can be used as a reporter in either native or split form, allowing for monitoring of a variety of cell-based events. The use of the β-galactosidase substrate DDAO galactoside (9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β-D-galactopyranoside) provides near-infrared fluorescence emission well separated from the typical background autofluorescence observed when the enzyme is expressed. iii) Secreted enzymes such as secreted alkaline phosphatase (SEAP) that can be used to detect secreted forms of FP or cellular responses with a green fluorescent readout. Illustrated is a fluorescent detection system for SEAP. [Figure 14-1] 14: Figures 14A-14E illustrate droplet sorting according to some embodiments. Figure 14A illustrates droplet flow with no electric field applied. Figure 14B illustrates an example of random droplet flow with no electric field applied. Figure 14C illustrates droplet sorting where an electric field is applied to select non-hit stream paths. Figures 14D and 14E illustrate droplet sorting where an electric field is applied to select hit stream paths. [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 illustrates detection and sorting of fluorescent droplets by applying an electric field according to some embodiments. Figures 15B-15E illustrate detection and sorting of droplets by alternating voltage potentials according to some 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] FIG. 16 shows a double emulsion droplet in an imaging flow cytometer according to some embodiments. [Figure 17-1] FIG. 17: FIGS. 17A-17F illustrate emulsion sorting by picking hit droplets from a microwell plate according to some embodiments. FIG. 17A illustrates a microwell plate where the dimensions of the microwells are chosen on the same size scale as the emulsion droplets for 1 droplet / well. FIG. 17B illustrates emulsion droplets loaded onto a microwell plate such that each well contains 1 emulsion droplet. FIG. 17C illustrates a microwell plate where single and multiple hit locations are identified. FIG. 17D illustrates a microwell plate where single and multiple hits are mechanically removed from specific wells. FIG. 17E illustrates single and multiple hits picked for analysis. FIG. 17F is a microscopic image of double emulsion droplets with and without fluorescent dyes 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-18D are microwell plates from which double emulsions are removed according to some embodiments. Figure 18A is a microwell plate containing a double emulsion. Figure 18B is a microwell plate containing a double emulsion and a capillary that is slightly larger than the double emulsion. Figures 18C and 18D show the defined volume that is aspirated to remove the double emulsion from the well. [Figure 18-2] (As stated above.) [Figure 18-3] (As stated above.) [Figure 18-4] (As stated above.) [Figure 19-1] FIG. 19: FIG. 19A shows quantitative PCR of PEGA resin, TentaGel® resin, and TentaGel® in polyacrylamide resin. Three samples are analyzed: a blank made 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. FIG. 19B shows PCR of TentaGel® resin with and without a thin-shell PEG40K coating (PEG chains of average molecular weight of 40 KDa). 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 equal amplification as control beads with the same DNA tags but without PEG. The overlapping curves clearly suggest that post-synthetic 40K PEG conjugation does not affect the amplification of DNA tags when compared to the no PEG control sample. Two samples with beads were run in quintuplicate, and MiliQ water was run in triplicate. [Figure 19-2] (As stated above.) [Figure 20-1]Figure 20: Figures 20A-B illustrate the effect of a thin PEG-coated shell on the dispersibility of compound-loaded TentaGel® beads in aqueous cell culture medium. A comparison of bead dispersions after 4 days of complete dispersion by probe sonication is shown, Figure 20A without PEG coating and Figure 20B with 40K PEG coating. The beads are loaded with fluorescein via a photocleavable linker as described in Figure 11A. After 4 days of standing, large bead clumps are observed for the beads without PEG coating, while for the beads with 40K PEG coating, the beads are primarily dispersed as monodisperses. [Figure 20-2] (As stated above.) [Figure 21] Figure 21 illustrates the effect of magnetic beads on PEGA beads. The time course of the drag of magnetic PEGA beads to the side is shown at times 1 second, 18 seconds, and 180 seconds from the left. At 180 seconds, most of the magnetic PEGA beads are collected at the side of the tube. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0073] There is a need for compositions, methods, devices, and systems that improve screening of libraries in droplets. For example, there is a need for compositions that increase molecular retention in droplets for compounds within a range of physiochemical properties. There is also a need for droplets that can achieve 1:1 or about 1:1 encapsulation efficiency. There is also a need for improved droplet imaging. There is also a need for improved monodispersity, biocompatibility, suspendability, and compressibility in aqueous media, low background autofluorescence, compatibility with a wide range of library chemistries in organic phases, and any combination thereof.

[0074] In cell screens for biologics in droplets, the continuous phase can be fluorinated oil due to its gas permeability, low cytotoxicity, and chemical inertness (C. Holtze et al. 2008). Emulsifiers that stabilize water in fluorinated oil emulsions include di- and triblock copolymers (US2010105112, Li et al. 2020, Christian Holtze et al. 2008). Retention of hydrophobic organic molecules relevant to 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 screens and / or compound-loaded bead encapsulation efficiencies (lambda, see MacConnell and Paegel 2017) are high.

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

[0076] The terminology used in the description of the various embodiments described herein is for the purpose of describing particular embodiments only and is not intended to be limiting. When used in the description of the various embodiments described and in the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term "and / or" as used herein means and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms "includes," "including," "comprises," and / or "comprising," as used herein, specifically indicate the presence of stated features, integers, steps, operations, elements, and / or components, but do 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 "fluorous 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 that contains carbon, fluorine, and preferably at least one heteroatom, and does not contain C-H bonds in the backbone.

[0080] As used herein and in the claims, the term "perfluorinated oil" means perfluorocarbons, perfluoroethers or partially fluorinated fluoroethers, perfluoroamines or partially fluorinated perfluoroamines.

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

[0082] As used herein and in the claims, the term "linked" or "linking" refers to direct and / or indirect linkage. Linkages can include covalent or non-covalent linkages, such as hydrogen bonds or ionic bonds. Linkages can be reversible (e.g., photocleavable linkers) or irreversible.

[0083] The term "functionalized handle" as used herein means an attachment point that allows for the attachment of one or more molecules, e.g., a barcode and / or a compound (e.g., a small molecule). The barcode can be a protein, peptide, enzyme, nucleic acid, or any other substance that can act as a barcode that allows for the identification of the bead (bead specific barcode, BSB) and the compound loaded on the bead (compound specific barcode, CSB). In an embodiment, the nucleic acid is DNA.

[0084] Provided herein are continuous phase formulations that stabilize and / or increase molecular retention in droplet emulsions. In some examples, the continuous phase formulations contain a fluorous dispersion oil and a droplet stabilizer comprising an emulsifier. In some embodiments, the fluorous dispersion oil can 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 to form Pickering emulsions (Gai et al., 2017; Tang et al., 2016). Such nanoparticles can be further modified to achieve compatibility with fluorinated oils with higher fluorine content than 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (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 the continuous phase formulation at a concentration 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 the continuous phase formulation at a concentration of 0.3% to 4% w / w, for example, 2% w / w. In some embodiments, the copolymers described herein are polydisperse copolymers.

[0087] In some embodiments, the nanoparticles described herein can be present in the continuous phase formulation. In some further embodiments, the nanoparticles are partially derivatized silica nanoparticles. In still further embodiments, the nanoparticles are present in the continuous phase formulation at a concentration 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 fluorous dispersion oil can contain perfluorocarbons, perfluorinated oils, hydrofluoroethers, or any combination thereof. In some embodiments, the fluorous dispersion oil comprises one or more oils and / or one or more hydrofluoroethers selected from the group consisting of perfluorocarbons and perfluorinated oils. In some embodiments, the fluorous dispersion oil comprises one or more oils and one or more hydrofluoroethers selected from the group consisting of perfluorocarbons and perfluorinated oils. In some embodiments, the fluorous dispersion oil comprises one or more oils or one or more hydrofluoroethers selected from the group consisting of perfluorocarbons and perfluorinated oils. In some embodiments, the fluorous dispersion oil comprises one or more oils selected from the group consisting of perfluorocarbons and perfluorinated oils. In some embodiments, the fluorous dispersion oil comprises one or more oils selected from one or more hydrofluoroethers. In some embodiments, the fluorous dispersion oil comprises one or more oils selected from the group consisting of perfluorocarbons and perfluorinated oils. In some embodiments, the fluorous dispersion oil comprises one or more oils selected from one or more hydrofluoroethers. In some embodiments, the total concentration of perfluorocarbons and / or perfluorinated oils in the fluorous dispersion oil is about 50% w / w or more. In some embodiments, the total concentration of perfluorocarbon and / or perfluorinated oil 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 perfluorocarbon 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 perfluorocarbon 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 fluorous dispersion oil is less than about 50% w / w. The selection of the particular fluorous dispersion oil, triblock and / or diblock copolymer, 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] Below are exemplary continuous phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. A: Triblock and / or diblock copolymers (0.5%-3% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w); B: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbon and / or perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w); and C: Triblock and / or diblock copolymers (0.5-3% w / w) and Pickering emulsifier (2-8% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w).

[0094] Below 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%-3% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w); B: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbon and / or perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w); and C: Triblock and diblock copolymers (0.5-3% w / w) and Pickering emulsifiers (2-8% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oils (greater than 50% w / w) and hydrofluoroethers (0-50% w / w).

[0095] Below 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%-3% w / w) dispersed in mixtures of perfluorocarbon and perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w); E: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbon 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 emulsifiers (2-8% w / w) dispersed in a mixture of perfluorocarbon and perfluorinated oils (greater than 50% w / w) and hydrofluoroethers (0-50% w / w).

[0096] Below 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%-3% w / w) dispersed in a mixture of perfluorocarbon or perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w); H: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbon 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 emulsifiers (2-8% w / w) dispersed in a mixture of perfluorocarbon or perfluorinated oils (greater than 50% w / w) and hydrofluoroethers (0-50% w / w).

[0097] Below are exemplary continuous phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. J: Triblock or diblock copolymers (0.5%-3% w / w) dispersed in a mixture of perfluorocarbon and / or perfluorinated oils (50% w / w or more) and hydrofluoroethers (0-50% w / w); K: Nanoparticles (e.g., partially fluorinated silica nanoparticles) (2-8% wt) dispersed in a mixture of perfluorocarbon and / or perfluorinated oils (50% w / w or more) and hydrofluoroethers (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 (greater than 50% w / w) and hydrofluoroether (0-50% w / w).

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

[0099] Below are exemplary continuous phase formulations for use in the compositions, sorters, uses, methods, devices, and systems provided herein. P: a triblock or diblock copolymer (0.5%-3% w / w) dispersed in a mixture of perfluorocarbon or perfluorinated oil (50% w / w or more) and hydrofluoroether (0-50% 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 (greater than 50% w / w) 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 copolymers 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 is at least C 10 Length of at least C 15Length of at least C 20 Length of at least C 25 Length of at least C 30 In another embodiment, the fluorophilic chain has a length of at least C 50 Length of at least C 75 Length of at least C 100 or longer. A non-limiting example is the structure -(C3F6O) 10 The fluorophilic component having the formula 30 It has a chain equivalent of 30 carbons. The fluorophilic moieties can be linear, branched, cyclic, saturated, unsaturated, and the like.

