WO2024091949A1 - Procédés et systèmes de criblage d'effecteur codé - Google Patents

Procédés et systèmes de criblage d'effecteur codé Download PDF

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WO2024091949A1
WO2024091949A1 PCT/US2023/077649 US2023077649W WO2024091949A1 WO 2024091949 A1 WO2024091949 A1 WO 2024091949A1 US 2023077649 W US2023077649 W US 2023077649W WO 2024091949 A1 WO2024091949 A1 WO 2024091949A1
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Prior art keywords
bead
effector
barcode
optical
examples
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PCT/US2023/077649
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English (en)
Inventor
Ramesh Ramji
Shahed KAY
Warren Stanfield Wade
Andrew Boyd MACCONNELL
Devon Michael CAYER
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1859, Inc.
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Publication of WO2024091949A1 publication Critical patent/WO2024091949A1/fr

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  • the present disclosure provides more efficient and higher-throughput sample screening methods and systems.
  • SUMMARY [0004] Presented herein are methods and systems for miniaturized high-throughput screening of encoded effector libraries using miniaturized compartmentalized systems.
  • the methods comprise providing or obtaining encoded effectors. Encoded effectors may be bound to solid supports.
  • the methods and systems presented herein further facilitate screening encoded effectors against one or more targets, in some cases, by performing assays, for the effects of the effectors on the targets to be assessed and/or profiled.
  • the methods and systems have vast applications in drug discovery, diagnostics, clinical applications, and beyond.
  • a bead comprising: a substantially homogeneous bead polymer material; an effector; and a barcode corresponding to and identifying the effector, wherein the bead is a spherical or semi-spherical bead made of the substantially homogenous polymer material, wherein the homogeneous polymer material is compatible with solid phase peptide synthesis (SPPS), and wherein the effector is synthesized on the bead.
  • SPPS solid phase peptide synthesis
  • the bead does not comprise a solid core made of a material different from the substantially homogeneous bead polymer material.
  • the bead does not comprise a core-shell structure.
  • the bead is a sphere, the sphere comprises an inner core and a peripheral section, and the inner core and the peripherical section are substantially composed of the same material.
  • the inner core is a polymer material.
  • the bead does not comprise a polystyrene core.
  • the inner core is a substantially soft material.
  • the inner core comprises functional groups.
  • the bead comprises functional groups for synthesizing effectors.
  • the substantially homogenous polymer material comprises functional groups for synthesizing effectors.
  • the inner core comprises functional groups for synthesizing effectors.
  • the functional groups for synthesizing effectors comprise amines.
  • amines may be protected by a protecting group. Examples of protecting groups may comprise F-moc, BOC, Azide, or Carbamide.
  • the bead further comprises functional groups for oligonucleotide synthesis.
  • the bead further comprises functional groups for oligonucleotide synthesis.
  • the functional groups for oligonucleotide synthesis comprise at least about 10 5 molecules per bead.
  • the functional groups for oligonucleotide synthesis comprise Azide.
  • the bead comprises an inner portion and an external surface, wherein the functional groups for oligonucleotide synthesis are substantially located on or near the external surface, and wherein the functional groups for synthesizing the effector are substantially near or inside the inner portion.
  • the barcode comprises one or more optical barcoding particles on the surface of the bead or inside the bead.
  • the optical barcoding particles comprise spectral properties in the short-wave infrared (IR) range.
  • the optical barcoding particles comprise a surface.
  • the optical barcoding particles comprise amphiphilic surfaces.
  • the surfaces of the optical barcoding particles comprise a surface coating.
  • the surface coating comprises Si.
  • the optical barcoding particle is a cylindrical optical barcoding particle. In some embodiments, the height of the cylindrical optical barcoding particle is at most about 0.5 ⁇ m. In some embodiments, the diameter of the cylindrical optical Attorney Docket No.56523-707.601 barcoding particle is at most about 5 ⁇ m. In some embodiments, the diameter of the cylindrical optical barcoding particle is at most about 2 ⁇ m. In some embodiments, the optical barcoding particle is detectable by fluorescence or luminescence [0011] In some embodiments, the optical barcoding particle comprises an excitation wavelength of from about 1000 to about 1100 nanometers (nm).
  • the optical barcoding particle comprises an excitation wavelength of from about 1060 to about 1070 nanometers (nm). In some embodiments, the optical barcoding particle comprises an emission wavelength of from about 1000 to about 2000 nanometers (nm). [0012] In some embodiments, the optical barcode comprises at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 unique spectral emissions. In some embodiments, the optical barcode comprises an emission bandwidth of less than about 3 nanometers (nm). In some embodiments, the optical barcode comprises an emission bandwidth of at most about 0.5 nm.
  • the optical barcode comprises at least about 1 million, 2 million, 10 million, 20 million, 30 million, 40 million, 40 million, 50 million, 80 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 10 billion, 20 billion, 30 billion, 40 billion, 50 billion, 60 billion, 70 billion, 80 billion, 100 billion, 200 billion, 300 billion, 400 billion, 500 billion, 600 billion, 700 billion, 800 billion, 900 billion, 1 trillion, 2 trillion, 3 trillion, or more unique optical signatures.
  • the volume of the optical barcode is at most about 0.05% of the volume of the bead.
  • the optical barcode comprises at least 1, 2, 3, 4, 5, or 6 optical barcoding particles.
  • the barcode comprises a nucleic acid molecule covalently bound to the bead or trapped inside the bead.
  • the barcode comprises a DNA, an RNA, a peptide, or a peptide nucleic acid (PNA).
  • the bead further comprises an optical barcode thereon or therein.
  • the bead remains substantially structurally intact during solid phase peptide synthesis (SPPS). In some embodiments, the bead remains substantially structurally intact during and after suspension in an organic solvent.
  • the bead diameter is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 micrometers ( ⁇ m) in water. In some embodiments, a coefficient of variation (CV%) of the diameter among a population of the beads is lower than about 20%. In some embodiments, the bead is encapsulated or compartmentalized in a compartment among a plurality of compartments. Attorney Docket No.56523-707.601 [0016] In some embodiments, the substantially homogeneous bead polymer material comprises a plurality of crosslinker monomers. In some embodiments, the plurality of crosslinker monomers is or comprises a monomer of the following structure: , where n is any integer.
  • the substantially homogeneous bead polymer material comprises a plurality of spacer monomers.
  • the plurality of spacer monomers is or comprises a monomer of the following structure: any integer.
  • the substantially homogeneous bead polymer material comprises a plurality of functional group monomers.
  • the plurality of functional group monomers is or comprises a monomer of the following structure: any integer.
  • the plurality of crosslinker monomers, the plurality of spacer monomers, and/or the plurality of functional group monomers comprise an amide.
  • the plurality of crosslinker monomers and/or the plurality of spacer monomers comprise a polyethylene glycol (PEG) group.
  • a method of screening an encoded effector comprising: (a) providing or obtaining a bead comprising an effector and a barcode corresponding to the effector, wherein the bead is made of a substantially homogeneous polymer resin; (b) encapsulating the bead in a compartment; (c) detecting a signal from the compartment; and (d) processing the compartment, the bead, or the barcode, based on the signal or a change thereof.
  • the compartment is a droplet or a well.
  • the effector is bound to the scaffold via a cleavable linker and is releasable upon cleavage of the cleavable linker
  • the method comprises exposing the bead to a stimulus to cleave the cleavable linker and release the effector into the compartment.
  • the compartment further comprises an assay reagent and a target, and the Attorney Docket No.56523-707.601 signal is indicative of the activity of the target in presence of the effector, as measured using the assay reagent.
  • a system comprising: a solid support comprising (a) an effector bound to the solid support via a cleavable linker, wherein the effector is releasable from the solid support upon cleavage of the cleavable linker; and, (b) one or more encoding particles embedded inside or on the surface of the solid support corresponding to and identifying the effector, wherein the one or more encoding particles have spectral properties in the short-wave infrared (IR) range.
  • the encoding particles comprises a spectral emission bandwidth of at most about 100 nm.
  • the solid support comprises a particle.
  • the particle comprises a diameter of at most about 30 micrometers ( ⁇ m).
  • the solid support comprises or is a bead.
  • solid support is encapsulated in a compartment.
  • the compartment comprises or is a droplet, a well, a nanopen, or a miniaturized channel.
  • the compartment is a droplet surrounded by an immiscible oil.
  • the droplet is generated with the aid of a droplet microfluidic device, and the system further comprises the droplet microfluidic device.
  • the compartment is a well in a miniaturized array platform.
  • the compartment is a microfluidic or miniaturized compartment comprising four sides, wherein the four sides comprise three closed sides and one open side.
  • a screening method comprising: (a) providing or obtaining a bead, wherein the bead comprises: (i) an effector bound to the bead via a cleavable linker, wherein the effector is releasable from the bead upon cleavage of the cleavable linker, thereby generating a released effector; (ii) one or more encoding particles embedded inside or on the surface of bead corresponding to and identifying the effector, wherein the one or more encoding particles comprise spectral properties in the short-wave infrared range and a spectral signature corresponding to the effector and identifying it.
  • the method further comprises (b) detecting a first signal indicative of the activity of the target in presence of the released effector; and, (c) detecting a second signal indicative of the spectral signature of the encoding particles.
  • the encoding particles comprise a spectral emission bandwidth of at most about 3 nm.
  • the first signal and the second signal are spectrally independent.
  • the first signal is in the visible range.
  • the second signal is not in the visible range.
  • the second signal is in the short-wave infrared range.
  • a bead comprising (i) an effector bound to the bead via a cleavable linker, wherein the effector is releasable from the bead upon cleavage of the cleavable linker; and, (ii) one or more encoding particles embedded inside or on the surface of the bead corresponding to and identifying the effector, wherein the one or more encoding particles have spectral properties in the short-wave infrared (IR) range.
  • IR short-wave infrared
  • FIGs.1A-1C schematically illustrate various embodiments of bead-bound encoded effectors using different encoding modalities such as optical barcodes, nucleic acid barcodes, and combinations thereof.
  • FIG. 2A provides a depiction of a library of encoded effector beads, wherein the effector is a fluorophore bound to the bead via a cleavable linker, also referred to as an effector- fluorophore.
  • FIG. 2B schematically illustrates encapsulating an encoded effector bead in a droplet compartment, wherein the effector is a fluorophore bound to the bead via a photocleavable inker (effector-fluorophore).
  • the workflow illustrates releasing the effector- fluorophore from the bead into the droplet upon photocleavage of the photocleavable linker by exposing the droplet to UV light.
  • FIG. 2C provides a depiction of the released encoded effector-fluorophore from FIG.2B.
  • FIG. 2D provides a depiction of the cleavage region or exposure region of a microfluidic device described herein.
  • a UV waveguide is inserted into a channel to expose droplets passing through the exposure region to UV light.
  • FIGs. 2E and 2F provide exemplary chemical reactions for activating molecules for photocleavage.
  • FIG. 3A provides an exemplary core-shell bead comprising an inner core and a peripheral section, wherein the inner core (e.g., a polystyrene core) is made of a material different from the peripheral section (e.g., a hydrogel such as polyethylene glycol).
  • the peripheral section of the bead comprises at least one type of functional groups for chemical synthesis.
  • FIG.3B provides an exemplary homogeneous bead comprising an inner core and a peripheral section, wherein the inner core and the peripheral section are made of the same material (e.g., a hydrogel), and wherein both the inner core and the peripheral section may each comprise at least one type of functional group for chemical synthesis.
  • Each type of functional group may be configured to be attached to a barcode and/or an effector.
  • FIG. 4 schematically illustrates an exemplary droplet microfluidic device for screening.
  • FIG. 5 provides an exemplary workflow for encoded effector screening using a droplet microfluidic platform.
  • FIG.6 provides an exemplary workflow for screening using optically or spectrally encoded beads or One Bead One Compound Spectrally Encoded Library Screening (OBOC- SEL) and decoding on the fly (DOTF) without the need for a physical sorting step through which structure activity relationship (SAR) datasets can be acquired.
  • FIG. 7 provides another exemplary workflow for screening One Bead One Compound Spectrally Encoded Library Screening (OBOC-SEL) and decoding on the fly (DOTF) without the need for a physical sorting step through which structure activity Attorney Docket No.56523-707.601 relationship (SAR) datasets can be acquired.
  • FIG. 8 provides another exemplary workflow for performing cocktail assays (testing one or more effectors/compounds) simultaneously in the same compartment on the same sample/target, wherein the compartment comprises one or more spectrally/optically encoded effector beads the synergistic effects of which on the same sample is being tested.
  • the bead may further comprise a bead barcode (e.g., an optical barcode such as a fluorophore/dye) allowing for bead localization (e.g., on time trace signals acquired) and counting and identifying that the effectors came from the same or different beads (multi-bead decoding).
  • a bead barcode e.g., an optical barcode such as a fluorophore/dye
  • FIG.9 illustrates an exemplary workflow of a split-and-pool method for generating spectrally/optically encoded effector libraires.
  • FIG. 10 illustrates an exemplary workflow of using a Bead Index Registry and Dispensing System (BIRDS) for generating beads pre-encoded with optical barcodes.
  • BIRDS Bead Index Registry and Dispensing System
  • FIG.11 schematically illustrates generating beads of the present disclosure using a droplet microfluidic device.
  • the beads may be according to any bead embodiment described in the disclosure
  • FIG.12 illustrates a bead-generation approach in which optical barcodes comprise a surface modification.
  • the optical barcoding particles are encapsulated in a monomer mixture for bead generation and driven to the surface of the bead by interfacial tension. This method localizes optical barcodes near the surface of the beads.
  • FIG.13 illustrates a bead-generation approach in which optical barcodes comprise a surface modification which renders their surfaces amphiphilic.
  • the optical barcoding particles are encapsulated in a monomer mixture for bead generation and driven to the surface of the bead by interfacial tension.
  • the bead comprises an inner core with a material different from its peripheral section.
  • the bead comprises a core-shell structure.
  • the core and the shell are immiscible phases. This method localizes optical barcodes near the surface (e.g., in the peripheral section) of the beads.
  • FIGs.14A-14C provide, respectively, view from the side, view from the top, and three-dimensional view of an exemplary miniaturized array for sample screening.
  • FIG. 15 schematically illustrates an exemplary workflow of a method for surface treatment of a miniaturized array platform to facilitate cell seeding or cell adhesion.
  • FIG. 16 depicts a miniaturized array comprising a plurality of wells each comprising a plurality of cells seeded therein and adhered to the bottom surface of the well.
  • FIG.17A depicts a flow cell comprising a plurality of wells immobilized on a solid substrate, each of the wells comprising one or more cells seeded therein, adhered to the bottom surface of the wells, and the direction of fluid flow into and out of the flow cell.
  • FIG. 16 depicts a miniaturized array comprising a plurality of wells each comprising a plurality of cells seeded therein and adhered to the bottom surface of the wells, and the direction of fluid flow into and out of the flow cell.
  • FIG. 17B schematically illustrates a plurality of miniaturized flow cells (e.g., similar to the flow cell shown in FIG. 17A).
  • the flow cells are immobilized on a solid substrate, each of the flow cells comprises one or more inlet and outlet port(s) connected to fluid lines such as tubes.
  • FIG. 18 illustrates an exemplary method for amplifying a primer to maximize cellular nucleic acid capture.
  • FIGs. 19A and 19B provide exemplary data demonstrating condensate/stress granule (SG) formation through liquid-liquid phase separation (LLPS) leading to fluorescence signal redistribution in space as detected from a static compartment via fluorescence microscopy using the methods and systems of the present disclosure.
  • SG condensate/stress granule
  • LLPS liquid-liquid phase separation
  • FIG.19C schematically illustrates LLPS and condensate formation in an assay.
  • FIG. 20 provides exemplary data demonstrating condensate/stress granule (SG) formation through liquid-liquid phase separation (LLPS) leading to fluorescence signal redistribution in space as detected from a droplet compartment via laser-induced fluorescence spectroscopy in a system comprising a droplet microfluidic device.
  • FIGs.21A and 21B provide an example of condensate detection and quantification (counting) using the methods and systems of the present disclosure.
  • FIGs. 22A and 22B provide examples of substantially homogeneous bead co- polymer material that can be used to construct the polymer beads disclosed herein.
  • FIG.22A demonstrates that the spacer and crosslinker monomer stoichiometry can be adjusted to alter the physical characteristics of the polymer bead.
  • FIG.22B demonstrates that the crosslinker length can be varied to alter density, swelling, and physical integrity of the polymer bead.
  • FIG.23A depicts images of exemplary polymer beads of the disclosure.
  • FIG.23B depicts the distribution of bead diameters of exemplary polymer beads of the disclosure.
  • FIG. 24 depicts images of exemplary polymer beads of the disclosure, swelled in different solvents.
  • a sample may comprise a biological/biochemical target, a cell, one or more cellular constituents inside cells or extracted from cells (e.g., cell lysates), deoxyribonucleic-acid (DNA), ribonucleic acid (RNA), messenger RNA, proteins, enzymes, cell-free samples, or other kinds of targets.
  • a biological/biochemical target e.g., cell lysates
  • DNA deoxyribonucleic-acid
  • RNA ribonucleic acid
  • messenger RNA e.g., messenger RNA, proteins, enzymes, cell-free samples, or other kinds of targets.
  • the methods may comprise screening the effects of one or more effectors against one or more samples in a high-throughput, low-material manner.
  • the methods and systems provided herein comprise devices such as miniaturized screening platforms, screening instrumentation (e.g., hardware), computer systems, software programs, reagents, and workflows, which may be used individually or in concert (e.g., using an integrated platform and workflow), to facilitate screening the samples which may in some cases comprise cells.
  • screening platforms may comprise droplet microfluidic devices.
  • screening platforms may comprise a plurality of wells (e.g., a well array system).
  • Sample screening may be performed for any application, in some cases, for diagnostics, drug discovery and/or development, or various combinations of both such as personalized medicine, precision medicine, and beyond.
  • sample screening may be performed for drug discovery purposes. For example, to screen one or more drugs, or a library of effectors on one or more samples comprising one or more targets such as to screen effectors to discover drug candidates (e.g., effectors) which may have an intended effect on the target.
  • the sample may be compartmentalized into the compartments of a system (e.g., wells of a plate and/or an array) or into compartments generated by a system (e.g., droplets generated by a microfluidic device or through bulk emulsification).
  • the methods and systems provided herein may be particularly useful for screening encoded effector libraries against targets in miniaturized systems such as droplet microfluidics and/or well array platforms.
  • screening may comprise high- throughput screening wherein large numbers of effectors are screened in a shorter period compared to preceding technologies.
  • the present disclosure provides methods and systems for screening encoded effector libraries in miniaturized and compartmentalized platforms.
  • Encoded effector libraries may be according to any encoded effector library described Attorney Docket No.56523-707.601 anywhere herein.
  • the miniaturized and compartmentalized screening system may be any screening system described anywhere herein which may comprise a droplet microfluidic device, a miniaturized well array platform, or another platform/device comprising a plurality of discrete or semi-discrete compartments/partitions described anywhere herein.
  • the present disclosure provides a set of bioanalytical toolkits which can be used individually and/or in various combinations to achieve various goals.
  • Encoded libraries may comprise bead-bound encoded effector (e.g., chemical effector, molecular effectors, compound/small molecule, peptide, macrocyclic molecules, polymers, RNA, DNA, genes or other types of effectors) libraries in which scaffolds such as beads are used as synthetic substrates and carriers for immobilizing, delivering, locating, tracking, extracting, or otherwise manipulating effectors in order to test or measure their effect on a sample or a target. Effectors and barcodes can be attached to the bead or encapsulated therein to be linked together in space, such that the barcode can provide information regarding the identity and/or the structure of the effector. The effector and barcode may be directly attached to one another.
  • an effector e.g., chemical effector, molecular effectors, compound/small molecule, peptide, macrocyclic molecules, polymers, RNA, DNA, genes or other types of effectors
  • scaffolds such as beads are used as synthetic substrates and carriers for im
  • the effector and barcode may be immobilized on the bead but not necessarily bound or attached to one another.
  • the scaffold e.g., bead resin
  • effector, and barcode may have many different modalities, versions, and embodiments, as described anywhere herein, which may be mixed and matched together and with the various embodiments of the screening/detection system to perform the methods of the present disclosure based on an intended application.
  • a scaffold e.g., a bead
  • a scaffold may comprise on or more effectors bound thereto.
  • the one or more effectors may be similar or different.
  • a scaffold may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different effectors attached thereto.
  • a scaffold e.g., a bead
  • the scaffold may further comprise one or more barcodes.
  • the one or more barcodes may be encoding the structures and/or identities of effector A and effector B.
  • the barcode may comprise one or more sequences each of which may encode each of the effectors. This embodiment may be referred to as “combinatorial effector screening”.
  • the methods provided herein may comprise screening encoded effector libraries which comprise a scaffold such as a bead.
  • the bead/scaffold may comprise a bead resin (e.g., hydrogel bead, core-shell bead, TentaGel bead, or any other suitable bead).
  • the bead may Attorney Docket No.56523-707.601 further comprise an effector covalently bound to the bead via a cleavable linker and a barcode (oligonucleotide such as DNA or RNA, peptide, peptide nucleic acid (PNA), one or more optical barcodes, or any combination thereof) corresponding to and for identifying the effector.
  • a barcode oligonucleotide such as DNA or RNA, peptide, peptide nucleic acid (PNA), one or more optical barcodes, or any combination thereof
  • the barcode may also be linked to the bead via a cleavable linker. In some cases, the barcode may be inside the bead. In some cases, the barcode may comprise or be an optical barcode (e.g., one or more fluorophore(s)/dye, optical particles, and/or both). In some examples, the effector on the bead can be released (e.g., selectively released) to assay its activity against a sample or target. In some examples, the target of a screen may be a protein contained in either a biochemical assay or expressed by a cell. [00060] Screening encoded effector libraries against targets present in live cells may be challenging.
  • the methods presented herein provide a plurality of miniaturized compartments/partitions such as droplet-based systems or arrays of wells (e.g., miniaturized wells, microwells, nanowells, picowells, or wells of any size).
  • the plurality of compartments may comprise a plurality of droplets, a plurality of wells, a plurality of fabricated pens (e.g., nano pens), a plurality of miniaturized constructs of miniaturized size, nanovials, container particles (e.g., lab on a particle nanovials).
  • the effective concentration of compounds released from a single bead of a given size inside the assay compartment can be increased (e.g., compared to releasing the contents of the same scaffold into a larger compartment). Additionally, the number of assay conditions that can be performed in a unit area can increase by miniaturization of the screening platform.
  • a miniaturized droplet-based microfluidic platform may be used to perform the screens.
  • platforms such as array-based platforms, well- based platforms, micro-raft arrays, and other compartmentalized screening platforms may be used.
  • Some cell types may be more suitably screened while adhered Attorney Docket No.56523-707.601 to a solid surface in presence of specific adhesion/signaling molecules or receptors. Such cells may function more properly when adhered to a solid/semi-solid surface. Such condition may be facilitated by seeding the cells in a static compartment such as a well.
  • a hydrogel matrix may be used to encapsulate the cell inside a well and/or in a droplet (e.g., in a microfluidic device) to provide a natural micro-environment for the cell.
  • a hydrogel matrix may provide support for cell adhesion in a screening platform.
  • an artificial tumor spheroid may be generated.
  • one or more cells e.g., suspension cells or adherent cells
  • the polymerizable monomer may be subjected to polymerization and gelation/solidification. This may generate an artificial tumor microenvironment made of a hydrogel material surrounding the cells suspended therein and may be referred to as a tumor spheroid.
  • the tumor spheroid may then be compartmentalized into a plurality of compartments such as droplets, wells, or any other compartment described anywhere herein.
  • the encoded effectors may be screened against the tumor spheroids.
  • the cells may be seeded on the surface of a hydrogel.
  • the cells seeded inside a hydrogel or on its surface may grow over time.
  • more than one cell type may be co-cultured using the described methods and systems and may be perturbed and/or screened.
  • the methods presented herein may further comprise or be useful for phenotypic image-based analysis and intracellular measurements and observations via live cell microscopy (e.g., high resolution fluorescence microscopy or confocal microscopy).
  • one or more beads can be encapsulated in a compartment such as a well with a cell or a population of cells.
  • the bead (scaffold) may comprise an effector (e.g., compound).
  • the effector can be released from the bead into the solution inside the compartment and interact with the cell(s).
  • the cell response to the effector can be measured to determine the potential effect of the released effector on the cell and/or a target therein.
  • the effector may perturb the cell, and the cell may be screened in presence and absence of the perturbation.
  • Perturbation may be at a genomic level, transcriptome level, translational level, protein function level, morphological level, or secretion, functional level, or any combination thereof.
  • perturbation may comprise gene therapy.
  • Perturbation may comprise perturbing transfection, translation, differentiation, homeostasis, spatial reorganization of the contents of the cell, cellular phenotype, or any combination thereof.
  • the methods of the present disclosure comprise performing an assay in a plurality of compartments and measuring a signal indicative of an effect (e.g., perturbation) of an effector on the sample or the contents of the wells which may comprise a cell or constituents Attorney Docket No.56523-707.601 of a cell (e.g., cell lysates or intracellular components, elements, organelles, molecules, or beyond).
  • a threshold may be defined to identify a condition or set of conditions as defined criteria for denoting an effector as a hit.
  • the hits or the population having defined criteria may be processed and/or sorted according to the signal at some point during or after a screen.
  • a threshold may not necessarily be defined for the signal.
  • Signals may be measured for a subset of, many, most, or all of the compartments of the screening platform and/or any contents therein (e.g., one or more scaffolds in the compartment, cells, particles, encoded effector), the data may be aggregated.
  • a plurality of signals such as images or digital/analog signals may be collected and/or aggregated from the compartments using computer systems, detectors, signal detection devices, hardware, and software.
  • hit definition or identification e.g., the conditions to be called hits, such as to have an effect on a sample, such as a cell, constituent of a cell, or a target in the sample or in the cell
  • the effects of the effectors encapsulated or trapped in the plurality of the compartments measured may be mapped for at least a subset of the compartments (e.g., wells), most, or all of the compartments.