[0101] In some embodiments, the fluorophilic component of the diblock copolymer includes heteroatoms (e.g., non-carbon, such as oxygen (e.g., divalent oxygen), sulfur (e.g., divalent or hexavalent sulfur), nitrogen (e.g., trivalent nitrogen)) 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 branching 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 can include those described in U.S. Patent Application Publication No. 2010 / 0105112 A1.

[0103] In some embodiments, the diblock copolymer is a copolymer of Formula 1 below, or a combination thereof: [ka] (In Formula 1, n and m are precise or average values ​​of the polydisperse building blocks, n is 35-45 and m is 2-24. In some embodiments, the precise or average molecular weight of the copolymer of Formula 1 is 1,000-10,000 Da. In some embodiments, n is about 38 and m is about 17. In some embodiments, n is about 38 and m is about 9.)

[0104] Triblock Copolymer In some embodiments, the triblock copolymer comprises a fluorophilic component. In some embodiments, the triblock copolymer comprises at least one fluorophilic component, for example, two fluorophilic components. In other embodiments, the triblock copolymer comprises at least one fluorophilic component, for example, two fluorophilic components, and a PEG group. In further embodiments, the triblock copolymer comprises at least one perfluorinated polyether (PFPE) chain, for example, two perfluorinated polyether (PFPE) chains, and a PEG group. The fluorophilic component of the triblock copolymers 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 is at least C 10 Length of at least C 15 Length of at least C 20 Length of at least C 25 Length of at least C 30 In another embodiment, the fluorophilic chain has a length of at least C 50 Length of at least C 75 Length of at least C 100 or longer. A non-limiting example is the structure -(C3F6O) 10 The fluorophilic component having the formula 30 It has a chain equivalent of 30 carbons. The fluorophilic moieties can be linear, branched, cyclic, saturated, unsaturated, and the like.

[0105] In some embodiments, the fluorophilic component of the triblock copolymer includes heteroatoms (e.g., non-carbon, such as oxygen (e.g., divalent oxygen), sulfur (e.g., divalent or hexavalent sulfur), nitrogen (e.g., trivalent nitrogen)) 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 branching branches extending from the main chain of the structure.

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

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

[0108] Subfluorinated Silica Nanoparticles Increasing the surface fluorophilicity of silica nanoparticles can improve their dispersibility in perfluorocarbons. In some embodiments, the fluorinated silica nanoparticles are partially derivatized. In some embodiments, the fluorinated silica nanoparticles can 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 the compositions, methods, uses, sorters, devices, and systems provided herein include subfluorinated silica nanoparticles of Formula 4 below and subfluorinated silica nanoparticles of Formula 5 below. [ka] 1H,1H,2H,2H-Perfluorooctyltriethoxysilane [ka] 1H,1H,2H,2H-Perfluorodecyltriethoxysilane

[0109] Fluorous Dispersion Oil In some embodiments, exemplary fluorous dispersion oils for use in the 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 fluorous dispersion oil and a droplet stabilizing agent comprising an emulsifier that is a diblock copolymer, a triblock copolymer, or a combination thereof. In some embodiments, the fluorous dispersion oil comprises: a mixture of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 1:1:1 w / w ratio or about a 1:1:1 w / w ratio, respectively; A mixture of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 2:2:1 w / w ratio or about a 2:2:1 w / w ratio, respectively; and a mixture of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 1:3:1 w / w ratio or about a 1:3:1 w / w ratio, respectively; and the emulsifier is selected from the following combinations of diblock and triblock copolymers: A diblock copolymer of formula 1 as described herein, and A triblock copolymer selected from the group consisting of compounds of formula 2 and formula 3 as described herein, and combinations thereof. It is.

[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] Fluorinated silica nanoparticles (100 nm).

[0113] Provided herein are methods for reducing cross-contamination between microdroplets. In some embodiments, the methods can include generating droplets in a continuous phase formulation, such as a continuous phase formulation comprising an oil and an emulsifier as described herein. In some embodiments, the emulsifier can be present at a concentration of about 0.3% to about 4% w / w, or about 2% w / w. In some embodiments, the emulsifier can be present at a concentration 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 can be a fluorous oil, a mineral oil, a silicone oil, or any other water-immiscible oil. In some embodiments, the oil can 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, the droplets can be generated by a syringe pump.

[0114] After droplet generation, the droplets can be collected (e.g., in an Eppendorf tube). The continuous phase can be exchanged with a fluorous dispersion oil, such as a mixture of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 1:3:1 ratio. In some examples, the volume of the second fluorous dispersion oil can be approximately the same as the volume of the droplets (e.g., for a 20 μL droplet, 20 μL of fluorous dispersion oil can be used). The exchange with fluorous dispersion oil can be performed more than once (e.g., a total of 2, 3, 4, 5, or more times). The droplets can achieve long-term compound retention, e.g., 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 are allowed to incubate for 30-60 min between one or more exchange steps. In some embodiments of the method for reducing cross-contamination between microdroplets, the continuous phase is exchangeable with a first fluorous dispersion oil to provide aqueous microdroplets suspended in the first fluorous dispersion oil. In some embodiments, the first fluorous dispersion oil is exchangeable with a second fluorous dispersion oil to provide aqueous microdroplets suspended in the second fluorous dispersion oil. In some embodiments, the first fluorous 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, such as those of Formula 6, dispersed in a mixture of perfluoro 2-butyltetrahydrofuran and 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500). [ka] fluorinated silica nanoparticles, for example, 8% wt of formula 6 [ka] fluorinated silica nanoparticles (100 nm). In some embodiments, the second fluorous dispersion oil can have an average fluorine content of >70 wt% and does not contain an emulsifier. In some examples, the volume of the fluorous dispersion oil can be approximately the same as the volume of the droplet (e.g., for a 20 μL droplet, 20 μL of fluorous dispersion oil can be used). The exchange with the fluorous dispersion oil can be performed more than once (e.g., a total of 2, 3, 4, 5, or more). The droplets can achieve long-term compound retention, e.g., 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 of compound retention. In some embodiments, the droplets can be incubated for 30-60 min between one or more exchange steps. In some embodiments, the second dispersion oil can be further exchanged with a third dispersion oil having an emulsifier as described above to provide stability for further droplet manipulation.

[0115] Compound Library Matrix The compositions, methods, uses, sorters, devices, and systems provided herein deliver coded library members in a single droplet. A matrix, such as beads, can be used as a carrier 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] Monodisperse hydrogel beads In some embodiments, the compound library matrix, such as beads, resins, hydrogels, etc., 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 to a wide range of organic reactions, loading capacity to achieve target compound concentrations in droplets, dispersibility in aqueous media when loaded with hydrophobic library compounds, compressibility to achieve quantitative 1:1 encapsulation in droplets in microfluidic devices, and low background autofluorescence to minimize assay interference in droplet-based screens. Quantitative (1:1) encapsulation of compressible compound beads enhances screening throughput. Without wishing to be bound by any theory, screening throughput enhancement is achieved by reducing statistics from double Poisson to single Poisson, i.e., using compressible beads so that every droplet contains an assay-relevant compound bead, whereas using incompressible compound beads that cannot be packed into the microfluidic channel results in many empty droplets in downstream sorter processing, reducing screening throughput. Furthermore, the use of bioassays that can tolerate multiple cells per droplet in combination with compressible beads will yield more assay-relevant droplets, since most of the droplets will contain at least one cell.

[0117] Provided herein is a method for preparing monodisperse PEGA resins via droplet polymerization. In some embodiments, the method can be performed using a microfluidic device capable of generating monodisperse droplets of desired size. In some embodiments, monodisperse droplets can be generated at very high throughput. Monodispersity can improve compound loading capacity and thus screening data consistency for droplet-based screens, allowing compound concentrations within droplets to be determined. In the methods provided herein, the size distribution of droplet-generated PEGA resins can be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the size distribution of droplet-generated PEGA resins can be within 5%.

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

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

[0120] In some embodiments, the monomer components of the PEGA resins exemplified in this disclosure are listed below. Exemplary compositions and methods for preparing amino-modified PEGA resins are described herein. It is understood that monomers containing other functional groups, such as those described herein, can be used. It is also understood that the methods described herein can be applied to the preparation of other PEGA resins, such as PEGA resins containing other functional groups.

[0121] In some embodiments, the acrylamide monomer 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 PEG for use in the compositions, methods, uses, sorters, devices, and systems provided herein may include a compound of formula 7: [ka] (In Formula 7, R1 and R2 are independently H or -CH3, n1 is a precise or average value, and n1 is from 1 to 108. In some embodiments, n1 is selected such that the bis-acrylamide PEG has an average molecular weight of from 250 to 5000 daltons, and in further embodiments, n1 is selected such that the bis-acrylamide PEG has an average molecular weight of from 800 to 2000 daltons.)

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

[0123] In some embodiments, the mono-acrylamidoethylenediamine for use in the compositions, methods, uses, sorters, devices, and systems provided herein is a compound of Formula 9. [ka] In formula 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 NH can independently be a functionalization handle for compound / DNA loading.)

[0125] In some embodiments, an example monomer molar ratio can 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 polydisperse 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 2D illustratively depicts compound-loaded beads 160 encapsulated in a hydrogel matrix 161. FIG. 2E illustratively depicts compound-loaded beads 160 coated with a hydrophilic polymer 162.

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

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

[0130] In some embodiments, compound libraries can be constructed on compound loading cores such as amino-modified PEGA resin 110 or TentaGel® beads prior to modification with a hydrophilic surface, e.g., prior to 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, e.g., 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 activated acid.

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

[0132] FIG. 5F illustrates an embodiment in which hydrogel compound-loaded core beads 110, e.g., amino-modified hydrogels described herein, e.g., PEGA, can be encapsulated in another layer of hydrogel, e.g., PEGA. In some embodiments, encapsulation can be performed in a microfluidic chip. In some embodiments, encapsulation can be performed with 1:1 encapsulation efficiency. The compound-loaded core beads 110, e.g., amino-modified PEGA resin, can be introduced into droplets of shell monomers 117, e.g., naive PEGA monomers, to form a shell, thereby physically encapsulating the library beads. A continuous phase formulation, e.g., continuous phase oil 118, is flowed through to form the core-shell beads. Polymerization can be performed with an initiator, e.g., APS in the aqueous phase and TEMED in the continuous phase. In some embodiments, polymerization can be initiated as the 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 to the core of the droplet, naive PEGA resin without functionalized handles is introduced to the periphery, and the layers are rapidly polymerized with TEMED to give the core-shell structure in a single experiment.

[0134] Core Shell Beads Provided herein are compositions, methods, uses, sorters, systems, and devices in which beads are encapsulated in a compressible hydrogel shell. In some embodiments, beads are encapsulated by in-droplet hydrogel formation. In some embodiments, in-droplet hydrogel formation is performed as described herein by a microfluidic device, such as a microfluidic device for preparing monodisperse PEGA resin. In some embodiments, the Poisson statistics of bead encapsulation and / or clumping in aqueous medium are improved by encapsulation. In some embodiments, the Poisson statistics of bead encapsulation and clumping in aqueous medium of polystyrene-PEG hybrid beads, such as TentaGel® beads, can be improved. In some further embodiments, the Poisson statistics of bead encapsulation and clumping in aqueous medium can be improved by encapsulation of polystyrene-PEG hybrid beads, such as TentaGel® beads, in a compressible hydrogel shell using in-droplet hydrogel formation utilizing a microfluidic device described herein, such as a microfluidic device for preparing monodisperse PEGA resin. In some embodiments, the hydrogel can be, for example, PEGA, polyacrylamide, alginate, agarose, collagen, or a combination thereof. In some further embodiments, the hydrogel can be polyacrylamide. The beads can be suspended in hydrogel monomers and encapsulated in the hydrogel in a microfluidic device. For example, 10 micron polystyrene-PEG hybrid beads, e.g., aminopolystyrene-PEG hybrid (e.g., TentaGel®) library beads, can be suspended in polyacrylamide hydrogel monomers and encapsulated in polyacrylamide in a microfluidic device to give a hydrogel shell, e.g., a 30 micron hydrogel shell or about a 30 micron hydrogel shell. Figures 20A-B illustrate the effect of a PEG-coated thin shell on the dispersibility of compound-loaded TentaGel® beads in aqueous cell culture medium.A comparison of bead dispersions after 4 days of complete dispersion by probe sonication is shown, FIG. 20A without PEG coating and FIG. 20B with 40K PEG coating. The beads are loaded with fluorescein via a photocleavable linker as described in FIG. 11A. After 4 days of standing, significant bead clumping is observed for the beads without PEG coating, while for the beads with 40K PEG coating, the beads are primarily dispersed as monodisperses.

[0135] Hydrogel-encapsulated library beads, e.g., hydrogel-encapsulated polystyrene-PEG hybrid library beads, can achieve the flexibility of compressible hydrogel beads such that the hydrogel-encapsulated beads can be encapsulated 1:1 or about 1:1 in droplets (e.g., microdroplets), while achieving the flexibility of performing combinatorial chemistry and DNA ligation according to traditional methods (see, e.g., (MacConnell et al., 2015, 2017)). Encapsulation can be measured, for example, by analyzing droplets with a hemocytometer / microscope chamber slide (e.g., iBidi or Countess) that can create a tightly packed monolayer of droplets to observe under a microscope where the number of encapsulated droplets can be compared to empty droplets.

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

[0137] Poisson statistics and clumping of bead encapsulation can be improved with any of the beads described herein, for example, polystyrene-PEG hybrid beads, such as M30102 TentaGel® M NH2, Rapp Polymere GmbH. Hydrogels capable of encapsulating beads include hydrogels described herein, for example, 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 structure: Acrylamide - [ka] and bis-acrylamide [ka]

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

[0139] Magnetic Monodisperse Hydrogel Beads 6A and 6B illustrate embodiments in which the hydrogel beads described herein can contain magnetic microparticles, e.g., magnetic core beads. In some embodiments, the magnetic beads can be suspended in the hydrogel generating mixture and droplets can be generated as described herein. FIG. 6A depicts an embodiment in which coated magnetic beads 120 are physically encapsulated in hydrogel beads 121. The coated magnetic beads can be suspended in the hydrogel generating mixture and droplets can be generated in which the coated magnetic beads are encapsulated in the hydrogel but not covalently linked thereto to provide magnetic hydrogel beads 121. FIG. 6B depicts an embodiment in which coated magnetic beads 122 containing amine groups are functionalized with acrylamide to form acrylic magnetic core beads 123. The acrylic magnetic core beads 123 can be suspended in the hydrogel generating mixture and droplets can be generated in which the coated magnetic beads are encapsulated in the hydrogel and covalently linked thereto to provide magnetic core hydrogel beads 124.

[0140] In some instances, the 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 forces, thereby increasing the efficiency of screening that can be performed, for example, by enriching for bead-encapsulated droplets. For example, during bead washing steps in compound synthesis, phase coding, etc., the beads can be separated by magnetic forces, thereby replacing more cumbersome separation steps such as filtration or centrifugation (Rana et al., 1999). In some embodiments, the magnetic bead-encapsulated droplets can be separated from non-encapsulated droplets, thereby reducing the number of droplets to be sorted (Ofner et al., 2017). In some embodiments, the magnetic core beads described herein can be fluorescent and can code their relative positions in a three-dimensional hydrogel grid (Meldal & Christensen, 2010).

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

[0142] In some embodiments, the silica magnetic beads can be suspended in the hydrogel generating mixture. The droplets can be generated as described herein. In some embodiments, the silica magnetic beads can be amino-modified. In some further embodiments, the 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 the hydrogel generating mixture. The droplets can be generated as described herein. The silica magnetic beads can have a low level of autofluorescence.

[0143] In yet another embodiment, carbon-coated cobalt nanoparticles (e.g., TurboBeads) (Grass et al., 2007) (TurboBeads LLC, Zurich, Switzerland) can be embedded in hydrogels as described herein. TurboBeads can be particularly highly magnetic, which can provide handling advantages.

[0144] In yet another embodiment, iron oxide magnetic nanoparticles (eg, TurboBeads) (Grass et al., 2007) (TurboBeads LLC, Zurich, Switzerland) can be embedded in the 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), enzymatic, pH, temperature, and / or redox sensitive linkers, can be selected for degradability upon 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 the monodisperse PEGA resins exemplified herein can replace the photolabile crosslinkers described in the literature (Kloxin et al., 2009; Raman et al., 2020) to generate monodisperse photolabile PEGA resins for the coded bead library synthesis and screening systems described herein.

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

[0147] Code 1 bead 1 compound library In some embodiments, the compound library can be assembled on coded beads. In some embodiments, the compound library can be assembled combinatorially on coded beads. Methods for assembling compound libraries are described in (MacConnell et al., 2015). In a code-one-bead-one-compound (eOBOC) library, each bead can have atto-femtomoles of the same compound loaded via a cleavable linker, preferably a photocleavable linker, and a unique tag, preferably a DNA tag, for the bead itself (bead-specific barcode, BSB) and for the combinatorially generated compound itself (compound barcode). The cleavable linker can alternatively be, but is not limited to, an enzyme-cleavable or pH-sensitive or reduction-sensitive (disulfide) linker. The tag can alternatively be, but is not limited to, an RFID, a fluorescent dye and / or bead, or a peptide mass tag, or a combination thereof.

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

[0149] 11A-11C illustratively depict embodiments of DNA-encoded bead library linkers and DNA barcode designs. FIG. 11A depicts an embodiment in which a split linker for compound loading and DNA coding via a photocleavable linker can contain 1) azidolysine for loading the DNA headpiece via strain-promoted click chemistry, and 2) an ortho-nitrobenzyl linker for photocleavage at 365 nm wavelength. FIG. 11B depicts an embodiment in which DNA coding can be achieved by clicking a DNA headpiece with a forward primer onto a compound bead, then ligating a first and second barcode for a bead-specific barcode, followed by standard split-pool combinatorial chemistry in a microtiter well plate with ligation for a building block-encoded double-stranded oligonucleotide, further closing with several identifier sequences, and finally a reverse primer (see, e.g., Paegel et al (MacConnell et al., 2015, 2017)).

[0150] The coded bead libraries described herein can be optionally prepared with different compound loadings and / or different compound combinations to allow for dose-response screens (qHTS) on the entire library. This method can add flexibility over optical dose-response methods (see, e.g., Paegel et al. (Price et al., 2016)) and can be particularly useful when partial cleavage of releasable linkers, e.g., enzyme or pH labile linkers, can be difficult.

[0151] 11C depicts an embodiment in which the linkers carrying the compound loading sites can be blocked at different doses and coded for different administration volumes. This dose-coding strategy can enable the pooling of dose-responsive beads for library preparation and further screening, thus adding a vast amount of structure-activity resolution while maintaining the ease of pool synthesis and screening.

[0152] Assay 12A-C depict embodiments in which an exemplary mix-and-read fluorescent reporter assay can be configured on a droplet-based screening platform, using various reporters to result in either gain or loss of signal. In some embodiments, the assay is a biochemical or IVTT assay in a droplet (FIG. 12A), fluorescent cells or beads in a droplet (FIG. 12B), secreted fluorescent reporters from cells in a droplet (FIG. 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 can either diffuse throughout the droplet (e.g., either biochemical cell-free assays and cell-based assays utilizing secreted reporter systems) or be contained within a cellular compartment detected via secondary reporter beads (e.g., cytokine secretion assays).

[0153] FIG. 13 depicts examples of reporters, such as fluorescent proteins (e.g., CFP, YFP, GFP, RFP, and related FPs (FIG. 13, section i) or fluorescent reporter enzymes, such as β-lactamase that provides ratiometric detection via a FRET substrate or β-galactosidase that provides near-infrared shifted fluorescence to limit interference from autofluorescent components (FIG. 13, section ii), that can be used to report on compounds that modulate expression via either transcriptional or post-transcriptional processes, or in the form of secreted reporters (FIG. 13, section iii).

[0154] In some embodiments, compounds that degrade a protein of interest (POI) can be assayed on the platform by fusing the POI to GFP. In the assay, either suspension cells (e.g., K562, KG1, U937, Jurkat 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 unfused RFP reporter as separate proteins at approximately equal levels in the cells with a ribosomal skipping sequence (P2A) incorporated between the two genes. If the POI contains a degron such as the IKZF3 tag (Sievers et al., 2018), the addition of a compound such as lenalidomide can induce degradation of the GFP fusion with loss of signal.

[0155] In the case of secreted reporters, the reporter, such as GFP, can be constructed as a secreted protein, or the reporter can be a secreted enzyme, such as secreted alkaline phosphatase (SEAP). The use of secreted reporters can have the advantage that rendering data processing and therefore sorting can be more straightforward and robust, since the signal is diffused throughout the droplet rather than at a focal point (cell).

[0156] In another example, viral infectivity into cells can be screened for, either for antiviral agents or for agents that increase the efficiency of virus-based therapy.

[0157] In some embodiments, the compositions, methods, uses, sorters, systems, and devices provided herein can be used to screen for agents that modulate protein-protein interactions, for example, utilizing the tripartite 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 a probe against a POI that contains a quencher of GFP fluorescence. Similar types of assays can be configured 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 photocleavage, and the image capture and / or methods provided herein can include UV photocleavage and / or image capture. Example 20 describes UV photocleavage and image capture.

[0160] The flexibility of this platform, including the IVTT system, may allow for the performance of assays that multiplex many compounds against many proteins and / or many targets. An example of such an assay is selective translation inhibition, where a sequence encoding the protein to be screened for translation inhibition is added next to a barcode sequence on the bead. In some embodiments, the assay involves selective stalling of human translation via small molecule engagement of the nascent ribosome chain (see, for example, Lintner et al. (2017) PLOS Biol. 15(3):e2001882). Protein expression can be achieved 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 gene encoding the protein.