  • a plurality of scaffolds e.g., beads or any other kind of scaffold or solid support mentioned anywhere herein
  • a plurality of scaffolds may be encapsulated or otherwise localized, loaded, dispensed (manually or automatically by a person, machine, or robot, or placed in a plurality of compartments according to any compartment described anywhere herein, such as droplets, wells, rafts, encapsulations, channels, or microfluidic confinements.
  • the compartment may further comprise a sample, a target, a reagent, assay probes, fluorophores, and any other components which may facilitate screening the sample/target in presence and/or absence of the effector(s)s.
  • the library of unique encoded effectors may comprise a predetermined number of unique and/or different encoded effectors (e.g., effectors with different structures and/or chemical properties and features). The different structure of the effector may lead to a different effect on the assay or the target. The number of unique encoded effector libraries may be designed according to the applications and objectives to be accomplished.
  • the library may comprise at least 2, 3, 4, 5, 6, 7, 89, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, Attorney Docket No.56523-707.601 200, 300, 400, 500, 600, 700, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 10000 or more unique encoded effectors.
  • the library may comprise at least 10 4 , 2 x 10 4 , 3 x 10 4 , 4 x 10 4 , 5 x 10 4 , 6 x 10 4 , 7 x 10 4 , 8 x 10 4 , 9 x 10 4 , or more unique encoded effectors.
  • the library may comprise at least 10 5 , 2 x 10 5 , 3 x 10 5 , 4 x 10 5 , 5 x 10 5 , 6 x 10 5 , 7 x 10 5 , 8 x 10 5 , 9 x 10 5 , or more unique encoded effectors.
  • the library may comprise at least 10 6 , 2 x 10 6 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 6 x 10 6 , 7 x 10 6 , 8 x 10 6 , 9 x 10 6 , or more unique encoded effectors.
  • the library may comprise at most 9 x 10 7 , 8 x 10 7 , 7 x 10 7 , 6 x 10 7 , 5 x 10 7 , 4 x 10 7 , 3 x 10 7 , 2 x 10 7 , 10 7 or less unique encoded effectors.
  • the library may comprise at most 9 x 10 6 , 8 x 10 6 , 7 x 10 6 , 6 x 10 6 , 5 x 10 6 , 4 x 10 6 , 3 x 10 6 , 2 x 10 6 , 10 6 or less unique encoded effectors.
  • the library may comprise 9 x 10 5 , 8 x 10 5 , 7 x 10 5 , 6 x 10 5 , 5 x 10 5 , 4 x 10 5 , 3 x 10 5 , 2 x 10 5 , 10 5 or a smaller number of unique encoded effectors.
  • the library may comprise 9 x 10 4 , 8 x 10 4 , 7 x 10 4 , 6 x 10 4 , 5 x 10 4 , 4 x 10 4 , 3 x 10 4 , 2 x 10 4 , 10 4 or less unique encoded effectors.
  • the library may comprise 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, or a smaller number of unique encoded effectors.
  • the encoded effector libraries are described in detail throughout the present disclosure.
  • the unique encoded effectors may be bound to solid supports or scaffolds (e.g., bead resin) via a cleavable linker.
  • the encoded effectors libraries may comprise or be a One Bead One Compound (OBOC) encoded effector library, such that each scaffold comprises a unique effector bound to the bead via a cleavable linker and releasable from the bead upon cleavage of the cleavable linker and encoded with an encoded on or in the scaffold which corresponds to and identifies the effector (e.g., as shown FIGs.1A-1C).
  • FIGs. 1A- 1C schematically illustrate exemplary embodiments of bead-bound encoded effectors. Encoding may comprise a variety of modalities as described anywhere herein.
  • FIG.1A illustrates a bead-bound encoded effector 100 which comprises a solid support 101 (in this case a scaffold, bead, or polymer bead resin according to any bead embodiment presented anywhere herein).
  • the bead comprises a nucleic acid barcode 102, which is covalently attached to the scaffold.
  • the nucleic acid barcode 102 comprises barcode subunits A, B, and C.
  • the barcode subunits correspond with effector subunits A, B, and C, which make up effector 103.
  • the effector 103 is linked to the bead 101 through a linker 104.
  • the linker 104 may be a cleavable linker, such a linker cleavable by electromagnetic radiation Attorney Docket No.56523-707.601 (photocleavable) or selectively cleavable by a cleaving reagent (chemically cleavable).
  • Cleavable linkers can be used to liberate effectors from a bead or other scaffold to allow the effector to interact with a sample (e.g., in a compartment).
  • FIG.1B illustrates a bead-bound encoded effector 110 comprising a solid support or bead 101, an effector 103 bound to the bead via a cleavable linker 104 and releasable upon cleavage of the cleavable linker.
  • the bead further comprises a nucleic acid barcode 102 corresponding to and identifying the effector 103.
  • the nucleic acid barcode may also be capable of capturing cellular nucleic acid molecules (e.g., mRNA) from cells in the sample as described elsewhere herein.
  • the bead 101 further comprises one or more optical barcoding particles 105 which together or collectively (in combination) comprise a unique optical or spectral signature corresponding to and identifying the effector 103.
  • the correspondence and identification may be facilitated during the synthesis of the encoded effector.
  • the barcodes e.g., the nucleic acid barcode, the optical barcode or both
  • the barcodes may encode a plurality of synthesis steps of the effector.
  • a database or manifest may be generated to record the synthesis steps of the effector and subunits thereof and may thereby be linked with the barcode subunits.
  • subunits may be sequences included in the nucleic acid barcode.
  • subunits of the barcode may be considered to be individual optical barcoding particles which together (in combination with one another) may make up a unique optical/spectral signature corresponding to and identifying the effector.
  • the bead may only comprise the optical barcoding particles as barcode(s) and not the nucleic acid barcode.
  • the optical barcode may be an alternative to the nucleic acid barcode. An example of this is shown in FIG.1C in which the bead-bound encoded effector 120 comprises optical barcoding particles 105 encoding the effector 103 but not a nucleic acid barcode.
  • An optical or spectral particle may comprise or be any particle made of any suitable material in any state of matter (e.g., solid, liquid, or beyond) which may be capable of receiving and/or responding to a stimulus (e.g., an energy such as electromagnetic energy/radiation, electromagnetic wave, light, heat, or another form of energy), for example to be excited, stimulated, and/or resonated, go through an increase in internal energy states and/or atomic vibrational states within the particle (e.g., due to the stimulus), and in some cases, to emit an optical or spectral signal as a result.
  • a stimulus e.g., an energy such as electromagnetic energy/radiation, electromagnetic wave, light, heat, or another form of energy
  • the optical or spectral signal emitted by an optical/spectral particle may form a unique optical/spectral encoding signature which can encode one or more effector(s), correspond to and identify it.
  • Attorney Docket No.56523-707.601 Correspondence and identification may relate to a structure, a concentration, a plurality of synthesis steps, cleavage dose/concentration, and other information about the effector (e.g., during a screen on an example target/sample).
  • the optical/spectral properties of the optical encoding particles and the signal emitted therefrom may comprise any suitable properties and characteristics involving any suitable mechanisms.
  • the optical barcode may get excited.
  • the particle may resonate to generate a signal.
  • the particle may be stimulated or excited by an energy source (electromagnetic waves, heat, acoustic) and resonate this energy internally.
  • the internal resonance is maintained through electron orbital conjugation, or through whispering gallery modes (WGMs).
  • WGMs whispering gallery modes
  • the particle may dissipate energy through vibration, mechanical forces, or quantum tunneling effects.
  • the particle may emit electromagnetic waves through collapse of resonance, or through a lasing cavity.
  • the energy of the emitted or lased electromagnetic waves is less than the energy of the stimulus.
  • the direction of photon emission depends on the orientation of the optical particle.
  • the signal from the optical barcoding particle may comprise a directionality or may be generated at a given direction.
  • the direction of the emitted signal from an optical particle may depend on the material properties of the optical particle.
  • Material properties of the optical particle may comprise chemical, physical, physiochemical, and other properties. In some cases, such properties may comprise porosity and internal structures and potential cavities which may be present inside the particle, and which may affect the properties (e.g., directionality and/or intensity) of the emitted signal.
  • signal emission from an optical particle may comprise or be in the form of whispering gallery modes (WGMs) which may emit a portion, some, or most of the signal in a plane.
  • the plane may be the plane of the cavity resonance, wherein cavity is an internal feature (e.g., physical property) in the optical particle.
  • the optical methods and systems provided herein may comprise microwave electronics.
  • Optical particle may comprise open dielectric resonators.
  • Open dielectric resonators may comprise circular optical modes and/or whispering gallery modes (WGMs).
  • WGMs may comprise closed circular beams supported by total internal reflections from boundaries of the resonators in the optical particles.
  • Screening and elucidation of the structure of the effector may be performed in a variety of ways as detailed elsewhere herein. This may depend on the application, the encoding modality used, the type of data and information that is to be obtained from the screen, and Attorney Docket No.56523-707.601 potentially/optionally other factors.
  • a barcode may be decoded after collecting the beads from a screen. For example, beads may be collected based on defined criteria (e.g., hit sorting based on a threshold defined for an assay signal), and sequenced (e.g., through NGS).
  • a barcode may be decoded or read during a screen (e.g., on the fly). This may be referred to as “decoding on the fly” or “DOTF” herein.
  • optical barcodes may be decoded on the fly and may eliminate the need for a physical sorting step. As such, a physical sorting step may in some cases be optional, not required, or not performed. This may accelerate the workflow for performing the methods of the present disclosure.
  • a screening device used for screening encoded effector libraries may comprise a sorting module.
  • a screening device used for screening encoded effector libraries may not comprise a sorting module.
  • a device may comprise a sorting module, but the sorting module, junction, or device may be idled and not used during a screen in case it is not needed. Eliminating the sorting step may allow for collection of more data during a screen. For example, instead of selecting a subset of the beads/compartments that meet defined criteria, sorting them, decoding them, analyzing them and so on, data may be recorded for most or all of the beads screened. This may allow for mapping out structure-activity relationships (SAR) more comprehensively.
  • SAR structure-activity relationships
  • the systems and methods provided herein may comprise providing, synthesizing, making, obtaining, and/or screening encoded effectors.
  • An encoded effector may comprise or be an effector that has been linked with, associated with, or barcoded with an encoding/barcode such that ascertaining a property of the encoding allows for readily determining the structure of the effector.
  • the terms encoding and barcode may be used interchangeably.
  • An effector can be any type of molecule or substance whose effect on a sample may be investigated.
  • the effector may comprise or be a compound, a protein, a peptide, an enzyme, a nucleic acid, a gene, or any other substance.
  • the encoding allows a user to determine the structure of the effector by measuring/detecting a property of the encoding.
  • each encoding moiety has a measurable property that, when measured, can be used to determine the structure of the effector which is encoded.
  • Encoding modalities may comprise nucleic acids, DNA, RNA, peptides, peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • encoding modalities may comprise optical barcodes, nanoparticles, luminescent materials, and/or quantum dot Attorney Docket No.56523-707.601 particles.
  • various combinations of encoding modalities may be used.
  • encoding modalities may comprise both nucleic acid molecules and optical barcodes.
  • encodings may comprise nucleic acid molecules such as DNA, RNA, or PNA.
  • the encoding may comprise a sequence unique to the structure of the effector, a sequence unique to the scaffold that is bound to, comprised therein, and/or both.
  • the sequence of the nucleic acid may provide information about the structure of its corresponding effector.
  • the encoded effectors are described by what kind of molecules is used in the encoding. For example, “nucleic acid encoded effectors” comprise an effector encoded by a nucleic acid.
  • the effectors and their corresponding encodings are bound to a scaffold.
  • the effector may be covalently bound to the scaffold via a cleavable linker.
  • the encoding may be covalently bound to the scaffold.
  • the effector may comprise a plurality of subunits which may be covalently bound to one another.
  • the encoding may comprise a plurality of subunits which may also be covalently bound to one another.
  • the effector and the encoding may form an effector/encoding pair linked in space bound to the scaffold, and in some cases, bound to one another. Alternatively, the effector and the encoding may be separately bound to the scaffold but not bound to one another.
  • the link between the pairing is not lost.
  • Many materials can be used as scaffolds, as any material capable of binding both the effector and the encoding may accomplish the desired goal of keeping the pair linked in space.
  • the scaffold may be a bead.
  • Various methods for preparing encoded effectors linked to scaffolds can be used. In some embodiments, the methods use orthogonal, compatible methodologies to create an effector and its encoding in a parallel synthesis scheme.
  • an exemplary workflow for the preparation of a scaffold containing an effector and encoding is described as follows: A first effector subunit is attached at an attachment point of a scaffold. The scaffold is then washed to remove unreacted and excess reagents from the scaffold. A first encoding subunit is then attached at another attachment point on the scaffold, and a wash step performed. Following this, a second effector subunit is then attached to the first effector subunit, followed by another wash step. Then, a second encoding subunit is attached to the first encoding subunit, followed by a wash step.
  • pre-synthesized compounds are loaded onto scaffolds which contain encodings.
  • the encodings may be pre-synthesized and loaded onto the scaffolds or are synthesized directly onto the scaffolds using methods analogous to the split and pool synthesis described above.
  • each scaffold comprises numerous copies of a unique effector and its corresponding encoding.
  • the encoding may comprise information related to the synthetic history (a plurality of synthetic steps) of the effector.
  • the scaffolds further comprise impurities in the effector and/or its encoding.
  • the subunits of the effector may comprise or be building blocks of a small molecule.
  • the effector may be a small molecule.
  • the effector may further comprise building block fragments.
  • impurities of the effector and its corresponding encoding occur due to damage during a screen, during manufacturing of the bead, effector, or encoding combination, or during storage.
  • impurities of the effector and its corresponding encoding are present due to defects in the methodologies used to synthesize the encoded effectors.
  • scaffolds as described herein can comprise a single encoder, an encoding and its impurities, or combinations thereof.
  • at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the effectors attached to a scaffold comprise an identical structure.
  • at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the encodings attached to a scaffold comprise a substantially identical structure or sequence.
  • Cleavable linkers can be used to attach effectors to scaffolds.
  • the effector is bound to a scaffold by a cleavable linker.
  • the cleavable linker is cleavable by electromagnetic radiation, an enzyme, a chemical reagent, heat, pH adjustment, sound, or electrochemical reactivity.
  • the cleavable linker is cleavable by electromagnetic radiation.
  • the cleavable linker is cleavable by electromagnetic radiation such as UV light.
  • the cleavable linker is a photocleavable linker.
  • the photocleavable linker is cleavable by electromagnetic radiation.
  • the photocleavable linker is cleavable through exposure to light.
  • the light comprises UV light.
  • the cleavable linker is cleavable by a cleaving reagent.
  • the cleavable linker must first be activated in order to be able to be cleaved.
  • the cleavable linker is activated through interaction with a reagent.
  • Attorney Docket No.56523-707.601 [00086]
  • the cleavable linker is a disulfide bond.
  • the cleavable linker is a disulfide bond
  • the cleavable reagent is a reducing agent.
  • the reducing agent is a disulfide reducing agent.
  • the disulfide reducing agent is a phosphine.
  • the reducing agent is 2-mercapto ethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol, a combination thereof, or a derivative thereof.
  • the cleavable linker and cleaving reagent are biorthogonal reagents.
  • Bioorthogonal reagents are combinations of reagents that selectively react with each other, but do not have significant reactivity with other biological components. Such reagents allow for minimal cross-reactivity with other components of the reaction mixture, which allows for less off target events.
  • the cleavable linker is a substituted trans-cyclooctene. In some embodiments, the cleavable linker is a substituted trans-cyclooctene and the cleaving reagent is a tetrazine.
  • the cleaving reagent is a tetrazine.
  • the cleaving reagent is dimethyl tetrazine (DMT). Further examples of tetrazine cleavable linkers and methods of use are described in Tetrazine-triggered release of carboxylic-acid-containing molecules for activation of an anti-inflammatory drug, ChemBioChem 2019, 20, 1541–1546, which is hereby incorporated by reference.
  • the cleavable linker comprises an azido group attached to the same carbon as an ether linkage. In some embodiments, the cleavable linker has the structure or . In some embodiments, the cleaving reagent is a reagent that reduces an azido group. In some embodiments, the cleaving reagent is a phosphine. In some embodiments, the cleaving reagent is hydrogen and a palladium catalyst. Attorney Docket No.56523-707.601 [00090] In some embodiments, the cleavable linker is cleaved by a transition metal catalyst. In some embodiments, the cleavage reagent is a transition metal catalyst.
  • the transition metal catalyst is a ruthenium metal complex.
  • the cleavable linker is an O-allylic alkene.
  • the cleavable linker has the structure .
  • a non-limiting example of such a catalyst is described in Bioorthogonal catalysis: a general method to evaluate metal-catalyzed reaction in real time in living systems using a cellular luciferase reporter system, Bioconjugate Chem.2016, 27, 376-382, which is hereby incorporated by reference.
  • the transition metal complex is a palladium complex.
  • the cleavable linker has the structure .
  • the number of effectors cleaved from the scaffold is controlled.
  • the number of effectors cleaved from a scaffold is controlled by controlling the amount of stimulus used to cleave the cleavable linker.
  • a “stimulus” is any method or chemical used to specifically cleave a cleavable linker.
  • the stimulus is a chemical reaction with a cleaving reagent.
  • the stimulus is electromagnetic radiation. In some embodiments, the stimulus is a change in pH. In some embodiments, the change in pH is acidification. In some embodiments, the change in pH is basification. [00092] In some embodiments, methods described herein comprise cleaving the cleavable linker with a cleaving reagent. In some embodiments, the methods comprise adding the cleaving reagent to an encapsulation comprising an effector bound to a scaffold through a cleavable linker. In some embodiments, the methods comprise adding the cleaving reagent to an encapsulation comprising an encoding bound to a scaffold through a cleavable linker.
  • the number of effectors cleaved from the scaffold is controlled by controlling the concentration of the cleaving reagent.
  • the concentration of the cleavage reagent is controlled in an encapsulation containing an encoded effector bound to a scaffold.
  • the concentration of chemical reagent used Attorney Docket No.56523-707.601 to cleave the cleavable linker is at least 100 pM, at least 500 pM, at least 1 nM, at last 10 nM, at least 100 nM, at least 1 ⁇ M, at least 10 ⁇ M, at least 100 ⁇ M, at least 1 mM.
  • the concentration of cleaving reagent used to cleave the cleavable linker is at most 100 pM, at most 500 pM, at most 1 nM, at most 10 nM, at most 100 nM, at most 1 ⁇ M, at most 10 ⁇ M, at most 100 ⁇ M, at most 1 mM, at most 10 mM, at most 100 mM, or at most 500 mM.
  • the cleaving reagent is added to a plurality of encapsulations.
  • the concentration of cleaving reagent added to the plurality of encapsulations is substantially uniform among individual encapsulations of the plurality. In some embodiments, the concentration of cleaving reagent used to cleave the cleavable linker in a plurality of encapsulations is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% identical in each individual encapsulation.
  • concentration of cleaving reagent used to cleave the cleavable linker in a plurality of encapsulations differs by no more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50- fold, or 100-fold among each individual encapsulation of the plurality.
  • the cleaving reagent is added to the encapsulation by pico- injection.
  • the encapsulation is passed through a microfluidic channel comprising a pico-injection site.
  • pico-injections are timed such that the rate of pico-injection matches the rate at which encapsulation cross the pico-injection site. In some embodiments, at least 80%, 85%, 90%, 95%, 98%, or 99% of encapsulations passing a pico-injection site receive a pico-injection. In some embodiments, the pico-injections are at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold smaller in volume than the passing droplets. In some embodiments, the cleaving reagent is added to the encapsulation by droplet merging.
  • the cleaving reagent is added from a stock solution to the encapsulation.
  • the stock solution is at least 2X, 5X, 10X, 20X, 30X, 50X, 100X, 500X, or 1000X more concentrated than the desired final concentration in the encapsulation.
  • methods and systems described herein comprise cleaving a photocleavable linker between an encoded effector and a scaffold.
  • the methods and systems described herein comprise exposing an encapsulation to electromagnetic radiation comprising an effector bound to a scaffold through a photocleavable linker.
  • the methods and systems described herein comprise exposing an encapsulation Attorney Docket No.56523-707.601 to light (for e.g., UV light) comprising an effector bound to a scaffold through a photocleavable linker.
  • light for e.g., UV light
  • the encapsulation is exposed to the light using a microfluidic device.
  • the photocleavable linker is cleaved by exposure to light (e.g., UV light).
  • the concentration of the number of effector molecules released from a scaffold is controlled by controlling the intensity and/or duration of exposure to UV light. Any suitable UV light intensity may be used.
  • the intensity of the UV light used of exposing and cleaving the cleavable linker may be from about 0.1 J/cm 2 to about 200 J/cm 2 . Any suitable UV power may be used. In some examples, the UV power for cleaving the cleavable linker may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 4000, 5000 mV. The light may be calibrated and optimized as needed. [00099] the cleavable linker may be cleaved by electromagnetic radiation.
  • the concentration of the number of effector molecules released from a scaffold is controlled by controlling the intensity or duration of electromagnetic radiation.
  • Any suitable photoreactive or photocleavable linker can be used as a cleavable linker cleaved by electromagnetic radiation (e.g., exposure to UV light).
  • a list of example linkers cleavable by electromagnetic radiation may comprise: o-nitrobenzyloxy linkers, o- nitrobenzylamino linkers, ⁇ -substituted o-nitrobenzyl linkers, o-nitroveratryl linkers, (v) phenacyl linkers, p-alkoxyphenacyl linkers, benzoin linkers, pivaloyl linkers, and other photolabile linkers. Further examples of photocleavable linkers are described in Photolabile linkers for solid-phase synthesis, ACS Comb Sci. 2018 Jul 9;20(7):377–99, which is hereby incorporated by reference.
  • the cleavable linker is an o-nitrobenzyloxy linker, an o-nitrobenzylamino linker, an ⁇ -substituted o-nitrobenzyl linker, an o-nitroveratryl linker, a phenacyl linker, p-alkoxyphenacyl linker, a benzoin linker, or a pivaloyl linker.
  • the photocleavable linker may be activatable by a stimulus before it is cleavable. For example, a first stimulus (light, heat, energy, chemical, or beyond) may be applied to activate the cleavable linker.
  • a second stimulus (light, heat, energy, chemical, or beyond) may be applied to cleave the cleavable linker.
  • UV exposure is an example of the second stimulus.
  • Activation by a chemical is an example of activating the photocleavable linker.
  • the number of effectors released can be controlled by controlling and modulating the stimulus.
  • Activatable photocleavable linkers that need to be activated before being cleaved through exposure to the second stimulus (e.g., UV light) may enable improved Attorney Docket No.56523-707.601 bead-handling, synthesis, storage, and preparation due to minimized or eliminated encoded effector release through the application of the second stimulus (e.g., incident UV exposure).
  • 2E provides an exemplary molecule configured to be transformed upon interaction with a reagent, such that it becomes activated for UV photocleavage (reference: J. AM. CHEM. SOC.2003, 125, 8118-8119; 10.1021/ja035616d).
  • the azide group functionally reduces the sensitivity of the photocleavable-linker moiety, such that linker is more stable, thus advantageous for handling and storing under ambient lighting.
  • the azide can be converted upon reagent treatment (HOF-CH3CN) to generate the photo-sensitive Nitro-benzyl motif (molecule depicted in the middle), wherein the product photocleavable-linker can be calibrated to release a known quantity of effector upon UV- exposure.
  • FIG. 2F provides another exemplary molecule configured to be transformed upon interaction with a reagent, such that it becomes activated for UV photocleavage (reference: J. Comb. Chem. 2000, 2, 3, 266–275).
  • the thio-phenol ester provides a stable covalent linker to compound (R).
  • An active cleavable linker may be cleaved by a stimulus (e.g., the second stimulus in case a first stimulus is required for activating the linker).
  • the stimulus for cleaving the cleavable linker may comprise a variety of modalities.
  • Examples of stimuli which can be used for cleaving the linker may comprise an enzyme, a protease, a nuclease, a hydrolase, a chemical, an energy, light (e.g., UV light), heat, electromagnetic radiation, or another kind of stimulus.
  • the cleavable linker may comprise or be a peptide, a nucleic acid molecule, a carbohydrate, a chemical moiety, a chemical bond and beyond.
  • the stimulus may be optimized in terms of intensity or power (e.g., intensity of an energy) or concentration (e.g., in case of a chemical stimulus such as an enzyme capable of cleaving the linker).
  • the methods of screening may comprise cleaving the cleavable linker and thereby releasing the effector in a compartment to interact with a sample.
  • the cleavable linker may be cleaved by any suitable stimulus described anywhere herein.
  • a chemical may be added to the compartment (e.g., droplet or well) by any suitable method (e.g., dispenser, robot, manually by a person, a microfluidic chip module, etc.) at a defined time point.
  • any suitable method e.g., dispenser, robot, manually by a person, a microfluidic chip module, etc.
  • pico-injection may be used to inject a chemical into a droplet after its formation to cleave the cleavable linker.
  • the cleaving reagent may be added to the wells of the array manually by a person or using a machine or robot.
  • the concentration of the released effector can be controlled by the intensity or concentration of the stimulus applied.
  • the activating reagent for activating the cleavable linker may comprise a disulfide reducing reagent.
  • the activating reagent comprises tetrazine.
  • the activating reagent may be added to the encapsulation by pico-injection.
  • the encapsulation may pass through a microfluidic channel comprising a pico- injection site, in case the encapsulation is a droplet in a microfluidic device.
  • Reagent addition e.g., pico-injections
  • the rate of reagent addition e.g., pico-injection
  • least 80%, 85%, 90%, 95%, 98%, or 99% of encapsulations passing a pico-injection site receive a pico-injection.
  • the pico-injections are at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 500- fold, or at least 1000-fold smaller in volume than the passing droplets.
  • the activating reagent is added to the encapsulation by droplet merging. [000106] Any suitable concentration of an activating reagent may be added to a compartment to activate a cleavable linker. In some examples, the concentration of the activating agent may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100 millimolar (mM) or less.
  • the concentration of activating reagent may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100 micromolar ( ⁇ M). In some examples, the concentration of the activating reagent used to activate the cleavable linker may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100 picomolar (pM) or less.
  • the effector may be released from the scaffold to move freely in the compartment (e.g., in the solution). Free movement may allow the effector to interact with the sample or target being interrogated. The effector may be released in a controlled fashion. This controlled release may allow for a predetermined and/or known dose of effectors to be released form the scaffold.
  • Such a procedure may allow for improved quantification and analysis of data (e.g., structure-activity relationship data or hits) from a screen, as dose response measurements can be detected or recorded. Additionally, releasing a known number of effectors across a library of effectors being screened may remove bias from the sample set.
  • Bias can occur in library screens using encoded scaffolds when individual scaffolds possess attachments of effectors that vary in amount among the scaffolds of the library. For example, one scaffold may contain 10 copies of an effector molecule, and another scaffold may contain 1000 copies of an effector molecule. Consequently, different Attorney Docket No.56523-707.601 concentrations of effector being screened against a sample or target may be released.
  • the effectors may be released to a determined concentration in a compartment.
  • the desired concentration may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 pM or higher.
  • the released effector concentration may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 nM or higher. In some examples, the released effector concentration may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 ⁇ M or higher. In some examples, the released effector concentration may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 mM or higher.
  • the released effector concentration may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mM or lower. In some examples, the released effector concentration may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 ⁇ M or lower.
  • the released effector concentration may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nM or lower. In some examples, the released effector concentration may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 pM or lower. The concentration of the effector released in the compartments may be substantially uniform across the compartments.
  • the concentration of the released effector among the compartments may vary at most about 50%, 40%, 30%, 20%, 10%, 8%, 9%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.
  • the compartment Upon assay set-up in a compartment (e.g., a droplet or a well of any kind as mentioned anywhere herein), the compartment may be incubation for a determined period.
  • the incubation time may be described anywhere herein. In some cases, incubation time may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 30, 40 minutes (min), or longer. In some cases, the incubation time may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hour(s) (hr).
  • the incubation time may be at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour(s) Attorney Docket No.56523-707.601 (hr) or less. In some cases, the incubation time may be at most about 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 minutes (min) or less.
  • the effectors of the present disclosure may be of any kind or modality.
  • the effector may be biochemical, chemical, a compound, a small molecule, a cell, a protein, a peptide, a biological moiety, a small molecule fragment, a nucleic acid, or another kind of effector. In some cases, an effector may be molecule capable of interacting with a target.
  • the effectors may comprise a handle that allows for attachment to a scaffold.
  • a handle may be a reactive functional group that can be used to tether the effector to an attachment site on a scaffold. This handle may be any functional group capable of forming a bond.
  • Example of handles may comprise sulfhydryl groups, CLICK chemistry reagents, amino groups, carboxylate groups, or other groups.
  • the effectors may comprise subunits (e.g., individual subunits). Subunits may be joined using various chemical reactions to form the full effector.
  • Iterative chemical processes may be used to generate the effectors, similar to methodologies used in peptide synthesis (e.g., solid-phase peptide synthesis (SPPS)). Similar methods can be used to create non-peptide effectors, wherein a first reaction may be performed to link two subunits, the two linked subunits may be subjected to a second reaction to activate the linked subunits, and a third subunit may then be attached, and so on. Any type of such an iterative chemical synthesis scheme may be employed to create the effectors used in the methods and systems provided herein. [000115] In some examples, the effectors may elicit a response from the target being interrogated.
  • SPPS solid-phase peptide synthesis
  • the response elicited can take any form and may depend on the sample being interrogated.
  • the response may be a change in expression pattern, apoptosis, expression of a particular molecule, or a morphological change in the cell.
  • the effector may inhibit protein activity, enhance protein activity, alter protein folding, or measure protein activity.
  • the effector may be a protein.
  • the protein may be naturally occurring or mutant.
  • the effector may be an antibody (AB) or antibody fragment.
  • the effector may be an enzyme, a binding protein, an AB or AB-fragment, a structural protein, an enzyme, a binding protein, a storage protein, a transport protein, or any mutant or combinations thereof.
  • the effector may be a peptide, a non-natural peptide, a polymer, or an unnatural amino acid.
  • the peptide may comprise a non-peptide region.
  • the peptide may be a cyclic peptide.
  • the peptide may comprise a secondary structure that mimics a protein.
  • the peptide or polymer may be made of a number of units (e.g., a number of amino acids).
  • the peptides may comprise a number of amino acids.
  • a peptide effector may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 39, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300 or more amino acids.
  • the peptide may comprise at most about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5 or a smaller number of amino acids.
  • the peptide may comprise from about 3 to about 10, from about 6 to 20, from about 9 to 60, from about 12 to 180 amino acids.
  • the effector may be a compound, an organic molecule, a drug-like small molecule, an organic compound, an effector comprising organic and/or inorganic atoms or molecules, an effector comprising one or more metal atoms or molecules, a small molecule, or a macro- molecule.
  • the compound may be an organic molecule.
  • the compound may be an inorganic molecule.
  • the compounds used as effectors may contain organic and inorganic atoms.
  • the compound may be a drug-like small molecule. In some embodiments, the compound may be an organic compound.
  • the compound may comprise one or more inorganic atoms, such as one or more metal atoms.
  • an effector/compound may be a completed chemical that is synthesized by connecting a plurality of chemical monomers to each other.
  • the effector may be a pre-synthesized compound loaded onto a bead after synthesis.
  • the compound may be a small molecule fragment. Small molecule fragments may be small organic molecules which are small in size and low in molecular weight. In some examples, the small molecule fragments may be less than about 500, 400, 300, 200, 100 Dalton (Da) or less in molecular weight (MW). [000119]
  • the effector may be an effector nucleic acid.
  • the effector nucleic acid may comprise a number of nucleotides.
  • the effector nucleic acid may comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more.
  • the number of nucleotides in the nucleic acid effector may be at least about 10 3 , 2 x 10 3 , 3 x 10 3 , 4 x 10 3 , 5 x 10 3 , 6 x 10 3 , 7 x 10 3 , 8 x 10 3 , 9 x 10 3 or more.
  • the number of nucleotides in a nucleic acid effector may be at least about 10 4 , 2 x 10 4 , 3 x 10 4 , 4 x 10 4 , 5 x 10 4 , 6 x 10 4 , 7 x 10 4 , 8 x 10 4 , 9 x 10 4 , or more. In some examples, the number of nucleotides in a nucleic acid effector may be at least about 10 5 , 2 x 10 5 , 3 x 10 5 , 4 x 10 5 , 5 x 10 5 , 6 x 10 5 , 7 x 10 5 , 8 x 10 5 , 9 x Attorney Docket No.56523-707.601 10 5 or more.
  • the number of nucleotides in a nucleic acid effector may be at least 10 6 , 2 x 10 6 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 6 x 10 6 , 7 x 10 6 , 8 x 10 6 , 9 x 10 6 or more. In some examples, the number of nucleotides in a nucleic acid effector may be at least about 10 7 , 2 x 10 7 , 3 x 10 7 , 4 x 10 7 , 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , 8 x 10 7 , 9 x 10 7 or more.
  • Nucleic acid barcodes [000120]
  • the effectors provided herein can be linked with encodings.
  • the effectors are linked with an encoding.
  • the encoding allows a user to determine the structure of the effector by determining a property of the encoding.
  • each encoding moiety has a measurable property that, when measured, can be used to determine the structure of the effector which is encoded.
  • the encoding is a nucleic acid.
  • the sequence of the nucleic acid may provide information about the structure and/or identity of the effector.
  • the encoding may comprise or be a nucleic acid barcode. The terms encoding and barcode may be used interchangeably.
  • the barcode may comprise a sequencing primer. Sequencing the nucleic acid encoding allows the user to ascertain the structure of the corresponding effector.
  • the barcode/encoding may comprise or be DNA.
  • An example barcode may comprise double-stranded DNA or single-stranded DNA.
  • the barcode/encoding may comprise or be RNA.
  • An example barcode may comprise double- stranded RNA or single-stranded RNA.
  • the barcode may comprise or be a peptide or a peptide nucleic acid (PNA).
  • PNA peptide or a peptide nucleic acid
  • the barcode encoding the effector may comprise or be a nucleic acid molecule of any suitable length.
  • the barcode may comprise a number of nucleotides.
  • the number of nucleotides in a barcode may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more.
  • the number of nucleotides may be at least 10 3 , 2 x 10 3 , 3 x 10 3 , 4 x 10 3 , 5 x 10 3 , 6 x 10 3 , 7 x 10 3 , 8 x 10 3 , 9 x 10 3 or more.
  • the number of nucleotides in a barcode may be at least 10 4 , 2 x 10 4 , 3 x 10 4 , 4 x 10 4 , 5 x 10 4 , 6 x 10 4 , 7 x 10 4 , 8 x 10 4 , 9 x 10 4 , or more. In some examples, the number of nucleotides in a barcode may be at least 10 5 , 2 x 10 5 , 3 x 10 5 , 4 x 10 5 , 5 x 10 5 , 6 x 10 5 , 7 x 10 5 , 8 x 10 5 , 9 x 10 5 or more.
  • the number of nucleotides in a barcode may be at least 10 6 , 2 x 10 6 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 6 x 10 6 , 7 x 10 6 , 8 x 10 6 , 9 x 10 6 or more. In some examples, the number of nucleotides in a barcode may be at least 10 7 , 2 x 10 7 , 3 x 10 7 , 4 x 10 7 , 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , 8 x 10 7 , 9 x 10 7 or more.
  • the encoding is made up of individual subunits that encode a corresponding effector subunit. Consequently, an entire encoding can specify which individual subunits have been linked or combined to form the effector.
  • each subunit may comprise up to 5, 10, 15, 20, 25, 30, 40, 50, or more individual nucleotides.
  • the full encoding sequence can comprise any number of these individual subunits. In some embodiments, the full encoding sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more encoding subunits.
  • the encoding/barcode may be a molecular weight barcode.
  • the molecular weight barcode may be a peptide.
  • molecular weight (MW) barcode may comprise a peptide with unnatural amino acids.
  • the molecular weight (MW) of the barcode may be at least about 1000, 5000, 10000, 15000 Daltons or larger.
  • the molecular weight barcode peptide may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more amino acids. In some cases, the molecular weight barcode peptide may comprise at most about 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2 or smaller number of amino acids.
  • Barcodes may be loaded onto and/or into the scaffold. In some examples, the barcode may comprise or be a DNA barcode. Alternatively or in addition, any suitable barcode mentioned anywhere herein may be used.
  • a scaffold (e.g., an example bead with a porous material as described anywhere herein, in some cases a 10-micron bead) may be loaded with a predetermined number of barcodes or copies of the barcode.
  • the bead may be a TentaGel bead.
  • any other kind of suitable bead may be used and loaded with the barcode and the effector.
  • the number of copies of the barcode loaded onto/into the scaffold may be at least about 10, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or more.
  • the number of copies of the barcode loaded onto/into the scaffold may be at most about 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 or less.
  • the barcode may comprise one or more barcoding sequences.
  • the barcode may comprise a bead-specific barcode (BSB) that identifies the bead/scaffold and may be used to count the scaffold.
  • BBB bead-specific barcode
  • Miniaturized screening platforms [000127]
  • the present disclosure provides a system comprising a plurality of partitions or compartments for screening (e.g., high-throughput screening in a miniaturized platform), which can be used to compartmentalize a sample into discrete volumes in open or Attorney Docket No.56523-707.601 closed partitions of various shapes, sizes, and materials.
  • a partition may comprise, be, or termed as a compartment, chamber, confinement, encapsulation, raft, or other type of partitions, such as to, for example, keep multiple discrete or semi-discrete sub-samples substantially separate from one another, and in some applications, test different perturbation conditions in each compartment/partition, in a parallelized and/or high-throughput fashion.
  • each compartment can act as an independent reaction vessel.
  • the terms compartment, partition, encapsulation, raft, chamber, microfluidic compartment may be used interchangeably.
  • the methods and systems provided herein can facilitate sample screening in a relatively short period of time (i.e., in high throughput) compared to conventional methods, and/or using small sample sizes.
  • the systems provided herein may comprise or be bench-time instruments which may effectively replace large HTS facilities. [000128]
  • the systems provided herein may comprise a plurality of partitions/compartments (e.g., wells or arrays) built in or immobilized on a solid support, surface, or substrate. Alternatively, the compartments may not be immobilized on a solid support.
  • the compartments may be suspended in a liquid, such as a droplet in oil emulsion (i.e., a plurality of droplets suspended in an immiscible oil fluid) generated using a microfluidic device or using a vortexer or shaking platform (e.g., bulk emulsification and/or particle-templated emulsification (PTE)).
  • a droplet in oil emulsion i.e., a plurality of droplets suspended in an immiscible oil fluid
  • a vortexer or shaking platform e.g., bulk emulsification and/or particle-templated emulsification (PTE)
  • PTE particle-templated emulsification
  • droplets may be performed in a variety ways.
  • the compartments may comprise or be droplets, wells, or combinations of both (e.g., droplets trapped in wells).
  • the methods and systems of the present disclosure may comprise obtaining, providing, and using encoded effector libraries.
  • Encoded effector libraries may be designed and synthesized using a variety of methods. Encoded effector libraries may further be prepared and integrated with a variety of screening platforms. Screening platforms may be according to Attorney Docket No.56523-707.601 the screening systems provided anywhere herein or other screening platforms beyond this disclosure which may be used for screening encoded effector libraries provided herein. [000131] Screening in a miniaturized platform may comprise screening an assay in a system provided herein. Screening systems may be high-throughput and miniaturized screening platforms configured to measure the activity of an assay. An assay may be configured to measure an activity or condition of a biological target, such as any kind of target mentioned anywhere herein. In some cases, samples may comprise live cells. In other cases, samples may not comprise live cells.
  • the condition of the sample may be screened using the screening methods, systems, and workflows provided herein.
  • the screening systems, the assays, and the effector libraries may be integrated into a workflow, such as to assess the effect of the members of the encoded effector library on the sample, through the assay, detected via the screening system.
  • assays may be performed in absence of effector libraries.
  • the methods, systems, system components, workflows, and all of their parts, pieces, and components may be used individually or in concert to achieve various goals.
  • encoded effectors may comprise or be molecules whose structures can be measured or identified by measuring a property of the corresponding encoding.
  • the members of the encoded effector library may be incubated with an assay in a plurality of compartments or encapsulations of a screening platform (e.g., wells of an array-based system) or generated by a screening platform (e.g., droplets generated by a microfluidic device or vortexing system).
  • a screening platform e.g., wells of an array-based system
  • a screening platform e.g., droplets generated by a microfluidic device or vortexing system.
  • the assay may generate a signal which can be detected by a screening system (e.g., a detector).
  • the effector may interact with the sample or a target therein and may have an effect or suspected effect on the target, the sample, and/or the signal measured from the assay (e.g., in each compartment).
  • the signal may be indicative of an effect of the effector on the assay, the target, or the sample.
  • the effector can be determined to have efficacy against the sample in inducing a particular response.
  • the systems and methods described herein, in some embodiments, utilize small encapsulations, such as droplets, wells, channels, miniaturized confinements, or any other type of compartment mentioned anywhere herein.
  • compartment and encapsulation may be used interchangeably.
  • at least a subset of the plurality of the encapsulations of a system or generated by the system may each individual carry out an assay.
  • the encapsulation may comprise a unique effector of the encoded Attorney Docket No.56523-707.601 effector library.
  • the encoded effector library may comprise a plurality of unique encoded effectors described in further detail elsewhere herein.
  • a plurality of unique and/or different encoded effectors may be screened in presence of a sample (e.g., against one or more targets) in a parallelized and high-throughput fashion, consuming small quantities of assay reagents, using the miniaturized systems provided herein.
  • the miniaturized systems may comprise automated benchtop instruments.
  • it may be intended (e.g., by a user or researcher) to achieve an effect from an effector. For example, it may be intended to find a drug for a disease target. It may be intended to find an effector which may decrease or increase an activity of a biological target in a sample.
  • the effect may be screened for using the methods and systems of the present disclosure.
  • the effects of a multi-member library e.g., a one-million-member library of encoded effectors
  • the effect may manifest in the signal detected from the compartments, using the screening platforms and assays of the present disclosure. If the intended effect is achieved, detected, or observed through the system, the compartment containing the effector may be selected by the system for further processing. Selection may be performed using computer systems, hardware, and software.
  • a rule may be defined on a software of the system which is in communication with the miniaturized screening chip through a computer.
  • the rule may comprise defining a threshold for the signal.
  • the effector and/or the compartment it is in may be selectively processed.
  • Selective processing may comprise pulsing, separation in space, sorting, and detecting a property of the barcode to elucidate the identity of the effector which led to the intended effect (e.g., the subject of the screen/search). Processing may comprise additional embodiments beyond sorting, as mentioned elsewhere herein.
  • Sample Screening in Droplet Microfluidic Platforms [000136] Provided herein are methods and systems for screening encoded effectors on samples using encapsulations (e.g., droplets or compartments). In some embodiments, methods and systems for screening encoded effectors on samples are capable of being performed in a high-throughput manner.
  • the methods and systems provided herein allow for screening large libraries of encoded effectors using small volumes, minimal amounts of reagents, and small amounts of the effectors being screened. In some embodiments, the methods and systems provided herein allow for uniform dosing of effectors in a library against samples. In some embodiments, the methods and systems described herein allow for measurement of cellular properties, behaviors, or responses, in a high throughput manner. In Attorney Docket No.56523-707.601 some embodiments, the methods and systems provided herein measure genomic, metabolomic, and/or proteomic data from cells screened against the encoded effectors.
  • the methods and systems provided herein allow for detecting synergistic effects of using multiple effectors against a particular sample. In some embodiments, the methods and systems provided herein allow for a library of mutant proteins to be screened for a desired activity or improvement in activity of a target.
  • Encoded effectors may be bound to solid supports, scaffolds, or beads. The effector may be bound to the scaffold via a cleavable linker and releasable upon cleavage of the cleavable linker.
  • the scaffold/bead may act as a solid support and keep the encoded effector molecules linked in space to their barcode(s).
  • the scaffold may be a structure with a plurality of attachment points that allow linkage of one or more molecules.
  • the encoded effector may be bound to or encapsulated in a scaffold.
  • the scaffold may be a solid support.
  • the scaffold may be a bead, a fiber, nanofibrous scaffold, a molecular cage, a dendrimer, or a multi-valent molecular assembly.
  • the scaffold/solid support may be a bead, polymer bead, a glass bead, a metal bead, or a magnetic bead.
  • the beads utilized in the methods provided herein may be made of any material.
  • the bead may be a polymer bead.
  • the bead may comprise a polystyrene core.
  • the beads may be derivatized with polyethylene glycol.
  • the beads may be grafted with polyethylene glycol (e.g., a polystyrene core grafted with polyethylene glycol).
  • the polyethylene glycol may contain reactive groups for the attachment of other functionalities, such as effectors or barcodes.
  • the reactive groups may comprise or be an amino or carboxylate group.
  • the reactive group may be at the terminal end of the polyethylene glycol chain.
  • the bead is a TentaGel® bead.
  • the polyethylene glycol (PEG) attached to the beads may be any size. In some embodiments, the PEG is up to 20 kDa.
  • the PEG is up to 5 kDa. In some embodiments, the PEG is about 3 kDa. In some embodiments, the PEG is about 2 to 3 kDa.
  • the PEG group is attached to the bead by an alkyl linkage. In some embodiments, the PEG group is attached to a polystyrene bead by an alkyl linkage. In some embodiments, the bead is a TentaGel® M resin. [000141] In some embodiments, the bead comprises a PEG attached to a bead through an alkyl linkage and the bead comprises two bifunctional species.
  • the beads comprise surface modification on the outer surface of the beads that are orthogonally protected Attorney Docket No.56523-707.601 to reactive sites in the internal section of the beads.
  • the beads comprise both cleavable and non-cleavable ligands.
  • the bead is a TentaGel® B resin.
  • the beads of the present disclosure may comprise any suitable size. The bead size may be optimize based on the application, the screening system, the effector, the barcode, the target, the assay, or other parameters involved in the integrated methods and systems.
  • the bead diameter may be at least about 1 nm, 10 nm, 100 nm, 200 nm, 500 nm, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 12 ⁇ m, 14 ⁇ m, 16 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 50 ⁇ m, 80 ⁇ m, 100 ⁇ m, 90 ⁇ m, 100 ⁇ m, 120 ⁇ m, 140 ⁇ m, 160 ⁇ m, 180 ⁇ m, 200 ⁇ m, 300 ⁇ m, or larger.
  • the bead size may differ based on the application. In some examples, the bead size may be at most about 300 ⁇ m, 200 ⁇ m, 160 ⁇ m, 100 ⁇ m, 80 ⁇ m, 50 ⁇ m, 40 ⁇ m, 30 ⁇ m, 20 ⁇ m, 10 ⁇ m, 8 ⁇ m, 6 ⁇ m, 4 ⁇ m, 3 ⁇ m 2 ⁇ m, 1 ⁇ m, 500 nm, 200 nm, 100 nm, 10 nm, 1 nm, or less.
  • the diameter of the bead may differ in different solvents. In some cases, the bead diameter may at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 micrometers ( ⁇ m) in water.
  • the effector may be covalently bound to the scaffold. In some examples, the effector may be non-covalently bound to the scaffold. In some examples, the effector may be bound to the scaffold through ionic interactions. In some examples, the effector is bound to the scaffold through hydrophobic interactions.
  • the bead mean diameter may be at least about 1 nm, 10 nm, 100 nm, 200 nm, 500 nm, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 12 ⁇ m, 14 ⁇ m, 16 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 50 ⁇ m, 80 ⁇ m, 100 ⁇ m, 90 ⁇ m, 100 ⁇ m, 120 ⁇ m, 140 ⁇ m, 160 ⁇ m, 180 ⁇ m, 200 ⁇ m, 300 ⁇ m, or larger.
  • the bead size may differ based on the application.
  • the bead size may be at most about 300 ⁇ m, 200 ⁇ m, 160 ⁇ m, 100 ⁇ m, 80 ⁇ m, 50 ⁇ m, 40 ⁇ m, 30 ⁇ m, 20 ⁇ m, 10 ⁇ m, 8 ⁇ m, 6 ⁇ m, 4 ⁇ m, 3 ⁇ m 2 ⁇ m, 1 ⁇ m, 500 nm, 200 nm, 100 nm, 10 nm, 1 nm, or less.
  • the bead mean diameter may be at least about 1 nm, 10 nm, 100 nm, 200 nm, 500 nm, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 12 ⁇ m, 14 ⁇ m, 16 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 50 ⁇ m, 80 ⁇ m, 100 ⁇ m, 90 ⁇ m, 100 ⁇ m, 120 ⁇ m, 140 ⁇ m, 160 ⁇ m, 180 ⁇ m, 200 ⁇ m, 300 ⁇ m, or larger.
  • the bead size may differ based on the application. In some examples, the bead size may be at most about 300 ⁇ m, 200 ⁇ m, 160 ⁇ m, 100 ⁇ m, 80 ⁇ m, 50 ⁇ m, 40 ⁇ m, 30 ⁇ m, 20 ⁇ m, 10 ⁇ m, 8 ⁇ m, 6 ⁇ m, 4 ⁇ m, 3 ⁇ m 2 ⁇ m, 1 ⁇ m, 500 nm, 200 nm, 100 nm, 10 nm, 1 nm, or less.
  • the size of the bead may differ in different solvents. In some cases, the bead mean diameter may at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 micrometers ( ⁇ m) in water.
  • the population of beads may comprise or be a plurality of beads according to the beads provided anywhere herein.
  • the beads may comprise a resin suitable for generating an encoded effector library, such that the bead is compatible with synthesizing effectors and barcodes thereon and/or therein.
  • the beads may be used for synthesizing encoded effector libraries.
  • the beads may comprise effectors and/or barcodes bound thereto.
  • the population of beads may comprise a bead diameter distribution.
  • the bead diameter distribution may be narrow.
  • the bead diameter of the bead population may be within a narrow range.
  • Bead monodispersity may lead to the homogeneity of effector loading (e.g., a low variability of loaded effectors) on the beads of the bead population.
  • Bead diameter distribution may be characterized using coefficient of variance (CV %).
  • CV % coefficient of variance
  • the CV of the bead population may be at most about 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 5%, 3%, 2%, 1%, or less.
  • CV of the bead population may be at most about 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 5%, 3%, 2%, 1%, or less.
  • a bead comprising or made of a substantially homogeneous bead polymer material, an effector, and a barcode corresponding to and identifying the effector.
  • the effector and the barcode may be according to any effector and barcode described anywhere herein.
  • the bead may be of any shape. In some cases, the bead may be a spherical or semi- spherical bead (e.g., bead resin). In some examples, the bead may be non-spherical. For example, the bead may comprise edges and planes. For example, a bead may be cubical, cylindrical, rectangular, or polyhedral. [000149] The bead may be made of a suitable material. In some cases, the bead may be a hydrogel bead. The bead may comprise or be made of a substantially homogenous polymer material.
  • the homogeneous polymer material may be compatible with solid phase peptide synthesis (SPPS).
  • SPPS solid phase peptide synthesis
  • the effector may be a chemical compound synthesized on the bead and/or inside the bead.
  • effector molecules may be synthesized within the entire bead sphere volume including on the surface of the sphere and in the interior volume of the sphere.
  • the effector molecules may be synthesized in the internal volume but not on the surface.
  • the effector molecules may be synthesized on the surface of the bead or in a shell near the surface of the bead, but not in the interior of the bead.
  • the bead may be customized based on the application.
  • the effector molecules may be localized to a selected location inside and/or on the bead (e.g., interior section, peripheral section, on the surface, or near the core). In some cases, such localization may be performed by partitioning the bead through diffusion-controlled modifications of Attorney Docket No.56523-707.601 reactive sites.
  • the bead may be polymerized with selective functional groups between the outer and inner layers of the bead matrix. [000150]
  • the bead may not comprise a solid/semi-solid core comprising a material different from the substantially homogeneous bead polymer material. In some cases, the bead may not comprise a core-shell structure.
  • the bead may not comprise a hard core or a core made of a material different from the shell.
  • the bead may not comprise a polystyrene core.
  • the bead may not be a polystyrene core grafted with a polymer such as Polyethylene glycol (PEG).
  • PEG Polyethylene glycol
  • the bead may be a bead other than TentaGel bead.
  • the bead may be substantially homogeneous.