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

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

[0163] [Table 2]

[0164] Droplet Sorting Fluorescence Activated Droplet Sorter (FADS) Provided herein are methods, systems, and devices for sorting droplets. Figure 2G illustrates an exemplary embodiment of a sorter in which droplets 134 can flow into a path (200) between two sets of electrodes (130 and 131) that can independently apply an electric field to sort droplets 203 into sorting channel 201 and droplets 204 into sorting channel 202. Droplets 134 can be detected and identified as hit or non-hit droplets before passing between the sets of electrodes (130 and 131).

[0165] The droplet sorter described herein is based on the surprising discovery that sorting with a dual electrode sorter, such as the dual electrode sorter described herein, can enhance the speed and / or reliability of sorting.

[0166] 14A-14E illustrate an exemplary embodiment in which there are two electrode sets 130 and 131. In some embodiments, the electrode set can be present just before the droplet path splits into two sorting channels 132 and 133. In some further embodiments, at least two electrode sets can be on different sides of the droplet stream. The sorting can be radio frequency sorting. FIGS. 14A-14B illustrate an embodiment in which a droplet 134 can transition into random channel 132 or 133 in the absence of an applied force, such as from an electric field. In the absence of an applied force acting on the droplet, the droplet can move randomly with equal or nearly equal resistance (FIG. 14B). FIG. 14C illustrates an embodiment in which an electric field is applied to the 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] 14D and 14E depict an embodiment in which droplets 134 can be guided into a stream flowing through a sorting channel 133 by applying an applied force, such as from an electric field, from electrode pair 131 (FIGS. 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 the non-hit stream can be deactivated at electrode pair 130. As shown in FIG. 14E, activation of the electric field for the hit stream can divert the target droplet 134 into that stream 133 when the target droplet arrives. This system can be implemented with the droplets described herein, the droplet generating oils and emulsifiers described herein, and combinations thereof. The droplet flow can be controlled, for example, by a pressure pump, a syringe pump, or any combination thereof.

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

[0169] In the methods, systems, and devices provided herein for sorting droplets, the droplets can be sorted by applying an electric field, a magnetic field, by changing the flow, or by any combination thereof. In some embodiments, two or more electromagnets can be positioned relative to the electrodes at the locations described herein. In some embodiments, the droplets are sorted by changing the applied electric field. A dual sorting system has been developed that allows droplets to be diverted by voltage. Non-hit droplets flow into the straight path at a lower voltage while preventing them from diverting into the bypass. In bypass sorting, the voltage is turned on while the non-hit path voltage is switched off.

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

[0171] In some embodiments, core-shell beads containing members of the coding library can be converted into water-in-oil droplets using preferred emulsion formulations described herein to have cavities housing cells, compounds released and incubated in discrete droplet compartments, and hydrogels carrying beads and cells that are extracted into aqueous buffers for off-droplet sorting using traditional flow cytometers (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-in-water (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. The sorting method can be performed with the droplets described herein, the droplet generating oils and emulsifiers described herein, and combinations thereof. Commercially available FACS machines can be used to sort double emulsion droplets at frequencies between 10-12 kHz (Brower et al., 2019). Figure 16 shows water-in-oil-in-water (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 17A-17F depict embodiments that can automatically detect and / or sort emulsion droplets. FIG. 17A depicts a grid 140 of partitions (e.g., a microwell plate containing wells 141) with each partition (e.g., well) 141 being equal or approximately equal to a size range of emulsion droplets. FIG. 17B depicts a grid 140 with emulsion droplets 142 arrayed in the wells 141 of the grid. In some embodiments, one droplet is arrayed in each partition or well 141. By arraying emulsion droplets, it is possible to spatially trap the droplets. Compared to flow-based FADS, this spatial control can result in a much wider range of readout possibilities over a given time frame, e.g., bright field, fluorescent or luminescent readouts, or any combination thereof. In addition to standard live and dead cell staining readouts and fluorescent biosensors, spatial control allows for high-resolution imaging for phenotypic readouts, e.g., cell or organelle morphology, localization of proteins of interest, etc. FIG. 17C depicts a grid 140 containing droplets 141. After application of one or more parameters (e.g., release of compounds from coded beads via a trigger such as electromagnetic irradiation (e.g., light, UV, UVA, e.g., about 365 nm), enzyme cleavage, incubation for peptide / protein expression using in-vitro transcription-translation, or simple incubation for reporters, pH change, reducing agents, or combinations thereof), the assay can be analyzed and hit droplets 143 and 144 can be identified. After defining the desired single or multiple groups, as illustrated in FIG. 17D, the hit droplets can be removed (e.g., mechanically removed) from wells 145 of microwell plate 140. FIG. 17E depicts the analysis of single and multiple hit group droplets 143 and 144, respectively. In some embodiments, the removal of droplets can be automated. Droplets can be removed, for example, by a micromanipulator (e.g., an automated robotic micromanipulator), by irradiation, or a combination thereof.FIG. 2H illustratively depicts an embodiment in which hit droplets 143 are identified in a 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 defined locations. In some embodiments, the droplets can be removed by irradiation (e.g., electromagnetic irradiation), which can burst the droplet and release its contents into the surrounding phase, allowing analysis of the contents. In some further embodiments, the irradiation is by a laser.

[0175] In some embodiments described herein, droplets (e.g., arrayed droplets) for use in the sorting methods, devices, and systems described herein can be denser than the continuous phase in which they are suspended. In some embodiments, the droplets can be denser than the oil phase in which they are suspended. If the droplets are denser than the liquid in the microplate, they can settle to the bottom of the well, facilitating imaging and / or analysis. In some embodiments, the droplets can be denser than the oil phase using fluorinated water-in-oil double emulsion droplets in water, or using water-in-oil droplets in which the oil phase is less dense than the aqueous phase. In an exemplary workflow, the arrayed emulsion droplets can be arrayed and automatically imaged based on defined hit criteria, and hit droplets can be automatically removed from the array for further analysis. Figure 17F shows a microscope image of an array in which double emulsion droplets with 146 and without 147 fluorescent dyes are arrayed in a microwell plate.

[0176] In some embodiments, the barcode associated with the coded bead can be identified. Identification of the barcode can include, for example, fluorescent microscopy / counter, RFID reader, mass spectrometry, DNA sequencing, or any combination thereof. In some embodiments, next generation sequencing and data science can be used to deconvolute the hits. In some embodiments, the sequence DNA barcode from the coded bead can be sequenced. For example, in some embodiments, PCR can be performed directly on the DNA linked to the bead.

[0177] In the context of droplet sorting and screening with NGS readouts, enrichment of unique bead-specific barcode (BSB) expression per compound-specific barcode (CSB) can be compared between assay hits sorted into "hit sort" (assay-active compounds in droplets) and "non-hit sort", and appropriate statistical models can be applied to assess the significance of enrichment. Using dataset randomization, an upper enrichment background limit for calling hits can be estimated.

[0178] The droplet sorting methods, systems, uses, devices described herein (e.g., double emulsion sorting by FADS, FACS, sorting of arrayed droplets) may include black hole quencher assays, secretion reporter assays, or combinations thereof. In some embodiments, droplet-based screening / sorting may include the use of an automated cell picker. In some embodiments, the automated cell picker may include a microwell array plate (hosting one microdroplet per microwell), a fluorescent microscope, an imaging setup for automated imaging of assay droplets and identification of desired droplets, a microcapillary-based droplet sampling device for automated picking and deposition of desired droplets into hit wells, or any combination thereof. In some embodiments, the continuous phase formulation includes 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 perfluorocarbon and / or perfluorinated oil 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-dimethyl-cyclohexane, octadecafluorodecahydronaphthalene (perfluorodecalin), and / or b) 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), perfluorotributylamine and perfluoro(dibutylamine). and / or c) 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 mixtures, methoxy nonafluorobutane and methoxy nonafluoroisobutane mixtures (HFE7100), methoxy nonafluorobutane, methoxy heptafluoropropane (HFE7000), HFE712, HFE649, HFE7100DL, HFE 71DE, HFE 71IPA, 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, octadecafluorodecahydronaphthalene (perfluorodecalin), and / or the perfluorinated oil is perfluoro-2-butyltetrahydrofuran, perfluoro-N-methylmorpholine (FC-3284), perfluorotripentylamine (FC-70), perfluorotributylamine (FC-43), perfluorotripropylamine (FC -3283), 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), a 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-dimethyl-cyclohexane, octadecafluorodecahydronaphthalene (perfluorodecalin), and 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), a mixture of perfluorotributylamine and perfluoro(dibutylmethylamine) (FC-40), and 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), a mixture of methyl perfluoroisobutyl ether / methyl perfluorobutyl ether (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, 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), 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-ethoxydodecafluorohexane (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 fluorous dispersion oil consists of perfluorohexane (FC-72), perfluorooctane, and 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500), each present in an amount ranging from 10 to 90% w / w.

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

[0188] In some embodiments, the triblock and / or diblock copolymers are fluorinated. In some embodiments, the triblock or diblock copolymers include a perfluoropolyether (PFPE) and a polyethylene glycol (PEG) (e.g., a perfluoropolyether (PFPE), a polypropylene glycol (PPG), and a polyethylene glycol (PEG), two perfluoropolyethers (PFPE) and a polyethylene glycol (PEG), or two perfluoropolyethers (PFPE), two polypropylene glycols (PPG), and a polyethylene glycol (PEG).

[0189] In some embodiments, the diblock copolymer has Formula 1 [ka] (wherein n and m are the precise or average values ​​of the polydisperse building blocks, the average molecular weight of the diblock copolymer is 1,000-10,000 Da, n is 35-45, and m is 2-24.) In some embodiments, the triblock copolymer has: [ka] where i, j, and k are precise or average values ​​of the polydisperse 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] where p, q, r, s, and t are precise or average values ​​of the polydisperse building blocks, the average molecular weight of the triblock copolymer is 2,000-20,000 Da, p and t are independently 35-45, q and s are each greater than 0, the precise or average value of the sum q+s is 3-6, and r is 1-23. The compound 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 which 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 stabilizing agent comprises fluorinated (eg, partially fluorinated with fluorinated alkyl side chains) silica nanoparticles.

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

[0193] In some embodiments, the fluorinated silica nanoparticles have the formula [ka] The compound includes one or more substituents selected from the group consisting of the substituents:

[0194] In some embodiments, the fluorinated silica nanoparticles are present in the continuous phase formulation at a concentration of about 2-8% 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 a 1:1 (w / w) ratio of perfluoro 2-butyltetrahydrofuran:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500). [ka] Fluorinated silica nanoparticles (100 nm).

[0197] In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further comprises coating the PEGA copolymer resin with a hydrophilic material (e.g., 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, the method for preparing a monodisperse PEGA copolymer resin further comprises linking a ligand and / or a barcode (e.g., a nucleic acid, DNA) to the PEGA copolymer resin, either reversibly (e.g., a photocleavable linker) or irreversibly (e.g., attachment to a functionalized handle). In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further comprises linking a ligand and a barcode (e.g., a nucleic acid, DNA) to the PEGA copolymer resin, either reversibly (e.g., a photocleavable linker) or irreversibly (e.g., attachment to a functionalized handle). In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further comprises linking a ligand or a barcode (e.g., a nucleic acid, DNA) to the PEGA copolymer resin, either reversibly (e.g., a photocleavable linker) or irreversibly (e.g., attachment to a functionalized handle).