  • the internal volume of the bead, the center of the bead, and the areas near the surface of the bead may be made of the same material, such as a hydrogel.
  • the bead may tolerate the conditions for chemical synthesis.
  • the core of the bead may be substantially comprised of or consisted essentially of a hydrogel.
  • the shell or peripheral portion of the bead may be substantially comprised of or consisted essentially of a hydrogel.
  • the hydrogel may comprise or be PEG.
  • the bead made of the soft material e.g., a bead not comprising a polystyrene core
  • may tolerate suspension in solvents such as water, DMA, DMF, ACN, DCE, Methanol, Dioxane, Diethylether, Ethanol, Isopropanol, and Acetone.
  • solvents such as water, DMA, DMF, ACN, DCE, Methanol, Dioxane, Diethylether, Ethanol, Isopropanol, and Acetone.
  • Tolerance may include being compatible with a condition.
  • the bead may remain substantially intact, uncompromised, undamaged, morphologically intact, properly shaped, structured, unresolved, and well-behaved during and after suspension in such solvents (e.g., both aqueous and organic solvents) as well as other conditions the bead experiences during encoded effector synthesis.
  • solvents e.g., both aqueous and organic solvents
  • the durability, resilience, and sturdiness of the structure may be facilitated by the soft material (e.g., substantially homogeneous polymer material or hydrogel) itself as opposed to a hard/polystyrene core at the center of the bead.
  • the bead may comprise swelling properties suitable for chemical synthesis according to the methods of the present disclosure.
  • the bead may be swelled in solvents such as water, Dimethylacetamide (DMA), Dimethylformamide (DMF), Acetonitrile (ACN), 1,2-Dichloroethane DCE, Methanol, Dioxane, Diethylether, Ethanol, Isopropanol, Dichloromethane (DCM), N-Methyl-2-pyrrolidone (NMP), Dimethyl sulfoxide (DMSO), Ethyl Acetate, and Acetone.
  • solvents such as water, Dimethylacetamide (DMA), Dimethylformamide (DMF), Acetonitrile (ACN), 1,2-Dichloroethane DCE, Methanol, Dioxane, Diethylether, Ethanol, Isopropanol, Dichloromethane (DCM), N-Methyl-2-pyrrolidone (NMP), Dimethyl sulfoxide (DMSO), Ethyl Acetate, and Acetone.
  • the bead may be at least about 5%, 10%, 15%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% swelled in each of the mentioned solvents, or more.
  • the diameter of the bead in each of the solvents may be at least about 5%, Attorney Docket No.56523-707.601 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% larger than its dry diameter.
  • the swelling percentage of the bead may be at most about 500%, 400%, 300%, 200%, 100%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5% or to a lesser extent larger.
  • the bead may be at least about 5%, 10%, 15%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or to a greater extent de-swelled or shrunk in a solvent such as water, DMA, DMF, ACN, DCE, Methanol, Dioxane, Diethylether, Ethanol, Isopropanol, DCM, NMP, DMSO, Ethyl Acetate, and Acetone.
  • a solvent such as water, DMA, DMF, ACN, DCE, Methanol, Dioxane, Diethylether, Ethanol, Isopropanol, DCM, NMP, DMSO, Ethyl Acetate,
  • the diameter of the bead in each of the solvents may be at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% smaller than its dry diameter.
  • the de-swelling or shrinkage percentage of the bead may be at most about 500%, 400%, 300%, 200%, 100%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5% or to a lesser extent smaller than its diameter when dry. Bead de-swelling may facilitate effector and/or barcode synthesis.
  • the soft material of the bead/bead resin may comprise functional groups for effector synthesis and the bead may be compatible with such chemical synthesis.
  • the polymer material may be made of monomers comprising functional groups for effector/barcode synthesis.
  • the polymer resin may be made of monomers which do not comprise functional groups.
  • functional groups may be decorated on the surface of the bead during or after the synthesis (e.g., if the monomers of the polymer resin themselves are non-functional). In some cases, a combination of both approaches may be applied.
  • the bead may be compatible with peptide synthesis (e.g., solid phase peptide synthesis (SPPS)).
  • SPPS solid phase peptide synthesis
  • the bead may remain substantially morphologically intact and/or unresolved during solid phase peptide synthesis.
  • the bead may not collapse or get dissolved.
  • the bead may not get damaged.
  • the bead may not crack.
  • the bead may not clump or aggregate to a significant degree.
  • the bead may remain swelled or substantially swelled during the synthesis or as long as the bead is in a solution.
  • the bead may be a sphere comprising an inner core and a peripheral section.
  • the bead comprises an external surface.
  • the peripheral section may be a shell near the surface of the bead which may be surrounding the inner core of the bead.
  • the peripheral section may comprise a thickness.
  • the thickness of the peripheral section of the bead may be a portion of the diameter of the bead.
  • the thickness of the peripheral Attorney Docket No.56523-707.601 section/shell may be at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40% of the bead diameter, or larger.
  • the thickness of the peripheral section/shell may be at most about 30%, 20%, 15%, 10%, 5%, 2%, 1%, 0.5%, 0.3%, 0.1% of the bead diameter or smaller.
  • the inner core may comprise or be a hard core (e.g., a core harder than the peripheral section of the bead).
  • the core may be a polymer, a gel, a hydrogel, polystyrene, a solid core, glass, a magnetic material, Iron, a hydrogel, silicon dioxide, silica, silicon, a crystal, crystalized silicon, europium, turbium, gold, silver or another material.
  • the inner core may be a solid core.
  • TentaGel beads comprise a polystyrene core.
  • the inner core and the peripherical section of the bead may be substantially composed of the same material (e.g., a soft polymer material comprised of a monomer unit which may in some cases comprise functionality for effector synthesis).
  • the inner core may be a polymer material.
  • the inner core may be a substantially soft material.
  • the inner core of the bead may not be a hard sphere.
  • the inner core of the bead may not be a hard core.
  • the inner core of the bead may not be a polystyrene core.
  • the inner core may comprise or be a hydrogel.
  • the inner core may be consisted essentially of a hydrogel.
  • the inner core may comprise or be PEG.
  • the inner core may comprise or be PEG-DA.
  • the inner core of the bead may comprise functional groups.
  • the bead may comprise functional groups for synthesizing effectors.
  • the substantially homogenous polymer material and/or the monomer building blocks thereof may comprise functional groups for synthesizing/conjugating effectors.
  • the inner core of the bead may comprise functional groups for synthesizing effectors.
  • the functional groups for synthesizing effectors may comprise Amine groups, hydroxyl groups, Alkyne groups.
  • the inner core may be functional.
  • the inner core may comprise effectors covalently bound thereto or therein.
  • the covalent bond may comprise a cleavable linker described anywhere herein.
  • the bead may further comprise functional groups for oligonucleotide synthesis or conjugation.
  • functional groups for oligonucleotide synthesis/conjugation may comprise or be Azide and/or hydroxyl groups.
  • Synthesis may comprise conjugation, coupling, covalent reactions, chemical ligation, attachment, metathesis, or other suitable synthesis methods and/or reactions.
  • the bead may comprise functional groups for effector synthesis (e.g., Amine groups) and functional groups for oligonucleotide synthesis/conjugation (e.g., Azide groups, alkyne groups, and/or hydroxyl group).
  • the functional groups for oligonucleotide synthesis may comprise at least about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or more molecules per bead.
  • the bead comprises an inner portion, an external surface, and a peripheral section.
  • functional groups for oligonucleotide synthesis may be substantially located on or near the external surface (e.g., in a peripheral section/shell of the bead), and the functional groups for synthesizing the effector may be substantially near or inside the inner portion. This feature may be advantageous for screening an encoded effector library.
  • This feature may decrease or eliminate non-specific binding of biological targets (e.g., in a compartment) to the effector, and/or avidity-driven effects which may interfere with the fidelity of effector screening.
  • containing the effectors in the inner portion of the bead may reduce non-specific effector-target interactions. It may reduce interactions between the effector and the target before effector release.
  • it may be intended for the effector to not interact with the target until the cleavable linker is cleaved and the released effector is generated upon releasing the bead-bound effector. Containing the effector in the inner portion of the bead by limiting the functionality to the inner portion of the bead may address this goal.
  • FIG. 3A An example bead according to the methods of the present disclosure is shown in FIG. 3A.
  • the bead 300 comprises an inner core 301 and a peripheral section 302.
  • the bead comprises functional groups for oligonucleotide synthesis 303 and functional groups for synthesizing the effector 304.
  • the functional groups are shown to be mostly on the surface of the bead.
  • the bead is shown to comprise a core 301 made of a material different from the material making the peripheral section 302.
  • An example of this may be TentaGel beads.
  • TentaGel beads comprise a polystyrene core (inner core 301) grafted with PEG-DA (the material of the peripheral section 302).
  • Functional groups may exist in the interior of the bead resin including in the peripheral section and in the inner core.
  • the number of functional groups in the inner core may be less than other bead embodiments in which the bead does not have the polystyrene core (e.g., as shown in FIG. 3B).
  • FIG. 3B Another example of a bead provided in the present disclosure is provided in FIG. 3B.
  • the bead 310 shown in FIG.3B comprises an inner section or inner core 301 made of a material substantially similar to the material making the peripheral section 302.
  • the bead materials that are substantially homogenous throughout the bead’s inner core and peripheral section may be made of monomer building blocks making up the polymer bead resin upon a Attorney Docket No.56523-707.601 polymerization reaction.
  • the polymer bead resin and/or the monomer building blocks thereof may comprise functional groups for oligonucleotide synthesis 303 and functional groups for effector synthesis 304.
  • the functional groups are comprised in the monomer building blocks.
  • the functional groups may be decorated on the bead resin at some point during the bead synthesis or thereafter. In some cases, a combination of both approaches may be implemented.
  • the monomer building blocks may be prepared and subjected to a stimulus (e.g., light, heat, or chemical) for the polymerization reaction to initiate.
  • the functional groups may be decorated on the surfaces of the bead and the peripheral section of the bead during the polymerization reaction.
  • the monomer building blocks of the polymer resin may or may not comprise the functional groups for oligonucleotide synthesis and/or for effector synthesis.
  • the bead of the present disclosure may comprise an effector.
  • the bead may further comprise a barcode that encodes the identity and/or structure of the effector.
  • the effector may be on or near the bead surface.
  • the effector may be in the inner section of the bead (e.g., near the center/core of the bead).
  • the barcode may comprise various embodiments and modalities and may be anywhere on/in the bead.
  • the barcode may be a nucleic acid molecule.
  • the barcode may comprise or be an optical barcode.
  • the optical barcode may comprise or be a particle.
  • the optical barcode may be on the surface of the bead or inside the bead.
  • the barcode may be a fluorescent or luminescent material, a material with surface plasmon resonance property.
  • the barcode may comprise or be a barcode particle.
  • the barcode may comprise a plurality of barcoding particles.
  • the barcode or barcoding particle(s) may comprise fluorescent or luminescent properties and may be detectable by fluorescence or luminescence. In some cases, more than one barcoding modality may be used on/in the bead.
  • a bead may comprise both a nucleic acid barcode and an optical barcode.
  • the effector on the bead may be any effector described anywhere herein.
  • the effector may be covalently bound to the bead through a cleavable (e.g., photocleavable) linker.
  • the effector may be a small molecule comprised of one or more (e.g., 1, 2, 3, 4, or more) subunits (e.g., building blocks of the effector).
  • the subunits of the effector may be encoded by the subunits of the barcode (e.g., sequences of the nucleic acid barcode).
  • the bead may be a spherical bead made with the aid of a microfluidic droplet generator.
  • the microfluidic device used for bead generation may comprise a flow focusing droplet generation junction. Examples of this include the droplet extrusion region 204 shown in FIG.4, or any other microfluidic droplet generation geometry.
  • a monomer mixture (dispersed phase) comprising one or more monomers may be introduced into a first inlet (e.g., an aqueous inlet) of a microfluidic droplet generator.
  • the one or more monomers may be building blocks or subunits of the bead resin which may be configured to form the polymer beads upon reacting (e.g., polymerization).
  • An oil (continuous phase) immiscible with the monomer mixture may be introduced into a second inlet (e.g., an oil inlet of the microfluidic droplet generator).
  • the monomer mixture and the oil may meet at the droplet formation region/junction of the microfluidic droplet generator.
  • the microfluidic droplet generator may generate spherical droplets comprising the monomer mixture. These droplets may be termed as the monomer droplets.
  • the components of the monomer mixture e.g., the monomers
  • the reaction taking place among the monomers of the monomer mixture may be any suitable polymerization reaction which bonds and connects the monomers together to form a polymer bead.
  • the polymerization reaction may be initiated upon application of a stimulus.
  • the stimulus may comprise application of an energy.
  • the energy may comprise heat, light, or both.
  • the energy may be light (e.g., UV light).
  • a droplet made from a monomer mixture may polymerize into a bead described anywhere herein upon exposure to UV. UV exposure may be performed according to any UV exposure method and system described anywhere herein.
  • the polymerization reaction may be initiated by a chemical or a change in PH.
  • the polymerization reaction may comprise a redox reaction.
  • the polymerization may initiate and complete its course within a set period.
  • the set period may be at least about 1 second (s), 10 s, 30 s, 40 s, 50 s, 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 10 min, 20 min, 30 min, 40 min, or above.
  • the emulsions/droplets may be broken such as by washing the beads with a proper solvent.
  • the polymerization reaction may be performed in an integrated microfluidic device, such as the microfluidic device shown in FIG.4, or any other microfluidic device provided herein which may comprise a UV exposure region.
  • the UV exposure region may be similar to or the same as the cleavage region 206 shown in FIG.4.
  • a UV-Waveguide may be inserted into the device in close proximity to the UV exposure region (e.g., cleavage region 206) to expose the monomer droplets to UV light Attorney Docket No.56523-707.601 (stimulus).
  • the stimulus in this case UV exposure, may initiate the polymerization of the monomer droplets, thereby generating polymerized droplets.
  • the polymerized droplets may be collected from an outlet port of the device.
  • the polymerized droplets may undergo one or more post-processing and/or washing steps to break the emulsions and remove oil and surfactant from the droplet-in-oil emulsion.
  • the excess/residual un-crosslinked monomers may be removed during the washing steps.
  • the polymerized droplets may convert into polymer/hydrogel beads.
  • the generated polymer/hydrogel beads may be according to the bead, and/or bead material resins described anywhere herein, and may be used for synthesized encoded effectors. In some embodiments, such beads do not comprise a polystyrene core.
  • the inner core of the bead and the peripheral sections closer to the external surface of the bead may consist of substantially the same material.
  • such material may be PEG [000168]
  • the UV exposure may not be performed in an integrated microfluidic circuit (e.g., such as a microfluidic device shown in FIG. 4).
  • photopolymerization may be performed in bulk. Bulk photopolymerization may comprise generating a plurality of monomer droplets (e.g., droplets consisted of a polymerizable monomer, such as PEG monomers or any suitable monomer described anywhere herein). The plurality of monomer droplets may then be exposed to a stimulus for initiating the polymerization reaction.
  • the stimulus may be light (e.g., UV light).
  • UV light may be exposed to the droplets in bulk.
  • the droplets may be collected from the microfluidic device (e.g., through the outlet port) into one or more collection containers (e.g., tubes, wells, any suitable container or compartment, or any combination thereof).
  • the monomer droplets may then be exposed to the stimulus (e.g., in the collection container, such as after exiting the chip).
  • the container e.g., as a whole or in part may
  • the stimulus may initiate the polymerization reaction.
  • the stimulus for initiating polymerization may comprise a chemical.
  • such chemical may act as a catalyst.
  • the catalyst may comprise cupper (Cu).
  • the stimulus for initiating polymerization may comprise heat.
  • Heat may be applied to monomer droplets in an integrated microfluidic device (e.g., on a microscope stage or using a conductive heater, Infrared Radiation (IR light) heater, a microwave, or other suitable heating device or method for thermal/heat transfer) of the present disclosure.
  • the droplets may be collected from the microfluidic device and exposed to the stimulus after collection. In some Attorney Docket No.56523-707.601 cases, the droplets may exit the chip through an outlet tubing.
  • the droplets may be exposed to the stimulus (e.g., heat, light, or other reasonable kind of stimulus while travelling through the outlet tubing).
  • the outlet tubing may be placed in a hot medium or heat source (e.g., hot water, hot oil, heated housing, or another type of heat source).
  • the outlet tubing may pass through a light region and be exposed to light.
  • the beads used for building the encoded effector libraries of the present disclosure is composed of a polymer material which is homogeneous inside the bead sphere.
  • the beady may not comprise a core and shell structure.
  • the bead may not comprise a core made of a material different from its shell.
  • the bead may not comprise a polystyrene core.
  • the bead may comprise PEG.
  • the bead may be a homogeneous PEG- containing polymer.
  • the bead resin may be consisted essentially of PEG.
  • the bead resin may be comprised of at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 95%, 98%, 99%, or 100% PEG.
  • the bead resin may comprise at most about 100%, 99.9%, 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5% or less PEG.
  • a plurality of beads may be used for encoded effector library synthesis.
  • Library synthesis may comprise split and pool combinatorial solid phase chemical synthesis (e.g., solid phase peptide synthesis (SPPS)) as described anywhere herein.
  • a bead used for library synthesis may comprise or be made of a material compatible with synthetic chemistry, chemical synthesis, solution phase chemical synthesis, solid phase chemical synthesis, solid phases synthesis (SPS) such as solid phase peptide synthesis (SPPS), native chemical ligation, catalytic/enzymatic reactions (e.g., ligation, peptide ligation, restriction enzyme digestion, polymerization reactions), and other chemical synthesis methodologies.
  • the bead may be compatible with conditions used for screening.
  • Chemical synthesis may comprise using solvents (e.g., organic and/or mineral solvents).
  • the beads may be suspended in solvents (e.g., organic solvents) during library (e.g., effector library) synthesis.
  • the material of the bead may be compatible with the organic solvents used for library synthesis.
  • the bead may remain substantially morphologically intact, solid, uncompromised, unresolved, integrated, and/or swelled during SPPS and/or in organic solvents.
  • the bead may be compatible with solvents such as Iso-propanol, Dioxane, THF, and/or TFA.
  • the bead may be compatible with water, DMA, DMF, CAN, DCE, DCM, Diethylether, Ethanol, and Acetone.
  • the bead may be compatible with organic solvents and do not get resolved or otherwise physically damaged during effector synthesis (e.g., Attorney Docket No.56523-707.601 synthesis of a small molecule on the bead through SPS).
  • the bead may be stable in a broad range of PH conditions. In some cases, the bead may be stable in PH 3-14.
  • the bead may be compatible with oligonucleotide synthesis and manipulation.
  • the bead may be compatible with DNA ligation, polymerization, restriction digestion, transcription, RNA reverse-transcription, and translation.
  • the beads may be compatible with enzymatic transformation reactions.
  • enzymes which may be used in performing reactions on, in, or in proximity of the beads may comprise using proteases, reductases, dehydrogenases, or other catalysts (e.g., biocatalysts and/or enzymes).
  • the bead may comprise functional groups or functional sites capable of synthesis of chemical compounds (e.g., effector compounds and/or barcodes).
  • functional groups may comprise or be amine groups.
  • the amount of functional groups on a bead may be at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900 fmol/bead, 1 pmol/bead, 10 pmol/bead, 100 pmol/bead, or more.
  • the number of functional sites on the bead may be at most 100 pmol/bead, 10 pmol/bead, 1 pmol/bead, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, 80, 50, 40, 30, 20, 10 fmol/bead or less.
  • the beads may be suspended in a solution, suspension, or emulsion.
  • the present disclosure provides a solution comprising a bead population.
  • the bead population may comprise a plurality of beads.
  • the plurality of beads may be according to any bead embodiment presented herein.
  • provided herein is an emulsion.
  • the emulsion may comprise a water in oil emulsion, buffer in oil emulsion, or monomer droplets surrounded by oil.
  • the emulsion may comprise a droplet or a plurality of droplets.
  • the emulsion may be a droplet in oil emulsion.
  • the droplets may be compartments for assay screening and/or encoded effector screening.
  • the droplets may be surrounded by an oil and surfactant.
  • the surfactant may insulate the internal volume of the droplet from the surrounding oil.
  • the droplet may comprise a bead according to the descriptions provided herein.
  • a bead provided herein may be encapsulated in a droplet.
  • a plurality of beads may be encapsulated in a plurality of droplets.
  • the droplets may be generated with the aid of a microfluidic device.
  • the droplets may be generated via bulk emulsification or vortexing. [000176] Provided herein is a method of bulk emulsification or vortex-based emulsification.
  • a plurality of beads may be provided in an aqueous solution in a container or compartment.
  • the container or compartment may be a tube of any size or Attorney Docket No.56523-707.601 shape, a confinement, or a platform (e.g., a platform comprising multiple compartments, such as any compartment mentioned anywhere herein, in some cases, a multiple well plate for generating and/or containing the emulsion therein).
  • the compartment or plurality of compartments may be a plurality of wells.
  • one or more (e.g., a plurality of) compartment(s) may be obtained or provided which may be part of a single unit or separate units.
  • a plurality of beads may be provided in an aqueous solution.
  • a compartment may further comprise a volume of oil which is immiscible with the aqueous solution.
  • the aqueous solution may comprise assay reagents and/or biological targets according to the descriptions provided anywhere herein which may be used to detect or quantify an activity of the biological target in presence or absence of a bead-bound encoded effector.
  • the compartment or plurality of compartments may be subjected to vortexing. Vortexing may break up the aqueous solution into a plurality of droplets (e.g., a droplet in oil emulsion).
  • the plurality of droplets may encapsulate the plurality of beads therein.
  • a subset of the plurality of droplets may each comprise at least 0, 1, 2, 3, or more beads encapsulated therein.
  • a subset of the plurality of droplets may each comprise at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 beads.
  • a subset of the plurality of droplets may remain empty.
  • the beads may act as a template for the droplets.
  • the presence of the beads in the aqueous solution may facilitate generating droplets that are substantially monodisperse or uniform in size (e.g., compared to droplets generated by vortexing in absence of beads).
  • the droplets containing beads may be used as compartments for performing screens using the methods and systems provided anywhere herein.
  • the compartments used for screening may comprise droplets.
  • the droplets may be hydrogel droplets.
  • a hydrogel droplet may be suitable for cell screening.
  • a hydrogel droplet may comprise an encoded effector synthesized on a bead according to the methods and systems of the present disclosure.
  • the hydrogel droplet may further comprise a cell. The cell may be in proximity of the bead, inside the hydrogel droplet.
  • the hydrogel droplet may provide a suitable matrix and environment for the cell.
  • the exemplary microfluidic device for performing the methods of the present disclosure is shown in FIG. 4.
  • the exemplary microfluidic device contains a first inlet 201.
  • the first inlet 201 is configured to accept an aqueous fluid, such as an aqueous assay reagent.
  • the exemplary microfluidic device also contains a second inlet 202.
  • the second Attorney Docket No.56523-707.601 inlet 202 is configured to accept another aqueous fluid. This may be the same or different as the aqueous fluid added to the first inlet 201.
  • the second inlet 202 may be configured to accept beads as provided herein, or the first inlet 201 may be so configured.
  • the exemplary microfluidic device shown in FIG.4 further comprises a third inlet 203 for carrier fluid (e.g. an oil immiscible with an aqueous fluid) in fluid connection with a droplet formation junction or extrusion junction 204.
  • carrier fluid e.g. an oil immiscible with an aqueous fluid
  • the inlet 203 in this example is connected to the droplet formation junction 204 by two channels, each reaching an aqueous stream channel at the same point on opposite sides of the aqueous stream channel.
  • the droplet formation junction 204 comprises a microfluidic channel that continues down the flow path towards cleavage region 206.
  • the cleavage region may also be referred to as the exposure region.
  • Near cleavage region or exposure region 206 is a fiberoptic waveguide 205 configured to deliver light (e.g., UV light) into the microfluidic channel of the cleavage region 206 for any reason (e.g., cleave a cleavable linker, polymerize a hydrogel, or otherwise stimulate the compartment for any intended purpose in the present disclosure)
  • the fiberoptic waveguide 205 may be embedded in the plane of the device such that the light emitted enters the microfluidic channel of cleavage region/exposure region 206 from the device plane.
  • the device also comprises an inlet for calibration fluid 207a in fluid connection with the cleavage region 106 and an outlet for calibration fluid 207b.
  • the inlet for calibration fluid 207a is configured to receive and deliver to the cleavage region 206 a fluid configured to normalize photon exposure within the cleavage region. After passing through the cleavage region 206, the calibration fluid exits through the outlet for calibration fluid 207b.
  • the cleavage region 206 is in fluid communication via a microfluidic channel to an incubation region 209.
  • the incubation region 209 contains a series of widened and deep chambers, each chamber connected to the next chamber in series by a microfluidic channel.
  • the configuration of these chambers affects the flow rate and residence time of the droplets formed at droplet formation region 204 through the device as well as a dispersion ratio of the incubation times of the droplets through the channel.
  • the chambers are configured to prevent trapping of droplets as they pass through incubation region 209.
  • Such configuration of the chambers is particularly important when using a carrier fluid that is denser than the aqueous droplets (e.g. 3-ethoxyperfluoro(2-methylhexane)).
  • this configuration is achieved by configuring the chambers and connecting channels to have only small difference in channel height between the chambers and the connecting channels.
  • the height of the chamber is about 80 ⁇ m and the height of the connecting channel is about 50 ⁇ m.
  • the height of the flow path does not change between the width of the chamber has been narrowed as the droplet approaches the connecting channel, thus facilitating the smooth transition of droplets from chamber to chamber without trapping.
  • Configured on either end of incubation region 209 are bypass shunts 208a and 208b.
  • the bypass shunts 208a and 208b are configured to allow a fluid coupled to the shunt to flow in or out of the main microfluidic channel.
  • incubation region 209 Positioned downstream of incubation region 209 is inlet for carrier fluid 210.
  • Inlet for carrier fluid 210 is in fluid communication with the main microfluidic channel of the device and is configured to deliver additional immiscible carrier fluid into the main microfluidic channel in order to space droplets as desired.
  • inlet for carrier fluid 211 Also in fluid communication with the main microfluidic channel is inlet for carrier fluid 211, which is configured to deliver droplet focusing oil into the main microfluidic channel. Downstream of inlets for carrier fluid 210 and 211 is detection position 216.