[0199] In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further comprises reversibly linking a ligand to the PEGA copolymer resin and irreversibly linking a barcode (e.g., nucleic acid, DNA) (e.g., attachment to a functionalized handle). In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further comprises irreversibly linking a ligand to the PEGA copolymer resin and irreversibly linking a barcode (e.g., nucleic acid, DNA) (e.g., attachment to a functionalized handle). In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further comprises irreversibly linking a ligand to the PEGA copolymer resin and reversibly linking a barcode (e.g., nucleic acid, DNA) (e.g., attachment to a functionalized handle). In some embodiments, the method for preparing a monodisperse PEGA copolymer resin further comprises reversibly linking a ligand to the PEGA copolymer resin and reversibly linking a barcode (e.g., nucleic acid, DNA) (e.g., attachment to a functionalized handle).

[0200] In some embodiments, the ligand is reversibly linked to a 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 comprises embedding magnetic particles in the PEGA copolymer resin by covalent linkage or by physical encapsulation. In such embodiments, the magnetic particles may have a size ranging from 1 nanometer to 10 microns in diameter.

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

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

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

[0205] In some embodiments of the method for preparing a monodisperse PEGA copolymer resin, the multiple 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 a monodisperse PEGA copolymer resin, the functionalized handle is selected from the group consisting of a protected amine (e.g., a Boc-protected amine or an Fmoc-protected amine), an unprotected amine (e.g., a free amine), an alcohol, an acid, an ester, an azide, an acetylene, and a tetrazine.

[0207] In some embodiments of the method for preparing a core-shell bead, the core bead is linked to a monomer that corresponds to a monomer of the plurality of monomers, in such embodiments, polymerization of the monomer comprises covalently linking the core bead to a hydrogel that encapsulates the bead.

[0208] In other embodiments, thin-shell (e.g., PEG-coated) core-shell beads are prepared by grafting a hydrophilic polymer coating onto a solid support. In some embodiments, the solid support is a polystyrene matrix, e.g., a low-crosslinked polystyrene matrix to which polyethylene glycol is grafted. In some examples, the hydrophilic polymer coating comprises PEG. In some examples, the PEG is attached by acetylene-azide click chemistry.

[0209] It is known that the swelling capacity (swelling degree) of the resulting network is affected by the amount of crosslinking (crosslinking density). The swelling capacity of the resin in both organic and aqueous media is particularly important when chemistry is performed in organic solvents while DNA ligation is performed in aqueous buffers. In photocrosslinking, a sufficient amount of crosslinking monomer is used to provide dimensional stability to the polymer beads so that they swell rather than dissolve in aqueous and / or organic media. Lower crosslinking levels usually provide greater 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 swelling degree as well as other process conditions. Therefore, the lightly (or lightly) crosslinked polystyrene matrix of the present disclosure with PEG grafting is chosen so that the swelling capacity of the polymer is sufficient in both aqueous and organic media.

[0210] In some embodiments, forming the core-shell beads comprises forming a mixture of core-shell beads and empty beads, in such embodiments, the method further comprises enriching the mixture of core-shell beads and empty beads (e.g., by flow cytometry) to obtain core-shell beads with one encapsulated core bead per core-shell bead.

[0211] In some embodiments, forming the core-shell beads comprises forming a plurality of core-shell beads that are sufficiently stable and compressible to enable packing of the core-shell beads in a microfluidic channel such that the core-shell beads are encapsulated in the microdroplet with super-Poisson encapsulation efficiency.

[0212] In some embodiments of the method for preparing the 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 comprises a ligand and / or a barcode, or the method further comprises coupling 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 comprises a ligand and / or a barcode, or the method further comprises coupling 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 comprises a ligand and a barcode, or the method further comprises coupling 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 comprises a ligand or a barcode, or the method further comprises coupling 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 comprises linking a ligand and / or a barcode to the hydrogel that encapsulates the core beads.

[0215] In some embodiments of the method for preparing core-shell beads, the method further comprises coupling a ligand and / or a barcode to the hydrogel that encapsulates the core beads. In some embodiments of the method for preparing core-shell beads, the method further comprises coupling a ligand or a barcode to the hydrogel that encapsulates the core beads. In some embodiments of the method for preparing core-shell beads, the method further comprises coupling a ligand and a barcode to the core beads. In some embodiments of the method for preparing core-shell beads, the method further comprises coupling a ligand to the core beads and coupling a barcode to the hydrogel that encapsulates the core beads. In some embodiments of the method for preparing core-shell beads, the method further comprises coupling a ligand to the hydrogel that encapsulates the core beads and coupling a barcode to the core beads.

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

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

[0218] In some embodiments, the core-shell beads comprise a core bead encapsulated by polyacrylamide, hi such embodiments, the core bead is a low cross-linked 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 comprises a sensor configured to detect a target composition having a selective property flowing through the inlet channel upstream of the junction, hi such embodiments, the controller is configured to switch the state of the 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 characteristic is selected from the group consisting of a threshold amount of fluorescent intensity, emission of a threshold range of fluorescent intensity, emission of a particular combination of a fluorescent signal and its threshold intensity, and emission of a particular combination of fluorescent signal intensities within a threshold ratiometric range.

[0223] In some embodiments, the controller is configured to return the first and second electrodes to the 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 both the first and second electrodes receive no voltage and the plurality of target compounds flow through the junction into both the first and second exit channels.

[0225] In some embodiments, the inlet channel has a diameter of between 1 and 200 microns.

[0226] In some embodiments, in the first state the second electrode receives no voltage, and in the second state the first electrode receives no voltage.

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

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

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

[0230] Example 1: Perfluoropolypropylene-polyethylene glycol (PFPE-PEG) [ka] An oven-dried 100 mL two-neck round bottom flask equipped with a stir bar and a fin denter was charged with Krytox157-FSH (a carboxylic acid terminated perfluoropolyether) (51.07 g, 7.60 mmol) in 50 mL HFE-7100. The reaction mixture was cooled to 0° C. under a 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 hr. The solvent was evaporated and placed under high vacuum for 18 hr to give the acid chloride 11 (average molecular weight=approximately 6739) as an opaque oil, which was used in the next step.

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

[0232] To remove unreacted amine, 6 mL of DCM was added to the reaction mixture followed by 6 mL of FC-72, 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 the organics, was discarded, and the bottom layer was evaporated to give an opaque oil.

[0233] To remove the PEG amine salt, the latter oil was washed with 1N HCl, DI water, followed by MeOH. The resulting oil was dissolved in 7 mL of HFE-7100 and 10 mL of THF was added, followed by 4 mL of MeOH. The resulting mixture was heated to 75 °C for 10 min and the top organic layer was removed. The bottom fluorous 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 to -84.77 (m), -131.29 (s), -133.19 to -133.79 (m), -145.16 to -146.81 (m).

[0234] Example 2: Perfluoropolypropylene-polyethylene glycol-perfluoropolypropylene (PFPE-PEG-PFPE) [ka] An oven-dried 100 mL two-neck round bottom flask equipped with a stir bar and a fin denter was charged with Krytox157-FSH (a carboxylic acid terminated perfluoropolyether) (51.07 g, 7.60 mmol) in 50 mL HFE-7100. The reaction mixture was cooled to 0° C. under a 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 h. The solvent was evaporated and the reaction was placed under high vacuum for 18 hr to give 11 (average molecular weight = ca. 6739) as an opaque oil, which was used in the next step.

[0235] An oven-dried 100 mL two-necked round bottom flask equipped with a stir bar and a fin denter was charged with poly(ethylene glycol) diamine 400 (156 mg, 0.389 mmol) in 10 mL / 5 mL THF / DMF and DIPEA (0.340 mL, 1.945 mmol) under N2 atmosphere. 11 (6553 mg, 0.972 mmol) in 10 mL HFE-7100 was added dropwise and heated to 60 °C for 30 min. Poly(ethylene glycol) methyl ether amine 500 (694 mg, 1.389 mmol) in 1 mL THF was added. The mixture was heated to 60 °C for 2.5 hr, cooled to room temperature, and stirred overnight under N2 atmosphere.

[0236] To remove unreacted amine, 10 mL of DCM was added to the reaction mixture followed by 10 mL of FC-72 (perfluorohexane) and the contents were transferred to a separatory funnel. The separatory funnel was shaken and left until two clear layers were obtained. The top layer, which contained the organics, was discarded. The bottom layer was evaporated to give an opaque oil.

[0237] To remove the PEG amine salt, the latter oil was washed with 1N HCl, DI water, followed by MeOH. The resulting oil was dissolved in 5 mL of HFE-7100 and 8 mL of THF was added. The resulting mixture was heated to 75°C for 10 min and the top organic layer was removed. 3 mL of MeOH was added and the resulting mixture was heated to 75°C for 10 min. The top layer was removed and the bottom fluorous 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 to -86.48 (m), -131.41 (s), -133.18 to -133.84 (m), -144.83 to -147.14 (m).

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

[0239] Figures 7A-7H show droplet retention of 100 μM fluorescein and 100 μM resorufin (in 1 M HEPES buffer) at time zero (top) and 1 hour (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 generation oil 1864006 (Bio-Rad) (Figures 7D and 7H). All showed good retention of the hydrophilic and negatively charged dye fluorescein (Figures 7A-7D), but all failed to retain neutral and more hydrophobic dyes such as resorufin (Figures 7E-7H).

[0240] By reducing the net dipole moment of the oil composition in the continuous phase, it is possible to limit the partitioning of organic molecules across the droplet boundaries while simultaneously achieving biocompatibility, including cell viability, as well as mechanical stability for droplet manipulation. The fluorophilicity of 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) (fluorine content 66%) was increased by wetting with co-solvent perfluorocarbon / perfluorinated oils (perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 1:1:1 w / w ratio, respectively, perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 2:2:1 w / w ratio, respectively, and perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 1:3:1 w / w ratio, respectively, resulting in increased fluorine content >75%), resulting in increased compound retention. The formulation contained 2% wt of copolymer of formula 1 (n is about 38, and m is about 9) and copolymer of formula 2 (i and k are about 38, and j is about 9-11) in a 1:1 ratio. No leakage of resorufin (100 μM) or fluorescein (100 μM) in HEPES buffer was observed. Figures 8A-8D show the molecular retention of fluorescein (green, Figure 8D) and resorufin (red, Figure 8C) in droplets in compositions containing a 1:3:1 w / w ratio of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500), respectively, and 2% wt of the block copolymer described above.

[0241] Conventional di- or triblock copolymer emulsifiers, including the commercial formulations described above (C. Holtze et al., 2008; Christian Holtze et al., 2008), when used with these highly non-polar hydrofluoroether / perfluorocarbon carrier oil systems did not provide adequate droplet stability (IFT γ>10 mN / m), whereas excellent droplet stability (>1 week) was achieved using the formulations described above containing 2% wt of the mixture of block copolymers.