  • the detection position 216 indicates the point on the device that the desired signal from the assay being run on the chip is detected.
  • the detection position 216 may be based on an alignment of an objective or fiber that directs an excitation light at the sample passing detection position 216 and an additional objective or fiber coupled to a detector configured to detect an emission from detection position 216.
  • the objective for the excitation light may be configured to also collect the emission.
  • the excitation source is reflected from detection position 216 through an inverted objective lens, where the emission is collected, columnated, and directed through optical fibers for quantification by a photomultiplier tube (PMT) or other detector.
  • PMT photomultiplier tube
  • the objective or fiber aligned at detection position 216 is not coupled to the device.
  • the detector or emission objective or fiber can be moved to adjust the detection positions 216 on the device in order to adjust the time between detection and sorting.
  • the detector or emission objective may also be moved for use in calibration of the device or initiation of the device, thus allowing a single light source to be used for multiple functions.
  • Downstream of inlets for carrier fluid 210 and 211 and detection position 216 is discrimination junction electrode 212.
  • the discrimination junction electrode 212 may be a dielectrophoresis electrode configured to propel droplets down outlet 214 if the droplet is determined to display a signal with a predefined criteria (e.g., above or below a given threshold, such as a threshold defined for hits or a subpopulation selected for post-processing) or to outlet/waste path 215 if the droplet is determined to lack a signal fitting into the predefined criterial/threshold according to any method described anywhere herein.
  • the discrimination Attorney Docket No.56523-707.601 junction electrode 212 is connected to a discrimination junction ground circuit, which is connected to the device at circuit connection point 213a. In some cases, an Optical Glue is displayed within the fiberoptic waveguide.
  • the Optical Glue helps to minimize scattering of the light from the fiberoptic wave guide.
  • beads may be provided with an aqueous fluid via an inlet (e.g., inlet 201, inlet 203, or inlet 218).
  • the beads are suspended in the aqueous fluid, and provided from a bead source.
  • a tubing or other channel (“bead tubing”) provides fluidic communication between the bead source and a microfluidic device described herein.
  • said bead tubing comprises a rigid material.
  • a vibration motor or other vibration generating device is configured vibrate the bead tubing so as to maintain the beads as being suspended within the bead tubing (e.g., suspended within an aqueous fluid), and/or help maintain the beads as being spaced apart from each other.
  • the beads may settle along the walls of the bead tubing.
  • the beads may also or alternatively agglomerate together thereby forming “clumps” of beads. In some instances, such “clumps” of beads lead to ineffectual screening of assays and/or readings from signal measurement.
  • a vibration motor or other vibration generating device is configured vibrate the bead tubing so as to prevent or reduce the beads from settling within the bead tubing (for example, settling along the walls of the bead tubing).
  • the vibration motor is configured to deliver high frequency vibration and/or low power (i.e., low amplitude of vibration of frequency).
  • the vibration frequency provided is optimize so as to prevent or reduce beads settling within the bead tubing, but also to prevent or reduce such vibration cascading to the flow profile of the beads within the bead tubing.
  • the vibration motor provides a vibration at a frequency of about 100Hz to about 200 Hz. In some embodiments, the vibration motor provides a vibration at a frequency of about 50Hz to about 300 Hz, or of about 25 Hz to about 500 Hz. In some embodiments, the vibration motor is coupled to the bead tubing. In some embodiments, the bead tubing passes through a channel within the vibration motor. In some embodiments, the vibration motor comprises a haptic motor.
  • beads settling out in the bead tubing results in an accumulation of beads within the bead tubing and may Attorney Docket No.56523-707.601 prevent beads from being disposed on the microfluidic chip.
  • such bead settling within a bead tubing is identified based on the detection and/or sorting region of the microfluidic chip, wherein no beads are detected with the corresponding measurements.
  • a feedback controller is provided and configured to modify the operation mode of a vibration motor based on no beads being detected.
  • a feedback controller in communication with such detection or sorting region sends a signal to the vibration motor to turn on, or adjust the vibration frequency, so as to “unsettle” the beads within the bead tubing.
  • the feedback controller may turn off the vibration motor or revert the vibration frequency to a predetermined value.
  • Near cleavage region 206 is a UV waveguide 205 configured to deliver light into the microfluidic channel of the cleavage region 206.
  • the UV waveguide is a fiberoptic wave guide.
  • the UV waveguide 205 is embedded in the plane of the device such that the light emitted enters the microfluidic channel of cleavage region 206 from the device plane.
  • the UV waveguide comprises a parabolic lens at an end closest to the cleavage region.
  • the parabolic lens is configured to columnate light inside the cleavage region.
  • the parabolic lens, or a curved lens minimizes the tendency for the light from the UV waveguide to be scattered.
  • the cleavage region is exposed to UV light projected normal to the circuit plane, exposing a defined area to UV where the compound is cleaved.
  • an Optical Glue 217 is provided with the UV waveguide.
  • the Optical Glue 217 helps to minimize light being scattered by UV waveguide.
  • near cleavage region 206 may be a pillar (not shown) configured to fix a fiberoptic manifold which can be configured to emit light from above the plane of the device into the microfluidic channel of cleavage region 206.
  • the device also comprises an inlet for calibration fluid 207a in fluid connection with the cleavage region 206 and an outlet for calibration fluid 207b.
  • the inlet for calibration fluid 207a is configured to receive and deliver to the cleavage region 206 a fluid configured to normalize photon exposure within the cleavage region.
  • the cleavage region 206 comprises a serpentine flow path. After passing through the cleavage region 206, the calibration fluid exits through the outlet for calibration fluid 207b.
  • the cleavage region 206 is in fluid communication via a microfluidic channel to an incubation region 209.
  • the chambers are configured to prevent trapping of droplets as they pass through incubation region 209.
  • Such configuration of the chambers is particularly important when using a carrier fluid that is denser than the aqueous droplets (e.g., 3-ethoxyperfluoro(2- methylhexane)).
  • the height of the chamber is about 30 ⁇ m to about 1,000 ⁇ m.
  • collection chambers 219 are optionally provided with this exemplary microfluidic device.
  • Configured on either end of incubation region 209 are bypass shunts 208a and 208b.
  • the bypass shunts 208a and 208b are configured to allow a fluid coupled to the shunt to flow in or out of the main microfluidic channel.
  • inlet for carrier fluid 210 Positioned downstream of incubation region 209 is inlet for carrier fluid 210.
  • Inlet for carrier fluid 210 is in fluid communication with the main microfluidic channel of the device and is configured to deliver additional immiscible carrier fluid into the main microfluidic channel in order to space droplets as desired.
  • inlet for carrier fluid 211 Also in fluid communication with the main microfluidic channel is inlet for carrier fluid 211, which is configured to deliver droplet focusing oil into the main microfluidic channel.
  • downstream of inlets for carrier fluid 210 and 211 is detection position 216.
  • the detection position 216 indicates the point on the device that the desired signal from the assay being run on the chip is detected.
  • the detection position 216 may be based on an alignment of an objective or fiber that directs an excitation light at the sample passing detection position 216 and an additional objective or fiber coupled to a detector configured to detect an emission from detection position 216.
  • the objective for the excitation light may be configured to also collect the emission.
  • the excitation source is reflected from detection position 216 through an inverted objective lens, where the emission is collected, columnated, and directed through optical fibers for quantification by a photomultiplier tube or other detector.
  • the objective or fiber aligned at detection position 216 is not coupled to the device.
  • the detector or emission objective or fiber can be moved to adjust the detection positions 216 on the device in order to adjust the time between detection and sorting.
  • the detector or emission objective may also be moved for use in calibration of the device or initiation of the device, thus allowing a single light source to be used for multiple functions.
  • Downstream of inlets for carrier fluid 210 and 211 and detection position 216 is discrimination junction electrode 212.
  • the discrimination junction electrode 212 may be a dielectrophoresis electrode configured to propel droplets down outlet 214 if the droplet is determined to display a desired signal or to outlet 215 if the droplet Attorney Docket No.56523-707.601 is determined to lack a desired signal.
  • the diameter of the droplets formed in the droplet generation junction may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 micrometers/microns (um) or larger. In some cases, droplet diameter may be at most about 100, 90, 80, 70, 60, 50, 40, 30, 20 microns or smaller.
  • the droplet may be of any suitable volume. In some cases, the droplet may be from about 1 picolitres to 500 picolitres. [000187] examples, the droplets are placed in an oil emulsion.
  • the oil comprises a silicone oil, a fluorosilicone oil, a hydrocarbon oil, a mineral oil, a paraffin oil, a halogenated oil, a fluorocarbon oil, or any combination thereof.
  • the oil comprises a silicone oil.
  • the oil comprises a fluorosilicone oil.
  • the oil comprises a hydrocarbon oil.
  • the oil comprises a mineral oil.
  • the oil comprises a paraffin oil.
  • the oil comprises a halogenated oil.
  • the oil comprises a fluorocarbon oil.
  • each encapsulation is within 5%, 10%, 15%, 20%, or 25% of the average size encapsulation within the plurality. In some embodiments, at least 80%, 85%, 90%, or 95% of the encapsulations are within about 5%, 10%, 15%, 20%, or 25% of the average size encapsulation within the plurality.
  • the droplets may be formed by any method. In some examples, a droplet is formed by flowing an aqueous stream into an immiscible carrier fluid. In some examples, the aqueous stream flows into an immiscible carrier fluid at a junction of microfluidic channels. In some embodiments, the junction is a T-junction.
  • the junction is a meeting of two perpendicular microfluidic channels.
  • the junction may be a meeting of any number of microfluidic channels.
  • the junction may be at any angle.
  • the aqueous stream may be formed by an upstream junction of two or more aqueous streams.
  • sample solutions and effector solutions are joined upstream of the aqueous stream junction with the immiscible carrier fluid.
  • the size of the droplets may be controlled by modulating a variety of parameters. These parameters include the geometry of the junction of two microfluidic channels, the flow rate of the two streams, the type of oil used, the presence of surfactants, the pressure applied to the flow streams, or any combination thereof.
  • a single encoded effector is present in a compartment of encapsulation of the present disclosure (e.g., a droplet or a well).
  • a single scaffold comprising an encoded effector and its encoding are present in an encapsulation.
  • a plurality of scaffolds, each scaffold comprising a different encoded effector and its respective encoding are present in a compartment.
  • encapsulations comprise biological samples.
  • encapsulations comprise single cells.
  • encapsulations comprise one or more cells.
  • the encapsulations comprise nucleic acids.
  • the encapsulations comprise proteins.
  • the percentage of droplets each containing exactly one bead may be at least 5% of the total droplets formed. In other examples, this percentage may be at least about 8%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 99.9%. In some cases, this percentage may be at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less of the total number of droplets formed per unit time.
  • the percentage of empty droplets among the total droplets formed may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some examples, the percentage of empty droplets among the total droplets formed may be at most about 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or less of the total number of droplets formed per unit time. In some examples, the percentage of droplets containing 2 or more beads may be at least about 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 10%, 20%, 30%, 40% or more of the total droplets formed.
  • FIG. 5 shows an exemplary workflow of an effector screen performed in a microfluidic device.
  • a nucleic acid encoded effector bound to a bead is placed in an inlet and merged with an additional aqueous stream, which, in some embodiments, contains a sample to be screened.
  • the merged fluids are driven through an “extrusion region” or “droplet formation region,” wherein beads and sample are encapsulated in droplets.
  • Droplets/encapsulations are discrete aqueous volumes surrounded by a continuous immiscible oil.
  • An effector is then cleaved from bead at the effector cleavage region or dosing region, which in some embodiments utilizes a light source to cleave a photocleavable linker.
  • the encapsulations containing cleaved effectors are then allowed to continue flowing along the Attorney Docket No.56523-707.601 flow path of the device through the incubation region, which in some embodiments contains widened or enlarged chambers to control flow rate or residence time of the encapsulations.
  • a detectable signal is generated by the assay (e.g., a cleavage of a fluorophore from an assay probe).
  • the signal may increase over time.
  • the signal can be measured dynamically throughout the device. Different regions in the device may correspond to a given incubation time (e.g., the duration of time which takes the encapsulation to arrive at that location). As such, the signal increase over time can be detected and characterized.
  • the signal may also be detected in a detection region of the device. In some embodiments, this detectable signal is a fluorescent signal, though any detectable signal can be employed.
  • This signal is then measured or detected at a detection region, which is in some embodiments equipped with a light source (e.g., a laser or LED) and a detector (e.g., a photomultiplier tube (PMT), a charged coupled device (CCD), or a photodiode) coupled to a sorting device (e.g., a dielectrophoresis electrode or any other sorting mechanism).
  • a detection region comprises an interrogation region, which is coupled to a sensor or an array of sensors. Based on the signal, the encapsulations are sorted into a waste outlet or a hit outlet. For example, the negative encapsulations may be defaulted into the waste stream.
  • the positive encapsulations may be deflected into a hit stream, and thereby separated from the negative encapsulations.
  • the encodings of the hits are amplified (e.g., by PCR or emulsion PCR) and the encodings sequenced (e.g., by next generation sequencing (NGS)).
  • NGS next generation sequencing
  • the sequenced encodings can then be decoded to reveal the effectors which had the desired activity.
  • each bead further comprises barcode unique to the bead itself (independent of the effector). Thus, in some embodiments, it is possible to ascertain if multiple beads bearing identical effectors were selected as hits within multiple encapsulations.
  • a compartment e.g., a droplet or a well.
  • a plurality of unique encoded effectors are screened.
  • Each unique encoded effector may comprise a unique effector the effect of which on a sample may be screened using any screening system provided herein.
  • screening the sample in presence and/or absence of encoded effectors may be performed in a droplet microfluidic device.
  • the droplet microfluidic device may comprise a droplet generation region/junction for forming droplets/encapsulations.
  • the droplet generation junction may comprise or be a microfluidic flow focusing junction or a microfluidic T-junction.
  • the microfluidic device may comprise one or more Attorney Docket No.56523-707.601 droplet generation junction, such as 1, 2, 3, 4, 5, 6, or more droplet generation junctions integrated in the droplet microfluidic platform.
  • Each droplet formation region may comprise a plurality aqueous inlet streams. Each aqueous inlet stream may be connected to a separate reservoir holding a liquid, through a tube and one or more tube connections.
  • the separate reservoirs may each comprise a sample, a portion of a sample, assay reagents, assay probes, fluorophores, targets, cells, and/or encoded effectors.
  • the materials held in separate reservoirs may be set up and adjusted to accomplish intended purposes for screening a sample in presence or absence of a plurality of unique encoded effectors.
  • a droplet generation junction of a microfluidic device may comprise 3 inlet aqueous streams.
  • the first aqueous inlet stream may comprise a first portion of an assay reagent.
  • the first aqueous inlet stream may comprise a target.
  • the second aqueous inlet stream may comprise a second portion of an assay reagent.
  • the second aqueous inlet stream may comprise a probe or materials which may result in a signal as a result of interacting with the target.
  • the assay will start once the two streams meet a microfluidic channel of the microfluidic device that is connected to the first and second streams and to the droplet generation region. Once the first and second streams meet and start mixing, the assay starts to take place, generating a signal which may continue to increase over time inside the formed droplet containing the assay materials.
  • the droplet may flow through the chip for the assay to be incubated. In some cases, the assay may be screened in presence of the encoded effectors.
  • the encoded effectors may be introduced through the first inlet or the second inlet.
  • the microfluidic device may comprise a third inlet stream for introduction of the encoded effectors.
  • the encoded effectors may be bound to beads according to the information presented elsewhere herein.
  • the third inlet stream may be holding a solution comprising a suspension of encoded effector beads.
  • the third aqueous stream may enter the device, co-flow along with the first and second streams, and reach the droplet generation junction.
  • the droplet generation junction may be a flow focusing droplet generation junction.
  • An oil phase immiscible with the aqueous solutions may enter the droplet generation junction and break up droplets which may contain assay reagents, encoded effectors, and/or both.
  • the oil may comprise a surfactant.
  • the percentage of the surfactant in oil may be at least about 0.5%, 1%, 2%, 3% (v/v), or more.
  • the surfactant may form as a barrier surrounding the droplet, reducing or eliminating material transfer from the aqueous environment of the droplet into the surrounding continuous oil.
  • a plurality of droplets may Attorney Docket No.56523-707.601 be formed at a predetermined frequency.
  • the droplets may encapsulate a plurality of different/unique scaffolds (beads comprising a plurality of different encoded effectors such that each bead comprises a unique effector encoded using an optical or a nucleic acid barcode).
  • a subset of the plurality of droplets may each comprise at least one scaffold/bead. Another subset of the plurality of droplets may be empty. Another subset of the plurality of droplets may comprise more than one scaffold, such as 2, 3, 4, 5, 6, or more scaffolds (e.g., multiple scaffolds).
  • the number of scaffolds (e.g., beads) entrapped in the droplets may be quantified using a parameter termed “droplet occupancy”. Droplet occupancy may characterize the percentage of droplets containing 0 beads, 1 bead, 2 beads, or more than 2 beads. Bead occupancy among the droplets may follow a Poisson distribution.
  • the barcode encoding the effector may comprise one or more optical barcoding particles on the surface of the scaffold/bead or inside it.
  • the optical barcoding particles may comprise spectral properties in the short-wave infrared (IR) range.
  • the optical barcoding particle may be detectable by fluorescence or luminescence.
  • the optical barcodes may be detected by line scan cameras or line scan spectrometers.
  • the optical barcoding particles may comprise an excitation wavelength of from about 1000 to about 1100 nanometers (nm).
  • the optical barcoding particle may comprise an excitation wavelength of from about 1060 to about 1070 nanometers (nm). In some examples, the optical barcoding particle may comprise an emission wavelength of from about 1000 to about 2000 nanometers (nm). [000200] In some examples, the optical barcode may comprise at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800, at least 1000, at least 1100, at least 1200, at least 1300, at least 1500, at least 1600, at least 2000, or more unique spectral emissions. In some examples, the optical barcode may comprise a narrow emission bandwidth.
  • the emission bandwidth may be at most about 500 nanometer (nm), 400 nm, 300 nm, 200 nm, 150 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2.5 nm, 2 nm, 1.5 nm, 1.2 nm, 1 nm, 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm, 0.3 nm, 0.2 nm, 0.1 nm, or narrower/less.
  • the emission bandwidth may be at least about 0.05 nm, 0.08 nm, 0.1 nm, 0.15 nm, 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 1.1 nm, 1.2 nm, 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 5 nm, 6 nm, 7 nm, 9 nm, 10 nm, 15 nm, 20 nm, Attorney Docket No.56523-707.601 30 nm, 50 nm or greater/broader.
  • the optical barcoding particles may comprise an emission bandwidth of at most about 0.5 nm.
  • the optical barcoding particles may comprise one or more optical barcoding particles.
  • the optical barcoding particles individually or in combination may comprise a unique optical signature.
  • one optical barcode may comprise one or more optical signature.
  • a bead/scaffold may comprise one or more optical barcoding particle each of which may comprise one or more unique optical signature(s).
  • the bead may comprise any suitable number of optical barcoding particles. In some examples, the bead may comprise at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more optical barcoding particles.
  • the bead may comprise at most about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 optical barcoding particle(s).
  • Each optical barcoding particle of the one or more optical barcoding particles may comprise at least 1, 2, 3, 4, 5, 6, 7, or more unique optical signatures.
  • the one or more optical barcoding particles, together or combination, may generate at least about 10, 20, 30, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10 4 , 2 x 10 4 , 3 x 10 4 , 4 x 10 4 , 5 x 10 4 , 6 x 10 4 , 7 x 10 4 , 8 x 10 4 , 9 x 10 4 , 10 5 , 2 x 10 5 , 3 x 10 5 , 4 x 10 5 , 5 x 10 5 , 6 x 10 5 , 7 x 10 5 , 8 x 10 5 , 9 x 10 5 , 10 6 , 2 x 10 6 , 3 x 10 6 , 4 x 10 6 , 5 x 10 6 , 6 x 10 6 , 7 x 10 6 , 8 x 10 5 , 9 x 10 5
  • the one or more optical barcoding particles may generate at least about 1 million, 2 million, 10 million, 20 million, 30 million, 40 million, 40 million, 50 million, 80 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 10 billion, 20 billion, 30 billion, 40 billion, 50 billion, 60 billion, 70 billion, 80 billion, 100 billion, 200 billion, 300 billion, 400 billion, 500 billion, 600 billion, 700 billion, 800 billion, 900 billion, 1 trillion, 2 trillion, 3 trillion, or more unique optical signatures.
  • the optical barcoding particles may be made of any suitable material.
  • the optical barcoding particles may comprise nanoparticles, quantum dots, fluorophore containing materials, a bead comprising an optical dye, nano-phosphorous particles, laser particles, and beyond.
  • the optical barcoding particles may comprise a surface.
  • the surfaces of the optical barcoding particles may comprise a surface coating.
  • the surface coating may comprise or be Si.
  • Attorney Docket No.56523-707.601 [000206]
  • the optical barcoding particle may comprise any suitable shape such as a sphere, a cylinder, a rectangular or cubical shape, a shape comprising any number of edges, a polygon, a hexagon, or any other suitable geometrical shape.
  • the optical barcoding particle may comprise or be a cylindrical optical barcoding particle.
  • the height of the cylindrical optical barcoding particle is at most about 20 ⁇ m, 15 ⁇ m, 10 ⁇ m, 9 ⁇ m, 8 ⁇ m, 7 ⁇ m, 6 ⁇ m, 5 ⁇ m, 4 ⁇ m, 3 ⁇ m, 2 ⁇ m, 1 ⁇ m, 0.9 ⁇ m, 0.8 ⁇ m, 0.7 ⁇ m, 0.6 ⁇ m, 0.5 ⁇ m, 0.4 ⁇ m, 0.3 ⁇ m, 0.2 ⁇ m, 0.1 ⁇ m, or less.
  • diameter of the cylindrical optical barcoding particle is at most about 20 ⁇ m, 15 ⁇ m, 10 ⁇ m, 9 ⁇ m, 8 ⁇ m, 7 ⁇ m, 6 ⁇ m, 5 ⁇ m, 4 ⁇ m, 3 ⁇ m, 2 ⁇ m, 1 ⁇ m, or less.
  • the diameter of the cylindrical optical barcoding particle may be at least about 0.1 ⁇ m, 0.2 ⁇ m, 0.3 ⁇ m, 0.4 ⁇ m, 0.5 ⁇ m, 0.6 ⁇ m, 0.7 ⁇ m, 0.8 ⁇ m, 0.9 ⁇ m, 1 ⁇ m, 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 11 ⁇ m, 12 ⁇ m, 13 ⁇ m, 14 ⁇ m, 15 ⁇ m, 16 ⁇ m, 17 ⁇ m, 18 ⁇ m, 19 ⁇ m, or larger.
  • the volume of the optical barcode is at most about 0.05% of the volume of the bead.
  • the barcode may comprise a nucleic acid molecule covalently bound to the bead or trapped inside the bead.
  • the barcode may comprise a Deoxyribonucleic acid (DNA), a Ribonucleic acid (RNA), a peptide, or a peptide nucleic acid (PNA).
  • a bead comprising a nucleic acid barcode may further comprise an optical barcode thereon or therein.
  • the bead of the present disclosure may remain substantially structurally intact during solid phase peptide synthesis (SPPS).
  • the bead may not get significantly damaged by being suspended in organic solvents used for effector synthesis for a prolonged period of at least about 5 minutes (min), 10 min, 20 min, 30 min, 60 min, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 72 hours or longer (e.g., under conditions required for library synthesis).
  • the bead remains substantially structurally intact during and after suspension in an organic solvent.
  • the bead diameter in any of the disclosed embodiments is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 micrometers ( ⁇ m) in water.
  • the bead diameter comprises a coefficient of variation (CV%) of the diameter among a population of the beads that is lower than about 20%, 10%, 5%, 3%, 2%, or less.
  • the bead of the present disclosure as described anywhere herein may be encapsulated or compartmentalized in a compartment among a plurality of compartments.
  • the compartment may be according to any compartment described anywhere herein (e.g., a droplet, a well, a miniaturized confinement, a nanopen, and beyond).
  • Attorney Docket No.56523-707.601 Provided herein are methods and systems for sample screening (e.g., high- throughput sample screening).
  • the methods and systems may comprise miniaturized compartmentalized systems of various kinds for screening samples, in some cases, against libraries of effectors.
  • a screen may be performed in a compartmentalized system provided herein.
  • a subset of the plurality of compartments may each comprise an encoded effector therein.
  • the effect of the encoded effector on a sample may be screened in the plurality of compartments.
  • a first signal may be detected from each compartment.
  • the first signal may be indicative of the assay activity.
  • the barcode of the encoded effector may be read/decoded during the screen, thereby generating a second signal.
  • the second signal may be indicative of the structure of the effector.
  • the first signal (indicative of assay activity) and the second signal (decoding the barcode and indicative of the structure of the effector) may be detected from the same compartment.
  • the first signal and the second signal may be linked together (e.g., informatically, such as in a database) thereby informatically linking the assay activity to the structure of the effector.
  • This approach may provide a comprehensive map of the effects of each effector structure on the activity of the same assay.
  • This method may be referred to as decoding on the fly (DOTF) or decoding in real-time.
  • this method may replace or eliminate sorting and/or post-processing steps such as sequencing.
  • the DOTF method may not comprise a physical sorting step.
  • a physical sorting step may also be implemented.
  • DOTF and sequencing may both be performed.
  • a scaffold e.g., bead
  • a bead may comprise one or more barcoding modalities.
  • a bead may comprise both a nucleic acid barcode and one or more optical barcodes (e.g., one or more optical barcoding particles).
  • One or more of the barcoding modalities may be decoded to elucidate the structure of the effector with or without sorting.
  • a bead may comprise an optical barcode, the optical barcode may be decoded on the fly by detecting the second signal described above.
  • the bead may comprise a nucleic acid molecule which can be sequenced.
  • the barcoding modality may be chosen or optimized based on the application.
  • a method of screening an encoded effector comprising providing or obtaining a bead comprising an effector and a barcode corresponding to the effector.
  • the bead may be made of a substantially homogeneous polymer resin.