[0242] The effectiveness of a 1:1:1 w / w ratio blend of perfluorohexane (FC-72):perfluorooctane:HFE-7500 containing 2% wt of copolymer of formula 1 (average value n is about 38 and average value m is about 9) for the retention of commonly used water-soluble dyes shown below (fluorescein derivatives, rhodamine derivatives, and resorufin) was determined. A range of physiochemical properties (Mol wt. 200->500 Da, TPSA 50-150 Å) were measured. 2 Excellent molecular retention in the droplets was achieved for compounds with hydrophobicity (logD -2 to +4, and net charge -1 to +1).

[0243] [Table 3]

[0244] [Table 4]

[0245] [Table 5]

[0246] Additionally, retention of resorufin (100 μM) and fluorescein (100 μM) was evaluated in droplets containing different buffer systems with various salt concentrations (PBS, 1×-10×, Tris 100 mM-1M, and HEPES 100 mM-1M) and assay medium (RPMI cell culture medium). No detectable leakage was observed and retention was independent of the buffer system used in a 1:1:1 w / w ratio formulation containing 2% wt of copolymer of formula 1 (average value n is about 38 and average value m is about 9) of perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500). Additionally, the nature of the buffer system had little effect on droplet stability.

[0247] Example 4: Cell viability in the presence of di- and / or triblock copolymer emulsifiers The effect of di- and triblock copolymers on cell viability was determined by encapsulating Jurkat cells in 100 micron droplets and incubating for 24 hours on the benchtop. 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) before encapsulation and the droplets were collected in Eppendorf tubes. The droplets were then disrupted using 1H,1H,2H,2H-perfluoro-1-octanol (PFO) at a final concentration of 10%. The aqueous layer containing the cells was collected, washed, stained with SYTOX RED (ThermoFisher S348969) at a 1:1000 dilution, and analyzed on 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 assessing residual versus coreless droplets under a microscope after repeated pipetting and centrifugation of the emulsion droplets at 16900rcf.Surface tension was determined by the pendant drop method (DSA 30, Kruss, Germany).

[0249] [Table 6]

[0250] Example 6: Replacement with an emulsifier-free continuous phase to minimize compound cross-contamination 2 μL of 100 micron diameter fluorescently labeled droplets (100 μM fluorescein and 100 μM resorufin in RPMI buffer) were mixed with 18 μL of 100 micron diameter unlabeled droplets (RPMI buffer). The oily layer containing emulsifier (Formula 1 (n is about 38 and m is about 9):Formula 2 (i and k are about 38 and j is about 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×. Droplets were incubated in eppi tubes for >5 days and leakage was monitored by fluorescence spectroscopy (CY3 10 ms and FITC 10 ms exposures).No leakage of resorufin or fluorescein into unlabeled droplets was observed (Figures 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 TBSET buffer, 30 μL 10% (w / v) APS (Sigma-Aldrich, A9164), 37.6 mg dimethylacrylamide (Sigma-Aldrich, 274135), 25.1 mg acrylamide-PEG-acrylamide 2KD (Biochempeg, 10060), 19.7 mg Boc-NH-PEG4-acrylamide (Biochempeg, 12310), and 787.6 mL water. The solution was filtered using a 0.2 micron syringe filter before loading the solution into a 1 mL syringe (Becton Dickinson, 309628). 2.5 mL carrier oil (RAN Biotechnologies, 008-FluoroSurfactant-2wtH-50G) and 10 μL 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 the droplet generation device by PE2 tubing (Scientific Commodities, BB31695-PE / 2). The aqueous solution was pumped at 900 μL / h and the oil at 1800 μL / h. The emulsion droplets were collected at the outlet of the microfluidics chip in an Eppendorf tube containing 50 μL 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 20% (vol / vol) PFO (Alfa Aesar, B20156) in HFE7500 (Novec7500) to break droplets. The beads in the aqueous phase were washed twice with 1% Span80 (Sigma-Aldrich, S6760-250ML) in hexane (Sigma-Aldrich, 227064-1L) and then three times with TBSET buffer. The beads were filtered through a 70 μm cell strainer (Corning, 352350) and stored in TET buffer at 4 °C for up to 6 months.

[0252] Example 8: Preparation of 10 μm TentaGel® and monodisperse 33 micron PEGA resin carrying fluorescein via a photocleavable linker To determine the loading capacity and photorelease compatibility of TentaGel® and PEGA resins, a standard solid-phase organic synthesis protocol with minor modifications was employed to load the fluorescein probe. Briefly, the Boc protecting group on freshly prepared 33 micron diameter monodisperse PEGA resin was removed by HCl treatment in methanol for 2-3 hours. The resin was washed by decanting the supernatant after centrifugation, and then acylated with an acid linker bearing fluorescein via a photocleavable linker using standard coupling conditions with DIC / HOAt, again washing the resin by decanting the supernatant and repeatedly exchanging the solvent. The thus prepared fluorescein-loaded 33 micron amino PEGA resin was kept in the dark and under refrigerated conditions until further use. [ka]

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

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

[0255] reagent 1.Tris-EDTA-Tween buffer (TET) a. 480mL nuclease-free water b. 5 mL 1 m Tris-HCl (pH 8.0) c. 10mL 0.5M EDTA d. 5 mL 10% (vol / vol) Tween-20 in nuclease-free water e. Filter the solution through a 0.2um membrane 2. Hydrogel Bead Wash Buffer (HBW) a. 980mL nuclease-free water b. 10 mL 1M Tris-HCl (pH 8.0) c. 200 μL 0.5 M EDTA d. 10 mL 10% (vol / vol) Tween-20 in nuclease-free water e. Filter the solution through a 0.2um membrane 3.2x Bead Concentration 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) 2.375mM KCl 3.5mM MgCl2 c.210μL 10%(vol / vol)Igepal CA-60

[0256] FIG. 10A shows the compressibility of the reference polyacrylamide resin for DropSeq (PAA) versus PEGA 1× rods as measured by texture analyzer, with the compressibility of PEGA 1× roughly matching the polyacrylamide reference gel (PAA).

[0257] FIG. 10B shows the tight packing of PEGA 1× resin in one channel and the quantitative 1:1 encapsulation into droplets.

[0258] Example 11: Cell viability in the presence of PEGA resin 1×10 6 Jurkat cells suspended in RPMI 20% FBS 1% Pen Strep at resin or cells / mL and resin suspended in water were mixed 1:1 in a 96-well microtiter plate in a final volume of 200 μL and then incubated at 37° C., 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 on a BD Fortessa flow cytometer. Figures 10C and 10D show the FACS results for the biocompatibility of 33 micron PEGA resin. PEGA resin had minimal effect on cell viability as determined by FACS after co-incubation in microtiter well plates for 24 hours.

[0259] Cells cultured in the presence of PEGA 32 μM 2000 PEG 1× survived similarly to cells cultured with 50% water, whereas cells cultured with PEGA 32 μM 3700 PEG 2× showed approximately a 70% decrease in 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 adapting 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, azidolysine, and glycine by sequential loading of each Fmoc-protected amino acid with DIC / Oxima / DIPEA in DMF. Each loading step was preceded by a capping step with 20% acetic anhydride solution in DMF, followed by Fmoc deprotection with 20% 4-methylpiperidine solution in DMF. The resin was then reacted with the reference compound derivatized with a photocleavable linker using DIC in a 1 / 1 mixture of DCM / DMF. The DNA headpiece was then loaded by click chemistry between the azide handle on the resin and the DBCO-derivatized headpiece. Finally, multiple simultaneous enzymatic ligations using T4 ligase were performed in Bis-Tris buffer amended with NaCl, ATP, and MgCl2 to fully encode the beads.

[0261] Example 13: Encapsulation of Fluorescein-Loaded TentaGel® in Polyacrylamide Hydrogel Beads, Droplet Encapsulation, and Intradroplet Light Release and Loading Capacity Determination. 0.15 mg of fluorescein-labeled TentaGel® (TG) beads (attached via a photolabile linker) were dispersed in 1.5 mL of hydrogel precursors (PAA / APS and water). The fluorescein-labeled TG beads were encapsulated in polyacrylamide hydrogels using a Dolomite microfluidic device. The aqueous lines contained the hydrogel precursors and TEMED were dispersed in oil (008-fluorsurfactant (RAN Biotechnologies) 2 wt% in HFE). 60 micron hydrogels were 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. Homogeneous diffusion of fluorescein from the TG into 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 TentaGel® in Polyacrylamide Hydrogel Beads 10 μl of TentaGel® in polyacrylamide beads was loaded into a countess slide (Countess Cell counting Chamber Slide C10228) and imaged under bright field at 4× magnification using an EVOS FLoid Imaging System (ThermoFisher Scientific 4471136). Beads in the images were manually counted and coencapsulation rates were calculated. Crude TentaGel® in polyacrylamide beads was enriched using a SONY SH800 FACS sorter configured with a 130 μM sorting tip at 9 PSI. Beads were sorted at a medium flow rate (6 / 10). All events flowing through the machine were analyzed for FITC (e.g., 488 nM em. 525 / 50 nm) and PE (e.g., 561 nm em. 600 / 60 nm) fluorescence. Beads were enriched with relatively higher FITC and PE fluorescence than other events analyzed. Figure 9I shows the scatter plot and gated populations. Subpopulations of beads were sorted at 10K beads / well into 96-well microtiter plates containing 100 ul 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 for TentaGel® in PEGA resin, TentaGel® resin, and polyacrylamide resin Figure 19 shows the amplification curves of the different samples against the standard curve (green). Three samples are analyzed: a blank made 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 show only low amplification compared to the blank sample, whereas the sample beads show unambiguous amplification compared to the blank and control, confirming the success of the enzymatic ligation. All samples were performed at least in triplicate. Based on the number of beads in the sample, the amount per bead can be estimated using the standard curve.

[0265] Example 16: UV photocleavage and image acquisition. A 25 μL aliquot of 100 micron water-in-oil droplets without mineral oil overlay (to avoid cracking / deterioration of the ibidi polymeric slide bottom) was loaded into one channel of an uncoated ibidi 100um channel slide (ibidi cat.#80661) and sealed with Kwik-Cast sealant (World Precision Instruments cat.#KWIK-CAST). The sample was placed on 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 prior to UV exposure. UV photocleavage was performed with a 10 msec interval of UV exposure (385 nm Colibri LED at 10% producing 20.7 mW at 365 nm) with the total number of cycles modified to yield a total exposure time of interest up to 10 sec (e.g., 100 cycles = 1 sec). A 10 msec 2% 488 nm Colibri LED excitation was also used to capture video of fluorescein emission from the beads into the droplets, making the total image acquisition time much longer than the sum of all cycles of the 10 msec burst due to switching times for the optics.

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

[0267] Example 17a: PEG coating of fluorescein-loaded TentaGel® beads to create thin-shell beads To a suspension of 20 million fluorescein-labeled TentaGel® (TG) beads (attached via a photolabile linker) in a 7:3 mixture of acetonitrile and 30 mM TEAA with 1% Pluronic F127 (a block polymer of PEG and PPG, also called Poloxamer or Poloxamer 407) at 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 6000rcf for 2 min, the supernatant was discarded, and 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 fridge until further use.

[0268] Thin shell beads with PEG10K coating were prepared similarly from 20 million fluorescein-labeled TentaGel® (TG) beads (attached via a photolabile linker) except using 26 mg of PEG 10K DBCO (BroadPharm #BP-22462) and a 1:1 mixture of DMSO and 30 mM TEAA pH 7.4 with overnight shaking.

[0269] Example 17b: UV Photocleavage and Image Acquisition of PEGylated Thin-Shell Fluorescein-Loaded TentaGel® Beads UV photocleavage and image acquisition of fluorescein probes with 10K or 40K PEG-loaded fluorescein-loaded TentaGel beads was confirmed using a similar protocol as described in Example 17. Approximate loading was determined to be ∼25 fmole per bead for 10K PEG-coated beads and ∼36 fmole per bead for 40K PEG-coated beads. Example 17c: PCR of PEGylated (thin shell) TentaGel® beads (Figure 19B) Using the same protocol as described in Example 15, the amplification of DNA tags by 40K PEG-coated null library beads was confirmed. Although the amplification of control beads without PEG is low, there is clear amplification when compared to the negative control MilliQ water. Sample beads with 40K PEG have equal amplification to control beads with the same DNA tag but without PEG. The overlapping curves clearly suggest that the conjugation of post-synthesis 40K PEG does not affect the amplification of DNA tags when compared to the control sample without PEG. Two samples with beads were run in quintuplicate, and MilliQ water was run in triplicate. 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 are microwell plates containing aqueous solutions loaded with water-in-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 is a microwell plate containing a double emulsion 150 to be removed. Figure 18B is a microwell plate into which a capillary 151, slightly larger than the double emulsion (150), is lowered. A volume of solution is aspirated to remove the double emulsion from the well (Figures 18C and 18D). Single detection emulsions were aspirated from the microwell plate by a CELLector tool from Molecular Machines & Industries (Eching, Germany) using a modified aspirating capillary tube designed for droplet aspiration from a grid without disturbing the adjacent droplets. The CELLector tool included a modified aspirating capillary tube designed for droplet aspiration from a grid versus aspiration of cells from a flat-bottom microwell plate.

[0271] Example 19: Next-generation sequencing (NGS) and deconvolution of a DNA-encoded one-bead one-compound library NGS and informatics for bead barcode counting and hit deconvolution were performed on a mock-coded library screen set consisting of a 1% mixture of positive and negative controls and approximately 99% of the 10K null library. Next generation sequencing and data science were used to deconvolute the hits. DNA barcodes from the coded beads were sequenced. Among other approaches, a method to perform PCR directly on the DNA linked to the beads is described herein.

[0272] Barcodes were recovered from the bead libraries via PCR and NGS workflow. PCR was performed using the KAPA HiFi HotStart ReadyMix PCR Kit (KAPA Biosystems KR0370). The bead libraries were combined in a 96:1 ratio and PCR reactions were performed on 2,500 beads in a 50 μl reaction. PCR products were purified using AMpure XP beads (Beckman Coulter Life sciences, A63882) and analyzed by Agilent D1000 ScreenTape system. Library preparation for NGS was prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina EB#e7545S / L Version 5.New England BioLabs Inc. according to 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 (paired-end 150 base reads are sequenced), NGS data in the form of pooled raw sequencing BCL files were demultiplexed into separate paired-end read (R1 and R2) fastq files (e.g., sample1.Read1.fastq and sample1.Read2.fastq) for each sample using Bcl2Fastq (Illumina), with Illumina adapter sequences trimmed in the process. Sequencing quality reports for each R1 / R2 fastq file pair were generated 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 downstream processing steps. Remnant Illumina adapter sequences were trimmed using BBTools / bbduk. Read pairs with identical sequences containing identical UMIs (unique molecular indexes) were combined using BBTools / dedupe to remove possible sequencing bias introduced by the PCR step, and deduplicate read pairs were reported as combined fastq files. Reads R1 and R2 were then merged into unique sequences with BBTools / bbmerge using default quality filters requiring a pairwise overlap of at least 145 bases. The majority of reads (75%) were preserved at this point.

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

[0276] Decoding / Hit Calling The preprocessed barcode sequences were decoded into a compound-specific barcode (CSB) portion (~39 bp) and an associated bead-specific barcode portion (BSB, ~45 bp), allowing up to 2 mismatches over the ~85 bp trimmed sequence. The majority (81%) of the preprocessed sequences map to the expected barcode at this mismatch level.

[0277] CSB+BSB pairs with read counts below a background read count of 2 (which can vary depending on sequencing read depth) were not considered.

[0278] The number of unique bead barcodes with a read count of 2 or more for each compound was counted, and the compounds (CSBs) were ranked by the number of unique beads.

[0279] Example 20: Acrylation of amino-functionalized polystyrene-coated iron oxide magnetic nanoparticles A 15 mL Falcon tube was loaded 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 a sonication bath for 1 h. 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 above washed magnetic nanoparticles were suspended in 3 mL DMF and treated with NHS ester functionalized PEG acrylate (Laysan Bio, ACRL-PEG-SVA-2000, 123 mg) in DMF (3 mL) followed by DIPEA (50 μL). The suspension was sonicated for 5 min and then mixed overnight on a shaker. The beads were washed 3× with DMF (3 mL each) followed by 3× with water (3 mL 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 of acrylated magnetic beads (150 mg / mL in water) prepared in Example 20 was added to 900 μL of the PEGA hydrogel mix described in Example 7. The suspension was chilled on ice and bath and probe sonicated at maximum amplitude (55 W) using a QSonica Q55 Sonicator Ultrasonic Processor (Qsonica, Vernon Hills, IL, USA) for four cycles of 15 s pulses. The suspension was then passed through a 20 um cell strainer (CellTrics 20 um, 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 500-600uL / hr for the dispersed phase and 1000uL / hr for the continuous phase. The generated droplets were collected into a total of two 1.5mL Eppendorf tubes, each of which was covered on top with 50uL of light mineral oil. The tubes were then incubated at 60°C for 18 hours.

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

[0283] Large hydrogel particles were removed by vortexing the Falcon tubes, then the large heavy particles were allowed to settle for approximately 30 seconds, and the supernatant was then transferred to one 15 mL Falcon tube. The thus collected supernatant containing the smaller suspended magnetic hydrogel beads was then collected on a 50 um mesh cell strainer (CellTrics, Sysmex, Kobe, Japan) by transferring the suspension with a syringe. The beads were washed 3x with 1% Pluronic F127 in 1x PBS (3 mL each). The beads were then collected in 1.5 mL Eppendorf tubes by resuspending these beads in 1% Pluronic F127 in 1x PBS, centrifuging, and removing the supernatant as necessary. The average size and %CV were determined by observing the magnetic beads under a microscope using 5 fields at 20x magnification. A total of 46 beads were analyzed, giving an average diameter of 120 μm with 16%CV.

[0284] Example 22: Assessment of Magnetic PEGA Beads The magnetic PEGA beads were then tested for their magnetic properties by placing them on a magnetic rack (MagJET Separation Rack, Thermo Scientific, Waltham, Mass.) By visual inspection, most of the hydrogel had collected on the side of the tube in approximately 3 minutes.

[0285] The above description has been provided with reference to specific embodiments for explanatory purposes. However, the illustrative discussion above is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teachings. The embodiments have been chosen and described in order to best explain the principles of the technology and its practical application. In order to do so, those skilled in the art may best utilize the technology and various embodiments with various modifications as may be appropriate for the particular use contemplated.

[0286] Although the present disclosure and examples have been fully described with reference to the accompanying drawings, it should be noted that various changes and modifications will become apparent to those skilled in the art, and such changes and modifications should be understood to be included within the scope of the present disclosure and examples as defined by the claims.

[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,KK,Brower,KK,Carswell-Crumpton,C.,Klemm,S.,Cruz,B.,Kim,G.,Calhoun,SGK,Nichols,L.,Fordyce,PM,Fordyce,PM,Fordyce,PM,& Fordyce,PM(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, KK, Carswell-Crumpton, C., Klemm, S., Cruz, B., Kim, G., Calhoun, SGK, Nichols, L., & Fordyce, PM (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, HB, Pedelacq, JD, Koraichi, F., Chaudhary, A., Ganguly, K., Lockard, MA, Favre, G., Terwilliger, TC, & Waldo, GS(2013). Reports.https: / / doi.org / 10.1038 / srep02854 Di Carlo,D.,Wu,C.,Destgeer,G.,& Ouyang,M.(2019).PARTICLE-DROP STRUCTURES AND METHODS FOR MAING ANDUSING THE SAME(Application No. 20190381497A1). https: / / doi.org / 10.1021 / acssynbio.7b00111 Duarte, JM, Barbier, I., & Schaerli, Y. (2017).Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries Gai, Y., Kim, M., Pan, M., & Tang, SKY(2017).Amphiphilic nanoparticles suppress droplet break-up in a concentrated emulsion flowing through a narrow constriction 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,A.L.,Fitzgerald,F.G.,Dang,V.Q.,Satz,A.L.,& Paegel,B.M.(2020).Off-DNA DNA-Encoded Library Affinity Screening. ACS Combinatorial Science.https: / / doi.org / 10.1021 / acscombsci.9b00153 Holtze,C.,Rowat,A.C.,Agresti,J.J.,Hutchison,J.B.,Angile,F.E.,Schmitz,C.H.J.,Koester,S.,Duan,H.,Humphry,K.J.,Scanga,R.A.,Johnson,J.S.,Pisignano,D.,& Weitz,D.A.(2008).Biocompatible surfactants for water-in-fluorocarbon emulsions.Lab on a Chip.https: / / doi.org / 10.1039 / b806706f Holtze, Christian, Guerra, R.E., Agresti, J., Weitz, D.A., Ahn, K., HUTCHISON, J.B., Griffiths, A., HARRAK, A. EL, MILLER, O.J., BARET, J.-C., TALY, V., RYCHELLYNCK, M., & MERTEN, C. (2008). Fluorocarbon emulsion stabilizing surfactants (U.S. Patent No. 9,012,390 B2). 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 (U.S. Patent Application Publication No. 20200190507A1). 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 fluorous dispersion oil, said fluorous dispersion oil having an average fluorine content of about 70 wt % or more; a droplet stabilizing agent comprising an emulsifier selected from the group consisting of a triblock copolymer, a diblock copolymer, a fluorinated silica nanoparticle, and combinations thereof; A continuous phase formulation for a stable emulsion comprising:

2. The fluorous dispersion oil is selected from the group consisting of: One or more oils selected from the group consisting of perfluorocarbons and perfluorinated oils, and / or one or more hydrofluoroethers, 2. The continuous phase formulation of claim 1 comprising:

3. the total concentration of the perfluorocarbon and / or the perfluorinated oil in the fluorous dispersion oil is about 50% w / w or more; and / or the concentration of said one or more hydrofluoroethers in said fluorous dispersion oil is less than about 50% w / w; the perfluorocarbon is a perfluoroalkane selected from the group consisting of perfluorooctane, perfluoroheptane, perfluorohexane (FC-72), perfluoro-1,3-dimethyl-cyclohexane, 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), 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); 3. The continuous phase formulation of claim 2.