  • the method may further comprise encapsulating the bead in a compartment, detecting a signal from the compartment, Attorney Docket No.56523-707.601 and processing the compartment, the bead, or the barcode, based on the signal or a change thereof.
  • the bead may comprise or be any bead embodiment described anywhere herein.
  • the compartment may be a droplet or a well.
  • the effector may be bound to the scaffold via a cleavable linker and be releasable upon cleavage of the cleavable linker, and the method may comprise exposing the bead to a stimulus to cleave the cleavable linker and release the effector into the compartment.
  • the compartment may further comprise an assay reagent and a target.
  • the signal may be indicative of the activity of the target in presence of the effector, as measured using the assay reagent.
  • the effector may be releasable from the solid support upon cleavage of the cleavable linker.
  • the solid support may further comprise one or more encoding particles embedded inside or on the surface of the solid support corresponding to and identifying the effector.
  • the one or more encoding particles may comprise spectral properties in the short-wave infrared (IR) range.
  • the encoding particles may comprise a spectral emission bandwidth of at most about 100 nm.
  • the solid support may comprise a particle. In some examples, the particle may comprise a diameter of at most about 30 micrometers ( ⁇ m).
  • the solid support comprises or be a bead. The bead may be any bead mentioned/described anywhere herein.
  • the solid support/bead may be encapsulated in a compartment.
  • the compartment may comprise or be a droplet, a well, a nanopen, or a miniaturized channel.
  • the compartment may be any kind of compartment described anywhere herein.
  • the compartment may be a droplet surrounded by an immiscible oil.
  • the droplet may be generated with the aid of a droplet microfluidic device, and the system further comprises the droplet microfluidic device.
  • the microfluidic device may be according to any suitable microfluidic device described anywhere herein.
  • the compartment may be a well in a miniaturized array platform.
  • the compartment may be a microfluidic or miniaturized compartment comprising four sides, wherein the four sides comprise three closed sides and one open side.
  • the compartment may be according to any compartment described anywhere herein.
  • a screening method comprising providing or obtaining a bead.
  • the bead may comprise an effector bound to the bead via a cleavable linker.
  • the effector may be releasable from the bead upon cleavage of the cleavable linker, thereby generating a released effector in the compartment.
  • the bead may further comprise one or more encoding particles embedded inside or on the surface of bead corresponding to and identifying Attorney Docket No.56523-707.601 the effector.
  • the one or more encoding particles may comprise spectral properties in the short- wave infrared range and a spectral signature corresponding to the effector and identifying it.
  • the method may further comprise detecting a first signal indicative of the activity of the target in presence of the released effector and detecting a second signal indicative of the spectral signature of the encoding particles.
  • the bead, barcode, encoding particles, the effector, and the rest of the elements used may be according to any embodiments described anywhere herein.
  • the encoding particles may comprise a spectral emission bandwidth of at most about 3 nm.
  • the first signal and the second signal may be spectrally independent.
  • the first signal may be in the visible range.
  • the second signal may not be in the visible range.
  • the second signal may be in the short-wave infrared (IR) range.
  • IR short-wave infrared
  • a bead comprising (i) an effector bound to the bead via a cleavable linker and releasable from the bead upon cleavage of the cleavable linker; and, (ii) one or more encoding particles embedded inside or on the surface of the bead corresponding to and identifying the effector.
  • the one or more encoding particles may comprise spectral properties in the short-wave infrared (IR) range.
  • IR short-wave infrared
  • FIG.6 provides an exemplary workflow for screening using optically or spectrally encoded beads or One Bead One Compound Spectrally Encoded Library Screening (OBOC- SEL) and decoding on the fly (DOTF) without the need for a physical sorting step through which structure activity relationship (SAR) datasets can be acquired according to the methods detailed herein.
  • FIG. 7 provides another exemplary workflow for screening One Bead One Compound Spectrally Encoded Library Screening (OBOC-SEL) and decoding on the fly (DOTF) without the need for a physical sorting step through which structure activity relationship (SAR) datasets can be acquired.
  • the detector acquires a first signal (assay signal) and a second signal (optical barcode signal or optical signature).
  • the term compound can be generalized to any kind of effector.
  • This exemplary workflow may be used to screen any kind of effector described anywhere herein.
  • FIG. 8 provides another exemplary workflow for performing cocktail assays (testing one or more effectors/compounds simultaneously in the same compartment on the Attorney Docket No.56523-707.601 same sample/target), wherein the compartment comprises one or more spectrally/optically encoded effector beads the synergistic effects of which on the same sample is being tested.
  • the bead may further comprise a bead barcode (e.g., an optical barcode such as a fluorophore/dye) allowing for bead localization (e.g., on time trace signals acquired) and counting and identifying that the effectors came from the same or different beads (multi-bead decoding).
  • a bead barcode e.g., an optical barcode such as a fluorophore/dye
  • FIG.9 illustrates an exemplary workflow of a split-and-pool method for generating spectrally/optically encoded effector libraires.
  • FIG. 10 illustrates an exemplary workflow of using a Bead Index Registry and Dispensing System (BIRDS) for generating beads pre-encoded with optical barcodes.
  • BIRDS Bead Index Registry and Dispensing System
  • FIG.11 schematically illustrates generating beads of the present disclosure using a droplet microfluidic device.
  • the beads may be according to any bead embodiment described in the disclosure.
  • the optical barcoding particles are randomly distributed in the beads.
  • density matching agents may be used to facilitate substantially uniform loading of the optical barcoding particles inside the beads, such that for example, the variability (e.g., coefficient of variance or standard deviation) in the number of particles encapsulated in the beads across the population is minimized.
  • the beads may each comprise about 3 to 5 particles with a narrow distribution of the number of particles encapsulated in the bead.
  • Variability may sometimes be caused by particle settling in a reservoir/container from which they are introduced into the microfluidic device (e.g., over time).
  • Variability may be caused by particle sedimentation, settlement, aggregation, or non- uniform spacing or distribution.
  • Density matching agents may help alleviate those factors to make up for a more properly suspended particle suspension leading to more uniform optically encoded beads (e.g., in terms of the number of particles they contain and the localization thereof inside the beads).
  • FIG.12 illustrates a bead-generation approach in which optical barcodes comprise a surface modification which renders their surfaces amphiphilic.
  • the surface of the bead may be amphiphilic.
  • the surface of the bead may further comprise Si coating.
  • Si coating may improve surface properties.
  • the surfaces may be modified in terms of any intended physical or chemical property.
  • the surfaces of the optical barcoding particles may be made amphiphilic using one or more surfactant(s) coating the surface of the optical barcoding particle.
  • Example surfactants for this purpose/application may comprise Attorney Docket No.56523-707.601 Tween, Span, PEGylated di or triblock copolymer surfactants.
  • the surfaces are made hydrophobic or hydrophilic.
  • the hydrophilicity of the optical barcoding particles may be modulated.
  • Such modulation may be made using coatings (e.g., surfactants or other chemicals/materials).
  • Surface coatings may, in some cases, comprise functionalities.
  • the surface of the optical barcoding particles may be coated with Si.
  • Si may make the surface of the optical barcoding particle hydrophobic.
  • Si may protect the optical barcoding particles (e.g., to maintain its integrity (e.g., chemical, structural, or physical integrity) or robustness during the encoded effector synthesis, screening, and the rest of the workflow it goes through).
  • the optical barcoding particles are encapsulated in a monomer mixture for bead generation and driven to the surface of the bead by interfacial tension. This method localized optical barcodes near the surface of the beads.
  • the optical barcoding particles may comprise a surface coating which leads to particle localization near the surface.
  • the material of the optical barcoding particle or a coating thereof may lead to its localization in a given location in/on the bead (e.g., closer to the surface, in the peripheral portion). Alternatively, some particle materials/coatings may lead to random distribution across the bead or clustering closer to the core of the bead.
  • FIG.13 illustrates a bead-generation approach in which optical barcodes comprise a surface modification which renders their surfaces amphiphilic, and the bead further comprises Si coating.
  • the optical barcoding particles are encapsulated in a monomer mixture for bead generation and driven to the surface of the bead by interfacial tension.
  • the bead comprises an inner core with a material different from its peripheral section.
  • the bead comprises a core-shell structure.
  • the core and the shell are immiscible phases.
  • This method localized optical barcodes near the surface of the beads.
  • the inner core of the bead may comprise Dextran
  • the peripheral section of the bead may comprise PEG.
  • the bead may be similar to any bead embodiment described anywhere herein.
  • the methods may comprise performing multiplexed dose- response studies simultaneously.
  • optical barcoding and DOTF may be used to perform dose-response studies.
  • an encoded effector library according to any embodiment described herein may be prepared.
  • the encoded effector library may comprise any unique number of effectors.
  • the effector loading e.g., the amount or concentration of an effector loaded on an individual bead
  • the efficiency or structures of the cleavable linker and/or cleavage thereof may be alternated among the beads, such that the effector dose released from those beads is alternated as they are exposed to the Attorney Docket No.56523-707.601 same stimulus.
  • more than one cleavable linker may be used, such as, 2, 3, 4, 5, 6, or more different cleavable linkers which may have different effector release properties and/or dynamics.
  • the library comprising members with variable effector loads and/or cleavable linkers may be exposed to the same stimulus.
  • the variable effector load and/or cleavage properties may lead to variable released effector concentration which may be encoded by the barcodes of the encoded effectors.
  • Effector loading/concentration may be encoded with a suitable barcode of any kind.
  • a library may be prepared in which the effector load comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more different effector load/concentration.
  • the barcode e.g., optical barcode or optical signature of the bead
  • This method may multiplex the assay and facilitate screening various effector concentrations during the same screen and decode them in real-time or on the fly (DOTF).
  • Data may be recorded in a database.
  • the database may comprise SAR data and dose-response data.
  • Methods and systems for sample screening in miniaturized arrays [000230]
  • the screens of the present disclosure may be performed in any suitable screening platform, in some cases, in miniaturized compartmentalized screening platforms. Any bead- bound encoded effector embodiment may be combined with any screening system disclosed. In some cases, screens may be performed in arrays. [000231] In some embodiments, the methods and systems of the present disclosure may comprise providing, obtaining, and/or utilizing array-based platforms.
  • Array-based platforms with a solid support in the bottom may be particularly advantageous for seeding cells (e.g., adherent cells) and/or screening them. For many cell lines, it may be more suitable to perform the screens under conditions in which the cells are adhered to a solid surface/support. The reason may be that seeding cells on a solid support may better mimic the natural state of the cells (e.g., conditions in vivo).
  • the effect(s) of a compound or effector may be tested on a predefined or specific target of any kind according to the information provided elsewhere herein. Alternatively, in some cases, the overall effects of a compound on a sample or a population of cells may be mapped without directly assessing a predefined/specific target.
  • the bottom substrate for the plurality of the partitions may be glass.
  • An example glass microscopy cover slip may comprise a standard microscope glass slide.
  • An example size of such microscope slide can be 75mm x 25mm x 1mm.
  • a Globe Scientific 1324 Glass Microscope Slide was Attorney Docket No.56523-707.601 used as the bottom substrate for the array device. The surface of the glass slide was treated with silane. A plurality of wells was then fabricated on the silanized glass slide according to the methods and examples described elsewhere herein.
  • Wells of a well-based platform and/or compartments of an array-based platform can be of any shape (circle, square, hexagon, or other shapes). Wells may also be referred to as miniaturized wells, microwells, nanowells, and picowells.
  • the diameter of the fabricated wells may be from about 200 to about 300 micrometers/microns ( ⁇ m).
  • the height or depth of the well walls e.g., the thickness of the material in which the wells are imprinted
  • the spacing between the wells may be from about 50 to about 100 microns.
  • the total internal volume of the wells may be from about 20 picoliters to about 100 nanoliters.
  • the well diameter may be about 200 ⁇ m and the well height (thickness of the well wall) may be about 50 ⁇ m. Accordingly, the internal volume of the well may be about 1.57 nanoliters (nL). In another example, the well diameter may be about 200 ⁇ m and the well height may be about 100 ⁇ m. Accordingly, the internal volume of the well may be about 3.14 nL. In another example, the well diameter may be about 200 ⁇ m and the well height may be about 200 ⁇ m. Accordingly, the internal volume of the well may be about 6.28 nL.
  • the diameter of the well may be about 300 ⁇ m, and the height of the well may be about 50 ⁇ m, 100 ⁇ m, or 200 ⁇ m. Accordingly, the internal volume of the well may be about 3.53 nL, 7.065 nL, or 14.13 nL, respectively. [000235] In some cases, the diameter of the well may be at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400 ⁇ m, or above. In some cases, the diameter of the well may be at most about 1 cm, 500 ⁇ m, 400 ⁇ m, 300 ⁇ m, 200 ⁇ m, 150 ⁇ m, 100 ⁇ m, or smaller.
  • the thickness or height of the well wall may be at least about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300 ⁇ m or greater. In some cases, the height of the well wall may be at most about 500 ⁇ m, 400 ⁇ m, 300 ⁇ m, 200 ⁇ m, 100 ⁇ m, 50 ⁇ m, 30 ⁇ m, 20 ⁇ m or smaller.
  • the volume of the well may be at least about 0.1, 0.2, 0.30.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 2, 2.2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 20, 30, 40, 50, 60, 100, 200, 300, 400, 500 nL, or greater.
  • the internal volume of the well may be at most about 900 nL, 800 nL, 700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100 nL, 50 nL, 30 nL, 20 nL, 10 nL, 5 nL, or less.
  • the plurality of compartments may comprise at least 2 compartments. In some cases, the plurality of compartments may comprise from about 10 compartments to about ten million compartments or more.
  • the plurality of compartments may comprise at least 2, 10, 20, 30, 40, 50, 60, 100, 200, 300, 400, 500, 1000, Attorney Docket No.56523-707.601 5000, 10,000, 20,000, 40,000, 60,000, 100,000, 1000,000, 10,000,000, 100,000,000 or more compartments.
  • FIGs.14A- 14C provide, respectively, view from the side, view from the top, and three-dimensional view of an exemplary miniaturized array for sample screening.
  • a plurality of compartments 1403 are built on a solid surface 1401.
  • the cells 1402 are adhered to the bottom of the wells and the walls 1403 are substantially non-adherent and impenetrable to the cells.
  • the cells are seeded and grown on the bottom of the wells in a monolayer.
  • the micro-array is open at the top (e.g., without a solid cap at the top and not sandwiched between two pieces of solid support/glass).
  • Materials can be freely introduced in and out of the partitions/wells using a variety of techniques such as directly pouring, pipetting, robotic handling, flowing through tubes, and beyond.
  • the solid substrate in the bottom of the compartments or wells can be made of any material and it can be the same as or different from the walls of the compartments.
  • the bottom substrate may be glass or plastic.
  • the walls may be made of any material.
  • the wall material may be a hydrogel or polymer.
  • the compartments or wells of a miniaturized well-based platform can be made of any suitable material.
  • the material of the wells may be preferred to be substantially impenetrable to liquids such as cell media, oil (e.g., fluorinated oil), and effectors of the present disclosure.
  • the mesh size can be small enough to prevent material diffusion therein, or such material transfer may be otherwise substantially blocked.
  • the wells e.g., microwells
  • the walls of individual wells may be substantially impermeable to any material that can convolute the dosing of one cell population with another and such mass transfer is preferred to be substantially blocked and/or prevented.
  • array platform/device material may be resistant to extracellular protein adsorption and/or cell growth.
  • the methods and systems of the present disclosure may be used to screen ion channels in cells in the array-based system.
  • the cells may be able to transmit ion channel signals to adjoining cells. Thus, if one population of cells is connected to another population of cells across two wells, it is possible that the signal from one cell population may trigger signals in the other.
  • array platform material may be resistant to ECM protein adsorption and cell growth, this may minimize the chance for cells growing between two wells.
  • preferred well wall materials may be malleable into a predetermined shape or feature at the correct resolution (e.g., a high resolution).
  • array platform (e.g., microarray) material may be substantially transparent or highly transparent to light at given wavelengths, such as ambient light, UV, visible, Near-infrared (NIR), Short Wave Infrared (SWIR) or other light wavelengths which may be suitable for the purposes of the present disclosure.
  • the material may be substantially transparent to a light with a wavelength in the range between 300 nm to 2500 nm.
  • substantially transparent may be for example, at least about 70%, 80%, 90%, 92%, 95%, 97%, 98%, 99%, or above 99% transparent. This characteristic may help avoid light scattering or light absorption by the array material, which may affect the sample inside the array such as by stimulating the sample and/or signal capture from the samples or the cells therein.
  • the material of the compartments may have low autofluorescence.
  • the materials of the well walls and the bottom substrate can be the same or different.
  • the wells were made of a polymerized hydrogel, using polyethylene glycol diacrylate (PEGDA) with a molecular weight of about 250 kDa as the monomer making up the polymerized hydrogel.
  • PEGDA polyethylene glycol diacrylate
  • the photoinitiator used for gelling/curing/solidifying the hydrogel was 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone mixed with the base in a 95% to 5% v/v ratio.
  • the surface of the glass bottom was treated with silane.
  • the surface of the wells were treated with extracellular matrix material (ECM) or a cell adhesive material such as vitronectin, fibronectin, or matrigel.
  • ECM extracellular matrix material
  • cell adhesive material such as vitronectin, fibronectin, or matrigel.
  • the material of the wells was a mix of 250 kDa PEGDA monomer with 750 kDa PEGDA in monomer at a defined ratio.
  • the ratio of the monomers, the concentrations of the monomers in the pre-polymer mixture, the molecular weights of each monomer in the pre-polymer mix, and the concentration of the photoinitiator, among other factors can be used to control and tune the mesh size of the polymerized hydrogel.
  • the pre-polymer mix may further comprise a spacer.
  • the material used as the spacer, its chemical structure, molecular weight, additional chemical and physical properties, and the concentration of the spacer in the pre-polymer mix can also be used to control and tune the chemical properties of the resulting polymerized hydrogel.
  • any Zwitterionic polymers such as (poly(phosphorylcholine), poly(carboxybetaine), poly(sulfobetaine), poly(trimethylamine N-oxide) could be used to fabricate the wells.
  • Another example for well material may comprise a Cyclic Olefin Polymer (COP) or Cyclic Olefin Copolymer (COC).
  • Other examples for well material comprise glass and plastic.
  • both the bottom and walls of the wells could be made of the same material (e.g., glass or plastic).
  • the bottom surface and/or walls may be made of any material.
  • PDMS polydimethylsiloxane
  • the bottom of the compartments/wells e.g., the solid surface in the bottom of the platform
  • a cell adhesive material e.g., the solid surface in the bottom of the platform
  • Any suitable cell adhesive material or chemical can be used.
  • the cell adhesive may be selected from the group consisting of Vitronectin, Matrigel, Fibronectin, Laminin, Poly-Lysine, and Extra-cellular Matrix (ECM) Protein, a Cell Adhesion Polymer (e.g., a synthetic cell adhesion polymer).
  • the well walls may be made of a solid, semi-solid, partially porous material.
  • the hydrogel may be at least partially hydrophobic.
  • Such material may comprise or be polymer, plastic, silica, hydrogel, or any combination thereof.
  • the material of the walls may comprise any level of wettability or hydrophilicity. In some cases, the material may be hydrophobic.
  • the well walls may be made of a material (e.g., hydrogel) with a molecular weight of from about 10 to about 2000 Daltons.
  • the well walls comprise a top surface.
  • the top surface of the well walls may be or may be rendered substantially non-adherent to cells.
  • the well walls may be made of a material that by itself is substantially non-adherent to cells.
  • the material of the wells may comprise or be glass through-holes or plastics (e.g., Cyclic Olefin Copolymer COC).
  • COC tissue-culture treated
  • the surfaces of the well walls e.g., a material such as COC
  • rendering the well top surface non-adherent to cells may be facilitated through surface treatment or coating.
  • the top surface of the well walls may be coated with a coating material.
  • FIG. 15 schematically illustrates an exemplary workflow of a method for surface treatment of a miniaturized array platform to facilitate cell seeding or cell adhesion.
  • the miniaturized array platform 1500 comprised a plurality of circular wells 1501 fabricated on a solid surface 1502.
  • the bottom surface of the platform was made of glass (e.g., a glass microscopy cover slip).
  • the well walls/exteriors 1503 were made of a hydrogel material Attorney Docket No.56523-707.601 (PEGDA).
  • PEGDA hydrogel material Attorney Docket No.56523-707.601
  • the surface of the glass slide was treated with silane.
  • a PDMS mold was used to fabricate the hydrogel wells on the glass slide according to methods described elsewhere herein.
  • a series of surface treatments were applied on the internal and external well surfaces to modify the surface properties, such as properties with respect to cell and extracellular matrix (ECM) adhesion to render the bottoms of the wells substantially adhesive to cell and the exteriors of the wells substantially non-adhesive to cells and ECM.
  • ECM extracellular matrix
  • water was used as a blocking liquid to fill in the wells and prevent the entry of coating material.
  • the wells were then capped with an oil immiscible with water (e.g., a standard microfluidics fluorinated oil such as Novec HFE7500 by 3M).
  • a hydrophobic surface treatment capable of rendering the top surfaces non- adhesive to cells and ECM e.g., fluorosilane polymer diluted in a solvent (Novec 1720, 3M) was added to the capping oil to treat the well exteriors.
  • ECM e.g., fluorosilane polymer diluted in a solvent (Novec 1720, 3M)
  • Such chemical was substantially hydrophobic, insoluble in water, low in surface tension, and configured to keep out dirt, dust, debris, cells, and particles away from the surfaces. Water inside the wells prevented the entry of the non-adhesive hydrophobic surface treatment material into the interior of the wells.
  • the hydrophobic surface coating was evaporated over time to form a thin, transparent, permanent coating on the well exteriors (shown in light green).
  • a charged surface treatment e.g., poly-D Lysine
  • the hydrophobic treatment prevented the charged surface treatment to substantially affect the well exteriors.
  • the well interiors were modified by the charged surface treatment to render the well interiors substantially adhesive to cells and ECM or prime the surface for the addition of another surface treatment layer comprising a cell adhesive material such as Vitronectin.
  • Vitronectin or another cell adhesive material was applied on the entire array.
  • the hydrophobic treatment on the top surfaces facilitated denaturing the cell adhesive material.
  • the cell adhesive material rendered the solid surface of the bottom of the wells adhesive to cells and ECM.
  • FIG. 16 provides an example image of cells seeded in a miniaturized array.
  • Adherent cells were seeded in the bottom of each well.
  • the bottom of the well comprised a surface treatment which facilitated or enhanced cell adhesion.
  • Surface treatments e.g., as described elsewhere herein
  • the walls and tops of the wells were treated with a material which minimized cell adhesion and cell penetration.
  • An assay was performed on the cells.
  • the cells seeded in the miniaturized array platform were used in combination with encoded effectors, assays, detection systems, and other methods and systems described anywhere herein.
  • FIG. 17A shows an exemplary flow cell 1700 comprising a plurality of compartments 1701.
  • a plurality of compartments (wells) 1701 were immobilized or built on a solid surface (e.g., glass) in the bottom of the flow cell 1700.
  • the flow cell was fabricated to facilitate flow introduction into the compartments (e.g., wells).
  • the top of the flow cell comprised a solid seal 1702 with one or more (e.g., two) openings 1703 to facilitate fluid flow.
  • FIG.17B shows four flow cells similar to flow cell 1700 immobilized on a solid support 1704.
  • Each flow cell 1700 has an inlet tube 1705 inserted into an inlet port 1706 and an outlet tube 1707 inserted into an outlet port 1708 to facilitate fluid flow into and out of the wells 1701.
  • a solution comprising cell culture media and cells can be introduced into the flow cell through an opening incorporated in the solid seal on top of the flow cell using various techniques such as pipetting, robotic handling, and beyond.
  • An assay can be performed in each compartment/well.
  • the assay may comprise using a scaffold, wherein the scaffold comprises an effector covalently bound to the scaffold via a cleavable linker and a barcode (e.g., nucleic acid or optical barcode) on/in the scaffold which corresponds to and identifies the effector, as described anywhere herein.
  • the cleavable linker can be cleaved by application of a stimulus to release the effector into the compartment to act on the cell.
  • Assay activity can be measured by measuring a signal from each compartment.
  • the compartments that demonstrate a predefined/given effect can be marked as having an effect (positive or hit) or not having an effect (negative or non-hit), for example, using a computer program or software in communication with the system through a computer.
  • post-processing may be performed on the hits.
  • post-processing may comprise selective polymerization as described elsewhere herein.
  • a polymerizable monomer can be introduced into the flow cell.
  • a digital mask and digital mirror device can be used to selectively polymerize select wells which were identified as non-hits to block liquid entry thereto.
  • the hit compartments can remain substantially open to fluid flow.
  • Liquid can be introduced into hit wells to extract the contents of the well (e.g., the hit scaffold to cleave the barcode from the scaffold, to add a secondary barcode to the barcode on the scaffold in order to mark or tag it as a hit, or to sequence the barcode of the scaffold inside the compartment and elucidate the identity or structure of the hit effector (e.g., compound or small molecule).
  • Any suitable system may be used to select wells and block liquid flow thereto.
  • the system may comprise robotics, laser printing, 3D printing, or any combination thereof.
  • the methods of the present disclosure may comprise identifying hits.
  • a hit may be an effector that has or is suspected of having an effect on a sample, a target, or a cell.
  • effectors that are found to be active against an intended target may be referred to as “hits”.
  • a scaffold on which a hit is identified may be referred to as a hit scaffold or positive scaffold.
  • a hit may be an effector for which activity against a target has found to be surpassing or below a threshold (e.g., predefined threshold or a hit identification condition defined during or after data acquisition/aggregation for the signal measured from the respective partition).
  • Activity or effect may be any effect described anywhere herein.
  • a compartment or partition which contains at least one hit may be referred to as a positive compartment.
  • the positive compartment may further comprise one or more non-hits.
  • a negative compartment may be a compartment that does not contain a hit. Whether or not the hit has a real effect, or it has been detected by mistake or because of an error, deficiency, artifact, by chance, due to an interruption in the screen, or any other reason during the screen (e.g., a false positive) can be determined by downstream validation assays.
  • Hits may comprise validated/real hits and false positives.
  • a validated hit has a real effect on the target, sample, or the cell.
  • a non-validated hit is a false positive.
  • hits may comprise or be high-interest events of unknown veracity.
  • hits may not be treated as bona-fide until validated in replicate tests afterwards.
  • further processing may be performed to elucidate the identity of the hits.
  • Such processing may comprise sorting the hit scaffolds.
  • Hit identification and/or processing can be performed in various ways.
  • Examples may comprise sorting and collecting the positive compartments/partitions (e.g., sort droplet, well, raft, or any other compartment such as by physically separating them in space or adding a membrane or barrier between them to partition them from one another), sorting and collecting the scaffolds/bead found/identified in the positive partitions (e.g., a partition containing at least one hit) with or without sorting the partitions themselves, for examples, extracting the hit scaffolds from the positive partitions and moving them to a separate container, or separating and sorting the barcodes of the hit scaffolds with or without sorting the scaffolds and partitions themselves.
  • Separating the barcode from the scaffold may comprise breaking or cleaving the bond between the barcode and the scaffold via a stimulus.
  • cleaving the barcode from the bead may be performed or catalyzed by an enzyme.
  • cleaving the barcode from the bead may be performed or catalyzed by an enzyme.
  • An example workflow may comprise Attorney Docket No.56523-707.601 providing or obtaining a plurality of compartments (e.g., a plurality of droplets in a droplet microfluidic platform or a plurality of wells of a well array on a solid support), introducing a plurality of scaffolds (e.g., beads) and a plurality of cells into the compartments such that a subset of the compartments each include at least one cell and at least one scaffold, the scaffold comprising a barcode and an effector attached thereto, wherein the barcode is corresponding to and identifying the effector.
  • a plurality of compartments e.g., a plurality of droplets in a droplet microfluidic platform or a plurality of wells of a well array on a solid support
  • a plurality of scaffolds e.g., beads
  • the scaffold comprising a barcode and an effector attached thereto, wherein the barcode is corresponding to and identifying the effector.
  • a plurality of cells may be pre-seeded in the plurality of the compartments, and a plurality of scaffolds may be introduced into the plurality of compartments to enter the compartments and interact with the pre-seeded cells.
  • a compartment may contain any number of cells and any number of scaffolds.
  • the cells and the scaffolds may be introduced into the plurality of compartments in any order.
  • the scaffolds may be introduced into the compartments before or after the cells.
  • the cells may be suspension cells floating in the compartments and not necessarily pre-seeded in the compartments or adhered thereto.
  • the effector may be attached to the scaffold (e.g., bead) via a cleavable linker (e.g., photocleavable linker) and be releasable from the scaffold via a stimulus (e.g., UV light) further described elsewhere herein.
  • the barcode may also be attached to the scaffold with a cleavable bond which may be cleaved via a stimulus (e.g., a chemical). In some cases, the barcode may be attached to the scaffold via a covalent bond that is not cleavable.
  • the compartment may further comprise assay reagents such as probes, reporters, and/or other materials for performing an intended assay in the compartment to measure the activity of the effector against the target (e.g., in or on the cell in some cases).
  • a signal can be measured from each compartment which may be a measure of the activity of the effector against/on the target.
  • the compartments may be labeled as either a positive compartment or negative compartment (e.g., based on the intensity of the signal).
  • the positive compartments may be referred to as hit compartment (e.g., a compartment containing one or more hits (i.e., hit effector or hit scaffold).
  • Compartments comprising a scaffold which has an intended effect on the assay or the target, based on the intensity of the signal, may be referred to as a positive compartment (a compartment bearing at least one hit).
  • a positive compartment may comprise more than one scaffold.
  • the presence of one positive scaffold/effector (a hit) is sufficient, and the additional scaffolds in the compartment may not necessarily all have an effect on the target or be hits.
  • a compartment marked as a negative compartment, which is a compartment not bearing a hit, may be sorted into a separate stream or space from the positive compartments.
  • the barcode encoding the effector is a nucleic acid molecule
  • the barcode may be decoded to elucidate the structure of the effector using a variety of techniques examples of which may comprise sequencing.
  • the barcode may be decoded or read to determine which effectors displayed the activity of interest against the target sample.
  • the methods presented herein may comprise the step of ascertaining which encodings/barcodes are present in the samples sorted based on the detection of the signal, in case sorting is performed.
  • the methods provided herein may not comprise a physical sorting step and barcodes may be decoded during the assay screen (e.g., decoded in real-time or on-the-fly).
  • the barcode/encoding may be a nucleic acid.
  • the terms encoding and barcode may be used interchangeably.
  • the method may further comprise the step of sequencing the barcode.
  • the barcode may be sequenced by next generation sequencing (NGS).
  • NGS next generation sequencing
  • the sequences may be compared to a reference (e.g., a reference database) to ascertain which effectors displayed the activity of interest in the screen.
  • the database may encode a plurality of synthesis steps of the effector (e.g., using the split-and-pool combinatorial synthesis).
  • Sequencing the nucleic acid barcode/encoding may comprise sequencing the encoding while the encoding is still attached to the scaffold. Sequencing the nucleic acid encoding may comprise cleaving the nucleic acid encoding from the scaffold before sequencing it. Sequencing the nucleic acid encoding may comprise cleaving the nucleic acid encoding from the scaffold prior to sequencing. Cleaving the nucleic acid encoding from the scaffold may comprise cleaving a cleavable linker with a stimulus (e.g., an energy or a chemical such as a cleaving reagent). Cleaving the nucleic acid encoding from the scaffold may comprise cleaving a cleavable linker with electromagnetic radiation.
  • a stimulus e.g., an energy or a chemical such as a cleaving reagent
  • the nucleic acid encoding may comprise a sequencing primer.
  • the sequencing primer may allow for facile amplification of the nucleic acid encoding.
  • the sequencing primer may be the same for each encoding. In some cases, the sequencing primer may differ among the encodings.
  • the sequencing primer may be upstream of the encoding.
  • the sequencing primer may be downstream of the encoding.
  • the methods and systems provided herein may utilize libraries of encoded effectors.
  • Libraries of encoded effectors may comprise a plurality of different effectors, each uniquely encoded by a known encoding modality, such as those described above.
  • Libraries may contain any number of encoded effectors.
  • the libraries comprise at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , or 10 16 unique effectors.
  • the libraries comprise at least about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , or 10 16 unique effectors.
  • libraries of encoded effectors are linked to scaffolds. These scaffolds may be referred to as “scaffold encoded libraries.” Scaffold encoded libraries comprise a plurality of encoded effector molecules linked to the scaffold. The scaffold acts as a solid support and keeps the encoded effector molecules linked in space to their encodings.
  • the libraries comprise at least 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , or 10 16 scaffolds. In some embodiments, the libraries comprise at least about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , or 10 16 scaffolds. [000264] Any of the methods or systems described herein for a single encoded effector may be utilized by a library of encoded effectors.
  • a method of screening a library of encoded effectors comprising using any of the methods previously described herein with a library of encoded effectors.
  • libraries of encoded effectors comprise a plurality of different encoded effectors.
  • libraries comprise multiple copies of substantially identical effectors or scaffold encoded effectors.
  • the barcode/encoding may comprise a variety of modalities.
  • the barcode may comprise nucleic acid molecules, optical barcodes, or both. Different barcoding modalities are described throughout the disclosure and may be combined or mixed-and-matched in a variety of ways based on the applications/screens to be performed using such barcodes.
  • nucleic acids from the sample may comprise transferring one or more nucleic acids from the sample (e.g., from a cell or cell nucleolus) to the encoding.
  • the transfer of nucleic acids from the sample to the encoding may allow for substantial information about the sample and the suspected effect of the effector on the sample or to be ascertained, for example, when the sample comprises a cell.
  • the transfer of the nucleic acids from the sample can allow for Attorney Docket No.56523-707.601 quantification of expressed protein by quantifying the amount of target mRNA, as well as provide global proteomic and genomic data about the cell. This data can be collected and compared to cells that did not receive a dose of the indicated effector.
  • a method for detecting sample nucleic acids in a nucleic acid encoded effector screen may comprises providing one or more cells, a nucleic acid encoded effector, and a nucleic acid encoding in a compartment.
  • the compartment may be incubated for a period of time to allow for the effector and the sample (e.g., a sample comprising a cell) to interact.
  • the interaction between the effector and the sample e.g., a cell
  • the interaction between the effector and the sample may produce a signal.
  • the period of time may be sufficient to allow for changes in transcription and/or translation to occur in the sample (e.g., a sample comprising a cell) in presence and/or absence of to the effector (e.g., in response to the effector).
  • the method may comprise transferring cellular nucleic acids from the sample (e.g., from a cell or constituent of a cell) to the nucleic acid encoding.
  • the cellular nucleic acids may be quantified by sequencing the nucleic acid encodings after the cellular nucleic acids have been transferred.
  • the expression fingerprint of the cell can be generated in response to treatment with the effector.
  • the method may further comprise detecting a signal produced through interaction between the effector and one or more cells, and sorting the encapsulation based on the detection of the signal.
  • the cell In order to release the cellular nucleic acids, the cell may be lysed.
  • the method may further comprise the step of lysing the cell. Lysing the cell may comprise adding lysis buffer to the compartment.
  • the lysis buffer may be added by pico-injection (e.g., in case the compartment is a droplet in a droplet microfluidic device). In case the compartment is a well, any suitable reagent addition method described anywhere herein (e.g., automatic dispensing by a robot, manual addition, or beyond) may be added.
  • the lysis buffer may comprise a salt.
  • the lysis buffer may comprise a detergent. Examples of the detergent may comprise SDS, Triton, or Tween.
  • the lysis buffer may comprise a chemical which causes cell lysis. [000269] Any type of cellular nucleic acid can be transferred to the nucleic acid encoding.
  • the method may comprise transferring one or more cellular nucleic acids from the sample to the nucleic acid encoding.
  • the nucleic acids may comprise or be mRNA.
  • the nucleic acids may be mRNA that express a protein of interest.
  • the nucleic acids may comprise or be genomic DNA.
  • the nucleic acids may be added as antibody-DNA constructs.
  • the nucleic acids added may be proximity ligation products.
  • the Attorney Docket No.56523-707.601 nucleic acids may proximity extension products.
  • a plurality of different cellular nucleic acids may be attached to nucleic acid encodings.
  • the nucleic acids transferred to the encoding may comprise a complementary sequence to a sequence on the encoding. This may allow for the ligation of the sample nucleic acid with the encoding nucleic acid via various methods. These methods may comprise annealing, ligating, chemically cross-linking, or amplifying the cellular contents on to the nucleic acid encoding the effector.
  • the nucleic acid encodings may comprise a sequence complementary to the nucleic acid of interest to be transferred to the encoding. This complementary sequence may allow for the nucleic acids to hybridize with the encoding, which in turn may allow for extension of the encoding with the cellular nucleic acid and/or vice versa.
  • additional reagents may be added to the compartment to facilitate the transfer of the nucleic acids to the encoding.
  • the additional reagents may comprise an enzyme that may facilitates the transfer of the nucleic acids.
  • the reagents for transferring the nucleic acids to the encoding may be added during the encapsulation step (e.g., loading the sample, the encoded effector, the reagents, and other components to the compartment).
  • the reagents for transferring the nucleic acids to the encoding may be added during an incubation step.
  • the reagents for transferring the nucleic acids to the encoding may be added after an incubation step.
  • the additional reagents to facilitate the transfer of the nucleic acids comprise an enzyme.
  • the enzyme is a polymerase, a ligase, a restriction enzyme, or a recombinase.
  • the enzyme is a polymerase.
  • the additional reagents comprise a chemical cross-linking reagent.
  • the chemical cross-linking reagent is psoralen.
  • a nucleic acid encoded scaffold is shown with the nucleic acid encoding bound thereto, wherein a plurality of cellular encodings (e.g., nucleic acid) are also shown to have been released from a lysed cell.
  • the nucleic acid encoded scaffold and cellular encodings are provided within an encapsulation.
  • the nicking site is identified on the nucleic acid encoding, along with a capture site.
  • the nicking site corresponds to a specific nucleotide sequence in the nucleic acid encoding.
  • the nucleic acid encoding is nicked at the nicking site.
  • nicking herein refers to a single strand of the encoding being displaced from the nucleic acid encoded scaffold.
  • an amplification enzyme may interact with the Attorney Docket No.56523-707.601 nicking site, thereby creating a new copy of the nucleic acid encoding (step 4), while the previously nicked nucleic acid encoding copy (encoded nucleic acid primer) is unbound and moves within the encapsulation, such that the encoded nucleic acid primer may interact with a released cellular encoding (e.g., cellular nucleic acid), as shown in step 5.
  • a released cellular encoding e.g., cellular nucleic acid
  • the encoded nucleic acid primer labels the cellular encoding.
  • the capture site of the encoded nucleic acid primer prescribes a targeted cellular nucleic acid.
  • an enzyme enables such labeling.
  • the encoded cell encoding is labeled with the encoded nucleic acid primer, while a created copy of the nucleic acid encoding is displaced from the scaffold, wherein the process returns to step 3.
  • the cell may be lysed in order to release the desired nucleic acids and to make the desired nucleic acids available for amplification.
  • the encapsulation further comprises a cell lysis buffer.
  • the lysis buffer is added by pico- injection.
  • the lysis buffer comprises a salt. In some embodiments, the lysis buffer comprises a detergent. In some embodiments, the detergent is SDS, Triton, or Tween. In some embodiments, the lysis buffer comprises a chemical which causes cell lysis. In some embodiments, cell lysis buffer is added to the encapsulation. In some embodiments, the cell lysis buffer is added to the encapsulation by pico-injection. [000275] In some embodiments, an amplification mix is used to amplify nucleic acid encodings to create additional primers for labeling cellular nucleic acids of interest in a screen. In some embodiments, the amplification mix is an isothermal amplification mix.
  • the isothermal amplification mix comprises reagents for loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase- dependent amplification (HAD), recombinase polymerase amplification (RPA), rolling circle replication (RCA), or nicking enzyme amplification reaction (NEAR).
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • HAD helicase- dependent amplification
  • RPA recombinase polymerase amplification
  • RCA rolling circle replication
  • NEAR nicking enzyme amplification reaction
  • the encapsulation further comprises reagents for isothermal amplification of the target nucleic acid.
  • the method comprises adding reagents for isothermal amplification to the encapsulation.
  • the reagents for isothermal amplification are targeted to the specific nucleic acid sequence.
  • the amplification mix comprises a nicking enzyme. In some embodiments, the amplification mix comprises a nicking-enzyme amplification mixture. In some embodiments, the nicking enzyme is an endonuclease. In some embodiments, the nicking enzyme is a restriction enzyme. In some embodiments, the amplification mix comprises a reverse transcriptase. In some embodiments, Attorney Docket No.56523-707.601 the amplification mix comprises an amplification enzyme. In some embodiments, the amplification enzyme comprises a polymerase. [000276] In some embodiments, the specific nucleotide sequence of interest can be amplified within the encapsulation.
  • the method comprises amplifying the cellular nucleic acid comprising the specific nucleotide sequence to produce amplified cellular nucleic acids.
  • amplifying the cellular nucleic acids is accomplished by PCR.
  • amplifying the cellular nucleic acids is accomplished by isothermal amplification.
  • cellular nucleic acids comprising the specific nucleotide sequence are amplified.
  • the amplified cellular nucleic acid is barcoded with the nucleic acid encoding the scaffold. Morphological changes in the sample [000277] The signal from the sample may be a morphological or visual change in the sample which can be measured by imaging the encapsulation.
  • detecting the signal comprises recording images of the sample in the encapsulation. In some embodiments, detecting the signal comprises recording a series of images of the sample in the encapsulation. In some embodiments, detecting a signal comprises recording a series of images of samples in encapsulations and superimposing the series of images of the sample. In some embodiments, detecting a signal comprises detecting morphological or visual changes in the sample measured by recording a series of images of the encapsulation. [000278] In some embodiments, morphology changes in a sample, such as one or more cells, can be detected by an imaging sensor, capturing trans illuminated light with a high-speed shutter, where composite video frames offers multiple full-cell images that can aid in shape determination.
  • morphology changes in a sample can be detected by an imaging sensor, capturing trans illuminated light from a high frequency pulsed light source, increasing temporal resolution and sharpening the perimeter of the cell.
  • morphology changes can be detected by fluorescence emission from a cell traversing a laser-light sheet excitation region.
  • the emission is captured by Avalanche Photodiode (APD) or charged coupled detector (CCD), in a one- dimensional array of pixels, binned by time, then restitched into a composite fluorescence- microscopy image.
  • APD Avalanche Photodiode
  • CCD charged coupled detector
  • detecting the signal comprises recording images of the sample, wherein the sample is a cell.
  • recording images of the cell provides information about cell morphology, mitotic stage, levels of expressed proteins, levels of cellular components, cell health, or combinations thereof.
  • the Attorney Docket No.56523-707.601 encapsulation comprises a detection agent.
  • the detection agent is an intercalation dye.
  • the intercalation dye is ethidium bromide, propidium iodide, crystal violet, a dUTP-conjugated probe, DAPI (4’,6-diamidino-2-phenylindole), 7- AAD (7-aminoactinomycin D), Hoechst 33258, Hoechst 33342, Hoechst 34580, combinations thereof, or derivatives thereof.
  • the detection agent highlights different regions of the cell. In some embodiments, the detection agent highlights a particular organelle.
  • the organelle is a mitochondrion, Golgi apparatus, endoplasmic reticulum, nucleus, ribosomes, cellular membrane, nucleolus, liposome, lipid vesicle, lysosome, or vacuole. In some embodiments, the organelle is a mitochondrion. In some embodiments, the organelle is the nucleus. [000280] Samples of any type can be utilized with the methods and systems provided herein. In some embodiments, the sample is a biological sample. In some embodiments, the sample comprises one or more cells, one or more proteins, one or more enzymes, one or more nucleic acids, one or more cellular lysates, or one or more tissue extracts.
  • the sample is a cell.
  • the cell is a eukaryotic cell.
  • the cell is a prokaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a bacterial cell.
  • the cell is a human cell.
  • the cell is a cancer cell.
  • the cell is SH-SY5Y, Human neuroblastoma; Hep G2, Human Caucasian hepatocyte carcinoma; 293 (also known as HEK 293), Human Embryo Kidney; RAW 264.7, Mouse monocyte macrophage; HeLa, Human cervix epitheloid carcinoma; MRC-5 (PD 19), Human fetal lung; A2780, Human ovarian carcinoma; CACO-2, Human Caucasian colon adenocarcinoma; THP 1, Human monocytic leukemia; A549, Human Caucasian lung carcinoma; MRC-5 (PD 30), Human fetal lung; MCF7, Human Caucasian breast adenocarcinoma; SNL 76/7, Mouse SIM strain embryonic fibroblast; C2C12, Mouse C3H muscle myoblast; Jurkat E6.1, Human leukemic T cell lymphoblast; U937, Human Caucasian histiocytic lymphoma; L929, Mouse C3H/An connective tissue
  • the sample may comprise any suitable number of cells from at least 1, 2, 3, 4, 5, 10, 100, 1000, 10000 or more cells seeded in each compartment.
  • Ion channel screen [000283] The methods and systems of the present disclosure may be used to screen cells. In some cases, screens may be performed on ion channels in cells.
  • the target may be one or more ion channels in one or more cell types.
  • the encoded effectors may be used to perturb ion channels and/or modulate their activity. The effect of the encoded effectors on ion channels may be tested using the screening systems present anywhere herein.
  • screening may be performed in an array-based system described anywhere herein, in cells seeded in the micro-array, such as any array and system shown in any one of figures FIG.14A, FIG.14B, FIG, 14C, FIG.15, FIG.16, FIG.17A, FIG.17B or other figures or sections in the present disclosure.
  • ion channels may be endogenous to the cells. In other cases, ion channels may not be endogenous to the cells. Ion channels may be mutant ion channels, such as an ion channel comprising a mutation. In some cases, mutations may cause the ion channel to be sensitive to stimulation (e.g., optical stimulation).
  • the ion channel may be sensitive to stimulation (e.g., optical stimulation) for another reason.
  • the ion channels may be stimulated as part of performing the methods of the present disclosure.
  • Various kinds of stimulation may be applied.
  • Stimulation may comprise electrostimulation, optical stimulation, chemical stimulation, any combination thereof, or other kind of stimulations.
  • the stimulation may comprise optical stimulation, electromagnetic radiation, UV-VIS, near-infrared radiation, UV radiation, stimulation with visible light, or any combination thereof. Any suitable light wavelength and intensity may be used to stimulate ion channels. Stimulation may be applied at any suitable frequency.
  • electrostimulation is performed on the cells using one or more electrodes which may be embedded in or used in conjunction with a screening system of the present disclosure.
  • the screening system used for ion channel screening with or without Attorney Docket No.56523-707.601 electrostimulation may be any screening system mentioned anywhere herein, such as a droplet microfluidic device (e.g., FIG.4) or an array-based system (e.g., FIG.14A, FIG.14B, FIG, 14C, FIG.15, FIG.16, FIG.17A, FIG.17B), or another suitable screening system.
  • the methods for ion channel screening may comprise for searching for an effector with an effect on an ion channel of a cell.
  • the effector can comprise any therapeutic modality.
  • an effector may be a small molecule compound, a biologic, a gene, a protein, a peptide, or any other effector mentioned in the present disclosure.
  • the effect may be inhibitory or agnostic.
  • the effector may be an inhibitor or an agonist.
  • the effector may increase or decrease the activity of the ion channel.
  • the effector may be inert and not have an effect on the ion channel.
  • Encoded effector libraries may be screened against cells to find effectors with a predetermined effect.
  • the ion channel may be a protein expressed by a cell.
  • One or more voltage sensors may be provided or obtained in a screening system of the present disclosure or as an add-on set of tools to be used in conjunction with the screening system.
  • the cells may be provided in the compartments (e.g., droplet or well), the cells may be stimulated for ion channels to be activated, the voltage sensors may be used to detect a signal indicative of the activity of the ion channel.
  • This method may be performed in presence and/or absence of an encoded effector which may be used to perturb the cell to modulate the activity of the ion channel.
  • a library of encoded effector libraries can be screened against ion channels of the cells to identify effectors with the predetermined effect. The encoded effectors and screening methods and systems are described in detailed throughout the entire disclosure.
  • the set of voltage sensor probes may comprise any suitable probe.
  • the set of voltage sensor probes comprise a FRET pair, a voltage-sensitive oxonol, a fluorescent coumarin, a DiSBAC compound, a coumarin phospholipid, a DiSBAC compound, a coumarin phospholipid, a DiSBAC2, DiSBAC4, DiSBAC6, CC1-DMPE, CC2-DMPE, a DiSBAC2(3), DiSBAC 2 (5), DiSBAC 4 (3), DiSBAC 4 (5), DiSBAC 6 (3), DiSBAC 6 (5), CC1-DMPE, CC2- DMPE, DiSBAC 6 , CC2-DMPE or any combination or derivative thereof.
  • the ion channel screened using the methods and systems of the present disclosure may be any kind of ion channel.
  • the ion channel may comprise or be a protein, sodium, calcium, chloride, proton, potassium ion channel protein, calcium ion channel protein, chloride ion channel protein, proton ion channel proteins, or other kind of protein.
  • An ion channel protein may comprise or be a voltage gated ion channel protein.
  • the voltage gated ion channel may comprise or be a protein, sodium, calcium, chloride, proton, potassium ion channel Attorney Docket No.56523-707.601 protein, calcium ion channel protein, chloride ion channel protein, proton ion channel protein.
  • the ion channel protein may be endogenous to the cell, an exogeneous ion channel protein, incorporated into the cell through a vector, expressed in the cells (e.g., after being incorporated into the cell by a vector), a gene encoding the ion channel protein transiently transfected into the cell, an overexpressed protein, or other kind of ion channel.
  • the screening method comprises detecting a signal from at least one member of the set of voltage sensor probes.
  • the signal may be electromagnetic radiation, luminescence, fluorescence, or another kind of signal. In some cases, the electromagnetic radiation may be emitted due to a FRET interaction.
  • the signal may be an increase, decrease, or change in electromagnetic radiation as compared to a compartment without the encoded effector. In another example, the signal may be an increase, decrease, or change in electromagnetic radiation as compared to the compartment before the stimulation of the ion channel.
  • Condensate detection [000291]
  • a change in the condition of the sample or target may be observed as a result of the progression of an assay, test, or experiment in presence or absence of an encoded effector.
  • the observed change may be a redistribution of the signal in space.
  • an assay may test phase condensation. A sample may go through a phase change during the course of an assay.
  • the condition of the sample and/or a change thereof may comprise liquid-liquid phase separation (LLPS) or phase condensation.
  • LLPS or phase condensation may result in formation of condensates in the sample.
  • the condensates may be an indication of a biological condition or activity.
  • condensate formation may be a measure of protein-protein interactions (ppi) in a sample or in a cell in a sample.
  • an assay may measure one or more protein-protein interactions through manifestation of a phase condensation (formation of condensates) in the sample.
  • such assays may be performed to study protein-protein interaction networks or protein-nucleic acid interaction networks (e.g., protein-RNA interaction network).
  • stressed- induced biomolecular condensates also referred to as “condensates” or “stress granules” may form during a screen, detected using the screening platforms presented herein.
  • the effects of members of an encoded effector library provided herein may be tested on stress granules, condensates, protein-protein interaction networks, and protein-RNA interaction networks.
  • the methods and systems provided herein may facilitate drug discovery and drug development for protein-protein-interaction networks or protein-RNA networks.
  • the methods and systems may comprise detecting and screening formation of stress granules (SG).
  • a stress granule may be a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to cells.
  • SG formation may be detected in cells.
  • SG formation may be detected in cell-free samples.
  • SGs may form or assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein- RNA interaction network.
  • LPS liquid-liquid phase separation
  • the central node of this network may comprise or be a moiety or molecule (e.g., G3BP1) which may function as a molecular switch which may trigger RNA-dependent LLPS in response to rise in intracellular free RNA concentrations.
  • G3BP1 may comprise one or more intrinsically disordered regions (IDRs) which may regulate its intrinsic propensity for LLPS. This propensity may be tuned by phosphorylation within the IDRs.
  • IDRs intrinsically disordered regions
  • SGs may show up as condensates in screening and may be referred to as condensates.