4. the fluorous dispersion oil consists of perfluorohexane (FC-72), perfluorooctane, and 2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500), each present in an amount ranging from 10 to 90% w / w; The fluorous dispersion oil is selected from the group consisting of: Perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 1:1:1 w / w ratio or about a 1:1:1 w / w ratio, respectively; Perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 2:2:1 w / w ratio or about a 2:2:1 w / w ratio, respectively; and Perfluorohexane (FC-72):perfluorooctane:2-(trifluoromethyl)-3-ethoxydodecafluorohexane (HFE-7500) in a 1:3:1 or about 1:3:1 w / w ratio, respectively; 4. The continuous phase formulation of claim 3, selected from the group consisting of:

5. The triblock or diblock copolymer is Perfluoropolyether (PFPE) and polyethylene glycol (PEG), Two perfluoropolyethers (PFPEs) and polyethylene glycol (PEG), or Two perfluoropolyethers (PFPEs), two polypropylene glycols (PPGs), and a polyethylene glycol (PEG); 5. The continuous phase formulation of claim 4 comprising:

6. a) the diblock copolymer has the formula 【Chemistry 1】 where n and m are the precise or average values ​​of the polydisperse building blocks, and the average molecular weight of the diblock copolymer is 1,000-10,000 Da, n is 35-45, and m is 2-24. and / or b) the triblock copolymer is 【Chemistry 2-1】 where i, j, and k are precise or average values ​​of the polydisperse 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 【Chemistry 3】 where p, q, r, s, and t are precise or average 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 average value of the sum q+s is 3 to 6, and r is 1 to 23.

6. The continuous phase formulation of claim 5 having a formula selected from the group consisting of:

7. the emulsifier comprises a diblock copolymer and a triblock copolymer in a ratio of about 1:1 to 1:9 (w / w), respectively; and the diblock and triblock copolymers are present in the continuous phase formulation at a combined concentration of about 0.3 to 4 wt %; 7. The continuous phase formulation of claim 6.

8. A water-in-oil (w / o) single emulsion comprising an aqueous dispersed phase and a continuous phase according to claim 1, or A water-in-oil-in-water (w / o / w) double emulsion comprising an aqueous dispersed phase and a continuous phase according to claim 1.

9. 9. The water-in-oil single emulsion of claim 8 or the water-in-oil-in-water double emulsion of claim 8, wherein the aqueous phase comprises an assay mixture and barcoded compound beads.

10. the beads are selected from the group consisting of hydrogel beads, magnetic hydrogel beads, divinylbenzene cross-linked polystyrene beads, polyethylene glycol grafted low cross-linked polystyrene matrix, magnetic beads, silica beads, glass beads, and ceramic beads; the beads are core-shell beads comprising polyacrylamide encapsulated beads, the core being a polyethylene glycol grafted low cross-linked polystyrene matrix; or The beads are thin shell beads coated with a hydrophilic polymer and the core is a low cross-linked polystyrene matrix grafted with polyethylene glycol.

10. A water-in-oil single emulsion or a water-in-oil-in-water double emulsion according to claim 9.

11. 11. The water-in-oil single emulsion or water-in-oil-in-water double emulsion of claim 10, wherein the diameter of the beads is about 1-70 microns or the diameter of the beads is up to 70% of the diameter of the droplets.

12. Dispersing a plurality of monomers in an aqueous buffer; combining the aqueous buffer and the plurality of monomers with a continuous phase formulation comprising an oil and an emulsifier, the oil being selected from the group consisting of fluorous oils, hydrocarbon oils, mineral oils, and silicone oils; forming at least one microdroplet from the aqueous buffer and the plurality of monomers; polymerizing the monomer in the at least one microdroplet to form a PEGA copolymer resin; Including, The monomers of the plurality of monomers are: Acrylamide, Bis-acrylamide PEG, and mono-acrylamide PEG with a functionalized handle, and / or mono-acrylamide diamine with a functionalized handle; 1. A method for preparing a monodisperse polyethylene glycol acrylamide (PEGA) copolymer resin, comprising:

13. the at least one microdroplet is formed in a microfluidic device; and forming the at least one microdroplet comprises forming a plurality of monodisperse microdroplets in parallel. The method of claim 12.

14. 14. The method of claim 12 or 13, further comprising coupling a ligand and / or a barcode to the PEGA copolymer resin.

15. 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. The method of claim 14.

16. 16. The method of any one of claims 12 to 15, further comprising embedding magnetic particles in the PEGA copolymer resin by covalent bonding or by physical encapsulation, wherein the magnetic particles have a size of 1 nanometer to 10 microns in diameter.

17. the monodisperse PEGA copolymer resin has a diameter of 1 to 100 microns; or the at least one microdroplet is a plurality of monodisperse microdroplets and the size distribution of the PEGA copolymer resin has a %CV of 5%; The method according to any one of claims 12 to 16.

18. the bis-acrylamide PEG has a precise or average molecular weight of 250 to 5,000 Daltons; and / or the mono-acrylamide PEG has a precise or average molecular weight of 150 to 5,000 daltons; The method according to any one of claims 12 to 17.

19. The bis-acrylamide PEG has the following formula 7: 【Chemistry 6】 (In formula 7, R １ is H or -CH ３ and R ２ is H or -CH ３ and precise or average n １ is 1 to 108) and The mono-acrylamide PEG has the following formula 8: 【Chemistry 7】 (In formula 8, R ３ ~R ６ are independently H or -CH ３ and R ７ is hydrogen, Boc, or Fmoc, and the exact or average n ２ is 1 to 109), or The mono-acrylamide diamine has the formula 9: 【Chemistry 8】 (In formula 9, R ８ and R ９ are independently H or -CH ３ and R １０ is selected from the group consisting of H, Boc, and Fmoc. having the structure The method according to any one of claims 12 to 18.

20. A method for preparing thin shell beads comprising grafting a hydrophilic polymer onto a compound load code core bead.

21. 21. The method of claim 20, wherein the grafted hydrophilic polymer is polyethylene glycol.

22. The method of claim 21, wherein the polyethylene glycol is grafted via strained cyclooctyne-azide click chemistry.

23. 23. The method of claim 21 or 22, wherein the polyethylene glycol is approximately 10,000 daltons or greater.

24. A method for producing a polymerizable composition comprising: 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 a core bead, the at least one microdroplet comprising the at least one core bead; and forming a core-shell bead by polymerizing the monomer in the at least one microdroplet to form a hydrogel encapsulating the bead; Core-shell beads obtainable by a method comprising:

25. A system for performing high throughput screening comprising a continuous phase formulation, a solid support and a sorter, said continuous phase formulation being defined as in claim 1, the solid support is selected from the group consisting of monodisperse polyethylene glycol acrylamide (PEGA) copolymer resins, core-shell beads, and thin-shell beads; 4. The method of claim 1, further comprising: 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 comprising an oil and an emulsifier, the oil being selected from the group consisting of fluorous oils, hydrocarbon oils, mineral oils, and silicone oils; forming at least one first microdroplet from the first aqueous buffer and a first plurality of monomers; and polymerizing the monomers in the at least one first microdroplet to form a PEGA copolymer resin, the monomers of the first plurality of monomers being selected from the following: Acrylamide, Bis-acrylamide PEG, and mono-acrylamide PEG containing a functionalized handle, and / or Mono-acrylamide diamines containing functionalized handles; and Prepared by and The core-shell beads are 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 a core bead, the at least one second microdroplet comprising the at least one core bead; and forming a core-shell bead by polymerizing the monomer in the at least one second microdroplet to form a hydrogel encapsulating the core bead; Prepared by or The thin shell beads are Grafting a hydrophilic polymer onto the core beads Prepared by A system for performing high throughput screening.

26. A system for performing high throughput screening according to claim 25, comprising: The continuous phase formulation comprises: at least one fluorous dispersion oil, said fluorous dispersion oil having an average fluorine content of about 70 wt % or more; a droplet stabilizing agent comprising an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof; the solid support is selected from the group consisting of monodisperse polyethylene glycol acrylamide (PEGA) copolymer resins, core-shell beads, and thin-shell beads; i) the monodisperse polyethylene glycol acrylamide (PEGA) copolymer resin comprises: 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 comprising an oil and an emulsifier, the oil being selected from the group consisting of fluorous oils, hydrocarbon oils, mineral oils, and silicone oils; forming at least one first microdroplet from the first aqueous buffer and a first plurality of monomers; and polymerizing the monomers in the at least one first microdroplet to form a PEGA copolymer resin, the monomers of the first plurality of monomers being selected from the following: Acrylamide, Bis-acrylamide PEG, and mono-acrylamide PEG with a functionalized handle, and / or mono-acrylamide diamine with a functionalized handle; and Prepared by and ii) the core-shell beads are 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 a core bead, the at least one second microdroplet comprising the at least one core bead; and forming a core-shell bead by polymerizing the monomer in the at least one second microdroplet to form a hydrogel encapsulating the core bead; Prepared by or The thin shell beads are Grafting a hydrophilic polymer onto the core beads Prepared by and The sorter comprises: An inlet channel; first and second outlet channels that merge into the inlet channel at a junction; first and second electrodes proximate first and second sides, respectively, of the junction, the first and second electrodes comprising: a first state in which the first electrode receives a greater voltage than the second electrode, thereby causing one or more target compositions to flow through the junction and into the first outlet channel; a second state in which the second electrode receives a greater voltage than the first electrode, thereby causing one or more target compositions to flow through the junction and into the second outlet channel; An electrode configured to have a controller configured to switch the first electrode and the second electrode between the first state and the second state; Including, the system.

27. A system for performing high throughput screening according to claim 25, comprising: The continuous phase formulation comprises: at least one fluorous dispersion oil, said fluorous dispersion oil having an average fluorine content of about 70 wt % or more; a droplet stabilizing agent comprising an emulsifier selected from the group consisting of triblock copolymers, diblock copolymers, fluorinated silica nanoparticles, and combinations thereof; the solid support is selected from the group consisting of monodisperse polyethylene glycol acrylamide (PEGA) copolymer resins, core-shell beads, and thin-shell beads; i) the monodisperse polyethylene glycol acrylamide (PEGA) copolymer resin comprises: 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 comprising an oil and an emulsifier, the oil being selected from the group consisting of fluorous oils, hydrocarbon oils, mineral oils, and silicone oils; forming at least one first microdroplet from the first aqueous buffer and a first plurality of monomers; and polymerizing the monomers in the at least one first microdroplet to form a PEGA copolymer resin, the monomers of the first plurality of monomers being selected from the following: Acrylamide, Bis-acrylamide PEG, and mono-acrylamide PEG with a functionalized handle, and / or mono-acrylamide diamine with a functionalized handle; and Prepared by and ii) the core-shell beads are 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 a core bead, the at least one second microdroplet comprising the at least one core bead; and forming a core-shell bead by polymerizing the monomer in the at least one second microdroplet to form a hydrogel encapsulating the core bead; Prepared by or The thin shell beads are Grafting a hydrophilic polymer onto the core beads Prepared by and The sorter comprises: 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 a desired droplet; An automated microcapillary-based droplet sampling device configured to sequentially select a plurality of desired droplets and deposit them into hit wells.

Images