  • the effectors of the present disclosure may be capable of affecting SG networks, SG formation in living organisms, protein-interaction networks, protein-RNA interaction networks, molecular switches, IDRs, phosphorylation in G3BP1 or IDRs thereof, and/or positive or negative cooperativity by extrinsic G3BP1-binding factors.
  • SG networks SG formation in living organisms, protein-interaction networks, protein-RNA interaction networks, molecular switches, IDRs, phosphorylation in G3BP1 or IDRs thereof, and/or positive or negative cooperativity by extrinsic G3BP1-binding factors may be relevant in one or more pathological conditions or diseases and may be targets for drug discovery.
  • the encoded effectors and screening platforms of the present disclosure may facilitate such drug discovery for the afore-mentioned targets.
  • RNP granules assemble by liquid-liquid phase separation (LLPS), which may occur when protein-laden RNAs that are dispersed in the cytoplasm or nucleoplasm (soluble phase) coalesce into a concentrated state (condensed phase).
  • LLPS liquid-liquid phase separation
  • the highly concentrated RNAs and RNA binding proteins (RBPs) may behave as a single organelle with liquid-like properties.
  • the constituents of membranelles organelles may remain in dynamic equilibrium with the surrounding nucleoplasm or cytoplasm and may form transiently or persist indefinitely.
  • Some RBPs, particularly those harboring low complexity Attorney Docket No.56523-707.601 domains (LCDs) undergo concentration dependent LLPS.
  • the methods and systems of the present disclosure may facilitate detection, screening, and perturbation of RNA- binding proteins (RBPs).
  • RBPs RNA- binding proteins
  • the effectors of the present disclosure may be screened for effects on RBPs.
  • the methods and systems of the present disclosure may facilitate drug screening, discovery, and development for affecting RBPs.
  • Condensates may show up as a change in signal locality, such as pixels, parts of pixels, or a plurality of pixels in an image which are brighter than the background. Fluorescence from the may aggregate, accumulate, or otherwise become brighter in the regions of the image where the condensates are formed. In some cases, this may also decrease the brightness of the background of the image.
  • a condensate may comprise a higher signal to background ratio compared to the areas of the image where condensates are not present. This effect may progress over time. More condensates may form in the sample/image over time.
  • a plurality of condensates may form in a sample over time. The plurality of condensates may comprise different sizes and intensities.
  • the number of condensates formed in a sample may be an indication of a condition of the sample.
  • the intensity of the condensates may be an indication of a condition of the sample.
  • a density of condensates (number of condensates per unit volume or per unit surface) may be measured, screened, and monitored over time.
  • the properties of the condensates such as the number of condensates, size of condensates, intensity of the condensates, and other detectable properties of condensates may be used to assess a condition regarding the sample.
  • an effect of an effector or effector library on a sample may be screened.
  • an effect of an effector on condensate formation may be screened.
  • the assay may assay a protein-protein interaction in a sample or a cell.
  • protein-protein interaction may be screened in a cell in presence and/or absence of an effector.
  • the effector may be any effector mentioned anywhere herein.
  • the effector may be an encoded effector described anywhere herein, for example, an encoded effector bound to a bead, such as shown in FIGs. 1A-1C.
  • the sample may be compartmentalized in any screening platform described herein.
  • the screening platform may be a droplet-based platform, such as shown in any one of FIGs.4-8.
  • the screening platform may be a well array platform such as shown in FIG. 14A, FIG. 14B, FIG, 14C, FIG. 15, FIG. 16, FIG. 17A, FIG. 17B or as described elsewhere.
  • the bead resin may be according to any bead embodiment (e.g., such as shown in FIG.3A or FIG.3B).
  • An effector may be cleaved from a bead and released into the compartment and allowed to interact with a sample in presence of an assay capable of detecting protein-protein Attorney Docket No.56523-707.601 interactions in the sample which may comprise a cell.
  • the protein-protein interaction may manifest as a change in fluorescence distribution and/or condensate formation. Fluorescence distribution or condensate formation may be monitored over time during assay incubation. Signals may be detected using any detector mentioned herein.
  • the properties of the condensates may be an indication of the activity of the assay and the protein-protein interactions being tested. The changes in the signals over time may indicate the change in the condensates.
  • the condition of the condensates and their various properties may be affected by an effector.
  • the effect of the effector on condensate formation may be detected using the methods and systems provided herein.
  • the condensates may be detected by imaging (e.g., fluorescence imaging).
  • condensates may be detected in a droplet-based screening platform provided herein.
  • the compartment/encapsulation is a droplet.
  • a condensate may be a bright three-dimensional (3D) region (e.g., a sphere brighter than the droplet) inside the droplet.
  • 3D three-dimensional
  • the condensates in the droplets may be detected using any suitable detector described anywhere herein, such as a system comprising any combination of a camera (fluorescent imaging system), image processing device, signal processing device, optical train, using laser induced fluorescence (LIF) and PMTs.
  • a camera fluorescent imaging system
  • image processing device image processing device
  • signal processing device optical train
  • LIF laser induced fluorescence
  • PMTs laser induced fluorescence
  • the settings, workflows, protocols, assay reagents, optical filters, and other conditions may be determined, set up, and integrated in each case to work properly individually and/or in concert toward the detection and screening goals (e.g., high-throughput screening of encoded effector libraries).
  • images of compartments may be captured.
  • a compartment may be a droplet in a microfluidic device.
  • the droplet may be imaged.
  • the condensates may be form during incubation in the microfluidic device and be present during the detection.
  • a droplet comprising an effector may be compared to a droplet not containing an effector.
  • the number of condensates in presence and absence of effectors may be compared.
  • the intensity of the condensates under different conditions e.g., presence of effector
  • the condensates may aggregate together to form larger and/or brighter aggregated condensates.
  • the formed condensates may aggregate on the beads of encoded effector libraries, thereby causing or increasing a fluorescence to be detected from the bead.
  • Such appearance or increase in bead fluorescence may be an indication of condensate formation and/or activity of the assay.
  • the effect of the effector on the activity of the biological event e.g., protein-protein interaction in a sample and/or in a cell
  • the compartment e.g., droplet or well
  • an effector changes the properties of condensates over time, compared to a control sample not containing an effector.
  • the condensates may manifest or appear as small spikes on a droplet digital signal trace detected by a system presented herein.
  • FIGs. 19A and 19B An example demonstrating a condensate formation assay in a static compartment (well) is shown in FIGs. 19A and 19B.
  • assay components for phase condensation were mixed.
  • Assay components may comprise a protein, RNA, and an enzyme.
  • the sample contained G3BP1 stress granule assembly factor 1 (+Green Fluorescence Protein), RNA, and Caprin1wt (Caprin1 wild type).
  • Parameters considered for assay development and set-up may comprise temperature, ionic strength of the buffer (salts and PH), viscosity, crowding agents, detergents, mixing or turbulence in the sample, and sample incubation time, which can be adjusted to optimize assay conditions for screening.
  • FIG. 19C shows a schematic illustrating the progression of the assay leading to Liquid-Liquid Phase Separation (LLPS).
  • SG represents stress granule formation.
  • the assay components G3BP1 FL (fluorescent), RNA, and Caprin1WT were added and incubated. In some cases, Caprin1WT may act in a catalysis capacity in the assay/reaction. Over time, the assay may progress, LLPS may occur in the compartment leading to the formation of small bubbles also referred to as condensates or stress granules.
  • FIG. 1 shows a schematic illustrating the progression of the assay leading to Liquid-Liquid Phase Separation
  • FIG. 19C schematically illustrates the increase in the number of condensates from right to left, marked with an arrow.
  • the reaction rate is catalyzed by the addition of Caprin1WT.
  • FIG. 19A a compartment comprising a sample containing SG components (as described with reference to FIG. 19C) was imaged over time using the methods and systems provided herein. The time span shown in this example was 30 minutes.
  • the assay can progress for any intended duration, such as 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 80 min, 90 min, 2 hr, 3 hr, 4 hr, 5 hr, or longer.
  • FIG.19B shows a plurality of condensates formed in a compartment, illustrated as bright spots in an image 1900 captured via fluorescent microscopy performed on a cell-free and bead-free sample in absence of an encoded effector in a compartment (well). The image was processed to quantify the fluorescent signal detecting the condensates.
  • a line scan is shown Attorney Docket No.56523-707.601 on image 2000 across which the intensity of the pixels of the image were measured.
  • the intensity of the signal in the plurality of regions across the line scan are shown in plot 1901.
  • X axis indicates the distance across the line scan.
  • the Y axis is the intensity of the signal at each point.
  • the background of the image comprises an intensity of about 80 (arbitrary units (AU).
  • the high-intensity areas peak around 160 AU and 180 AU.
  • the distance across the X axis is a measure of the size of the condensate.
  • the intensity of the peaks is a measure of the brightness of each condensate.
  • Graph 1901 indicated two signal peaks corresponding to the two condensates across the line scan in image 1900. [000303]
  • Various factors may affect condensate formation in an assay and its dynamics over time.
  • Such factors may comprise temperature, PH, Ionic Strength of the assay buffer, buffer viscosity, crowding agents, detergents, mixing, and static or dynamic conditions of the compartment.
  • a well in an array-based system is static.
  • a droplet may be dynamic.
  • a droplet may move and shake as it travels through a microfluidic device.
  • Such factors may increase mixing and turbulence or semi-turbulence inside the droplet.
  • Such dynamic conditions may affect and alternate the assay conditions and may be accounted for and adjusted during assay development and assay optimization.
  • FIG. 20 An example of condensate detection using a droplet-based platform is shown in FIG. 20.
  • the droplet-based platform and workflow used in this example was similar to the system and workflow schematically illustrated in any one of FIGs. 4-8.
  • FIG. 20 demonstrates the successful detection of condensates (stress granules (SG)) formed under dynamic flow conditions in a droplet microfluidic system.
  • X axis represents time (min).
  • Y axis represents digital signal measured using a PMT in the system. This PMT is arbitrarily marked as PMT1 on the system used. The signal was measured in Volts [V].
  • the signal was detected using a Field Programmable Gate Array (FPGA) sensor.
  • FPGA Field Programmable Gate Array
  • the FPGA sensor in this example was the detector, or a part of the detection system. Signals were recorded over time (T1, T2, T3, T4, T5, and T6). These time points were measured across the loops of the incubation line (assay flow path) of the droplet-based microfluidic device shown in FIG. 4. The incubation time for each device region was known at the time of the experiment. As such, the progression of the assay and condensate formation in the droplets of a droplet microfluidic Attorney Docket No.56523-707.601 device and flow path were detected and monitored over time, across the device. This experiment was performed in absence of cells and in absence of beads.
  • the droplet trace 2001 in T1 shows a droplet in early regions/loops in the device at an early time point (approximately T1 ⁇ 0 min). Condensates are not present in droplet trace 2001 as they have not formed yet. This is because the assay has not progress yet (T0). After 2 minutes, another series of signals were captured (T2). Droplet trace 2002 detected in time point T2 shows an appearance of a small spike in the droplet trace, an early indication of a formation of a dim and small condensate. Droplet trace 2003 captured at T3 clearly indicates a spike demonstrating the formation of a condensate in the droplet.
  • FIGs.21A and 21B provide additional examples of condensate detection in a static miniaturized compartmentalized system.
  • FIG. 21A shows a plurality of condensates (stress granules) formed in a static compartment.
  • the number of condensates may be counted as an indication of the activity of the assay over time. For example, the number (quantity) of the condensates may increase over time.
  • FIG. 21B shows a plot presenting the relative frequency of condensates of various diameters.
  • the sample may be exposed to an effector.
  • the effector may affect the number of condensates formed over time. For example, a greater number of condensates may be formed in absence of a compound, compared to in presence of the compound. That may indicate that the effector has successfully reduced the number of condensates formed, and therefore has been active against the target or Attorney Docket No.56523-707.601 the assay.
  • the methods and systems of the present disclosure facilitate screening condensate detection in presence and absence of drug candidates such as using the bead-bound encoded effector libraries and the miniaturized screening platforms.
  • Exemplary protocol implementing the Bead Index Registration and Dispensing System for encoded effector synthesis using optically pre-encoded beads 1) Start: prepare a pool of Optically Pre-encoded Resin in Tube (conical) (e.g., using the workflow illustrated in FIG.10) 2) Mix, flow through BIRDS, read barcode, dispense into wells in filter-plate a) Index every LASE-barcode signature for every bead entering that well b) count beads per well 3) Conduct synthesis in each well, where each well is often a different Building Block (BB) of a small molecule effector.
  • BB Building Block
  • Some wells may contain identical BBs. Some wells may use a different method. a) correlate optical Bead-barcode to Plate-well location, thereby correlating optical Bead- barcode to BB structure and/or method used. 4) Pool beads from wells into Tube (conical) 5) Mix, flow through BIRDS, read barcode, dispense into wells in filter-plate a) index every LASE-barcode signature for every bead entering that well b) count beads per well 6) Conduct synthesis in each well, where each well is often a different BB. Some wells will contain identical BBs. Some wells may use a different method.
  • Exemplary polymer materials for constructing beads of the disclosure for encoded effector synthesis [000307] Provided herein are beads comprising a substantially homogenous polymer material.
  • the beads may comprise a plurality of spacer monomers, a plurality of crosslinker monomers, and/or a plurality of functional group monomers.
  • FIGS.22A and 22B depict examples of co-polymer materials that can be used to construct the beads disclosed herein.
  • FIG.22A demonstrates that the spacer monomer and crosslinker monomer stoichiometry can be adjusted to optimize the physical characteristics of the final bead polymer material.
  • FIG.22B demonstrates that the crosslinker length can be varied to Attorney Docket No.56523-707.601 optimize density, swelling, and physical integrity.
  • the spacer monomer, the crosslinker monomer, and/or the functional group monomer comprises an amide.
  • the spacer monomer and/or the crosslinker monomer comprises PEG.
  • the length of the PEG (e.g., in the crosslinker monomer) can be variable, e.g., to adjust the physical characteristics of the final bead polymer material (e.g., density, swelling, physical integrity).
  • the crosslinker monomer comprises or is any integer.
  • the spacer monomer comprises or is , where n is any integer.
  • the functional group monomer comprises or is , where n is any integer.
  • the amine of the functional group monomer has a protecting group (e.g., F-moc, BOC, etc.). In some cases, the amine of the functional group monomer has an alkyl moiety.
  • the beads and polymer materials provided herein are merely exemplary and are not intended to be limiting. Any polymer material that is compatible with solid phase peptide synthesis, as described herein, is contemplated.
  • FIG.23A depicts images of exemplary polymerized beads produced using the monomers described herein.
  • FIG 23B depicts the distribution of bead diameters measured from the images shown in FIG.23A.
  • FIG.24 depicts images of exemplary polymerized beads produced using the monomers described herein, and swelled in various solvents (e.g., such as solvents used in solid phase peptide synthesis).
  • FIG.24 demonstrates that the bead size was relatively consistent and the beads did not collapse in different solvents, Attorney Docket No.56523-707.601 demonstrating that the polymer beads are compatible with solid phase peptide synthesis.
  • a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
  • description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6.
  • the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
  • the term “a sample” includes a plurality of samples, including mixtures thereof.
  • determining means determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
  • in vivo is used to describe an event that takes place in a subject’s body.
  • ex vivo is used to describe an event that takes place outside of a subject’s body.
  • ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.
  • An example of an ex vivo assay performed on a sample is an “in vitro” assay.
  • Attorney Docket No.56523-707.601 [000319] The term “in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained.
  • In vitro assays can encompass cell-based assays in which living or dead cells are employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
  • hit refers to an effector that has been screened against a sample and returned a positive result.
  • the positive result may depend upon the nature of the screen being employed, but may include, without limitation, an indication of efficacy against a target being interrogated.
  • hits may be high-interest events of unknown veracity.
  • hits may not be treated as bona-fide until validated (e.g., in replicate tests) afterward.
  • Downstream validation assays may be performed to validate the hits or identify them as false positives.
  • screen refers to performing an assay using a plurality of effectors in order to determine the effect various effectors have on a particular sample.
  • sequencing refers to determining the nucleotide sequence of a nucleic acid. Any suitable method for sequencing may be employed with the methods and systems provided herein. The sequencing may be accomplished by next generation sequencing. Next generation sequencing encompasses many kinds of sequencing such as pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, second- generation sequencing, nanopore sequencing, sequencing by ligation, or sequencing by hybridization. Next-generation sequencing platforms are those commercially available from Illumina (RNA-Seq) and Helicos (Digital Gene Expression or "DGE").
  • Next generation sequencing methods include, but are not limited to those commercialized by: 1 ) 454/Roche Lifesciences including but not limited to the methods and apparatus described in Margulies et al., Nature (2005) 437:376-380 (2005); and US Patent Nos. 7,244,559; 7,335,762; 7,211,390; 7,244,567; 7,264,929; 7,323,305; 2) Helicos Biosciences Corporation (Cambridge, MA) as described in U.S. application Ser. No.1 1/167046, and US Patent Nos.7501245; 7491498; 7,276,720; and in U.S. Patent Application Publication Nos.
  • barcode refers to a nucleic acid sequence that is unique to a particular system.
  • the barcode may be unique to a particular method or to a particular effector.
  • the nucleic acid encodings of the methods and systems provided herein are analogous to barcodes in that they are unique nucleic acid sequences that can be used to identify the structure of a given effector.
  • the length of a barcode or nucleic acid encoding should be sufficient to differentiate between all the effectors in a given library.
  • flow means any movement of liquid or solid through a device or in a method of the disclosure, and encompasses without limitation any fluid stream, and any material moving with, within or against the stream, whether or not the material is carried by the stream. For example, the movement of molecules, cells or virions through a device or in a method of the disclosure, e.g.
  • a flow through channels of a microfluidic chip of the disclosure, comprises a flow.
  • a flow may be used to provide a flow, including without limitation, pressure, capillary action, electro-osmosis, electrophoresis, dielectrophoresis, optical tweezers, and combinations thereof, without regard for any particular theory or mechanism of action, so long as molecules, cells or virions are directed for detection, measurement or sorting according to the disclosure.
  • An “inlet region” is an area of a microfabricated chip that receives molecules, cells or virions for detection measurement or sorting.
  • the inlet region may contain an inlet channel, a well or reservoir, an opening, and other features which facilitate the entry of molecules, cells or virions into the device.
  • a chip may contain more than one inlet region if desired.
  • the inlet region is in fluid communication with the main channel and is upstream therefrom.
  • An “outlet region” is an area of a microfabricated chip that collects or dispenses molecules, cells or virions after detection, measurement or sorting.
  • An outlet region is downstream from a discrimination region and may contain branch channels or outlet channels.
  • a chip may contain more than one outlet region if desired.
  • An “analysis unit” is a microfabricated substrate, e.g., a microfabricated chip, having at least one inlet region, at least one main channel, at least one detection region and at Attorney Docket No.56523-707.601 least one outlet region. Sorting embodiments of the analysis unit include a discrimination region and/or a branch point, e.g. downstream of the detection region, that forms at least two branch channels and two outlet regions.
  • a device according to the disclosure may comprise a plurality of analysis units.
  • a “main channel” is a channel of the chip of the disclosure which permits the flow of molecules, cells or virions past a detection region for detection (identification), measurement, or sorting.
  • the main channel also comprises a discrimination region.
  • the detection and discrimination regions can be placed or fabricated into the main channel.
  • the main channel is typically in fluid communication with an inlet channel or inlet region, which permits the flow of molecules, cells or virions into the main channel.
  • the main channel is also typically in fluid communication with an outlet region and optionally with branch channels, each of which may have an outlet channel or waste channel. These channels permit the flow of cells out of the main channel.
  • a “detection region” is a location within the chip, typically within the main channel where molecules, cells or virions to be identified, measured or sorted on the basis of a predetermined characteristic.
  • molecules, cells or virions are examined one at a time, and the characteristic is detected or measured optically, for example, by testing for the presence or amount of a reporter.
  • the detection region is in communication with one or more microscopes, diodes, light stimulating devices, (e.g., lasers), photo multiplier tubes, and processors (e.g., computers and software), and combinations thereof, which cooperate to detect a signal representative of a characteristic, marker, or reporter, and to determine and direct the measurement or the sorting action at the discrimination region.
  • the detection region is in fluid communication with a discrimination region and is at, proximate to, or upstream of the discrimination region.
  • a “carrier fluid,” “immiscible fluid,” or “immiscible carrier fluid” or similar term as used herein refers to a liquid in which a sample or assay liquid is incapable of mixing and allows formation of droplets of the sample or assay liquid within the carrier fluid. These terms are used interchangeable herein and are meant to encompass the same materials.
  • Non- limiting examples of such carrier fluids include silicon-based oils, silicone oils, hydrophobic oils (e.g., squalene, fluorinated oils, perfluorinated oils), or any fluid capable of encapsulating another desired liquid containing a sample to be analyzed.
  • An “extrusion region,” “droplet extrusion region,” or “droplet formation region” is a junction between an inlet region and the main channel of a chip of the disclosure, which permits the introduction of a pressurized fluid to the main channel at an angle perpendicular Attorney Docket No.56523-707.601 to the flow of fluid in the main channel.
  • the fluid introduced to the main channel through the extrusion region is “incompatible” (i.e., immiscible) with the fluid in the main channel so that droplets of the fluid introduced through the extrusion region are sheared off into the stream of fluid in the main channel.
  • a “discrimination region” or “branch point” is a junction of a channel where the flow of molecules, cells or virions can change direction to enter one or more other channels, e.g., a branch channel, depending on a signal received in connection with an examination in the detection region.
  • a discrimination region is monitored and/or under the control of a detection region, and therefore a discrimination region may “correspond” to such detection region.
  • the discrimination region is in communication with and is influenced by one or more sorting techniques or flow control systems, e.g., electric, electro-osmotic, (micro-) valve, etc.
  • a flow control system can employ a variety of sorting techniques to change or direct the flow of molecules, cells or virions into a predetermined branch channel.
  • a “branch channel” is a channel which is in communication with a discrimination region and a main channel. Typically, a branch channel receives molecules, cells or virions depending on the molecule, cell or virion characteristic of interest as detected by the detection region and sorted at the discrimination region.
  • a branch channel may be in communication with other channels to permit additional sorting.
  • a branch channel may also have an outlet region and/or terminate with a well or reservoir to allow collection or disposal of the molecules, cells or virions.
  • forward sorting or flow describes a one-direction flow of molecules, cells or virions, typically from an inlet region (upstream) to an outlet region (downstream), and in some instances without a change in direction, e.g., opposing the “forward” flow.
  • molecules, cells or virions travel forward in a linear fashion, i.e., in single file.
  • a “forward” sorting algorithm consists of running molecules, cells or virions from the input channel to the waste channel, until a molecule, cell or virion is identified to have an optically detectable signal (e.g., fluorescence) that is above a pre-set threshold, at which point voltages are temporarily changed to electro-osmotically divert the molecule or to the collection channel.
  • optically detectable signal e.g., fluorescence
  • the term “reversible sorting” or flow describes a movement or flow that can change, i.e., reverse direction, for example, from a forward direction to an opposing backwards direction. Stated another way, reversible sorting permits a change in the direction of flow from a downstream to an upstream direction.
  • sorting algorithms for sorting in the microfluidic device can be implemented by different programs, for example under the control of a personal computer.
  • Sorting algorithms for sorting in the microfluidic device can be implemented by different programs, for example under the control of a personal computer.
  • a pressure-switched scheme instead of electro-osmotic flow. Electro-osmotic switching is virtually instantaneous and throughput is limited by the highest voltage that can be applied to the sorter (which also affects the run time through ion depletion effects).
  • a pressure switched-scheme does not require high voltages and is more robust for longer runs.
  • the system is then run backwards at a slow (switchable) speed from waste to input, and the molecule, cell or virion is switched to the collection channel when it passes through the detection region.
  • the molecule, cell or virion is “saved” and the device can be run at high speed in the forward direction again.
  • a device of the disclosure that is used for analysis, without sorting, can be run in reverse to re-read or verify the detection or analysis made for one or more molecules, cells or virions in the detection region. This “reversible” analysis or sorting method is not possible with standard gel electrophoresis technologies (for molecules) nor with conventional FACS machines (for cells).
  • emulsion refers to a preparation of one liquid distributed in small globules (also referred to herein as drops or droplets) in the body of a second liquid.
  • the first liquid which is dispersed in globules, is referred to as the discontinuous phase
  • the second liquid is referred to as the continuous phase or the dispersion medium.
  • the continuous phase is an aqueous solution and the discontinuous phase is a hydrophobic fluid, such as an oil (e.g., decane, tetradecane, or hexadecane).
  • an emulsion is referred to here as an oil in water emulsion.
  • an emulsion may be a water in oil emulsion.
  • the discontinuous phase is an aqueous solution Attorney Docket No.56523-707.601 and the continuous phase is a hydrophobic fluid such as an oil.
  • the droplets or globules of oil in an oil in water emulsion are also referred to herein as “micelles”, whereas globules of water in a water in oil emulsion may be referred to as “reverse micelles”. [000338]
  • the term “about” a number refers to that number plus or minus 10% of that number.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La divulgation concerne des procédés et des systèmes de criblage d'échantillons qui peuvent impliquer le criblage d'échantillons en présence et/ou en l'absence d'effecteurs (par exemple, des bibliothèques d'effecteurs/composés codés) pour leurs effets potentiels sur l'échantillon ou une cible, dans certains cas, un échantillon comprenant une cellule ou un constituant d'une cellule, une cible (par exemple, une enzyme) dans une cellule ou une cible dans un échantillon acellulaire (par exemple, dans un dosage biochimique avec une cible purifiée extraite d'une cellule). Une variété de modalités de codage comprenant des codes-barres d'acide nucléique et des codes-barres optiques sont divulguées. Les procédés et les systèmes présentés ici peuvent comprendre des systèmes miniaturisés pour le criblage d'échantillons à haut débit avec des applications vasculaires comprenant, mais sans y être limitées, un criblage cellulaire et une analyse de perturbation cellulaire telle qu'un criblage de bibliothèque d'effecteurs codés.
PCT/US2023/077649 2022-10-24 2023-10-24 Procédés et systèmes de criblage d'effecteur codé WO2024091949A1 (fr)

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US63/418,915 2022-10-24
US202263419623P 2022-10-26 2022-10-26
US63/419,623 2022-10-26

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