METHODS AND SYSTEMS FOR IN-SILICO AND EMPIRICAL SCREENING

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/421,891, filed November 2, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Sample screening may often be a time consuming and expensive process requiring large quantities of precious samples. Miniaturizing may facilitate efficiently performing sample screening in a high-throughput manner, using small sample volumes, at a high speed. Such methods and systems may be highly valuable and have vast applications in research, diagnostics, and drug discovery. There are several limitations with conventional technologies. The present disclosure provides more efficient and higher-throughput sample screening methods and systems.

SUMMARY

[0003] In some aspects, provided herein is a method comprising: (a) providing or obtaining a computational representation of a target and a computational representation of an effector; (b) calculating one or more parameters indicative of a predicted interaction between the computational representation of the target and the computational representation of the effector; and (c) when the predicted interaction meets a defined criterion, synthesizing the effector on a scaffold, wherein the effector is bound to the scaffold via a cleavable linker and releasable from the scaffold upon cleavage of the cleavable linker. In some cases, the calculating of (b) comprises using a computational program to calculate the one or more parameters. In some cases, the scaffold further comprises a barcode corresponding to and identifying the effector. In some cases, the barcode comprises a nucleic acid molecule or a molecule with detectable spectral or optical properties. In some cases, the method further comprises performing an empirical screen to measure a signal indicative of an interaction between the target and the effector, thereby generating an empirical dataset. In some cases, the interaction, the predicted interaction, or both are influenced by a property of the effector, and wherein the property of the effector is selected from the group consisting of a subunit of the effector, a structure of the effector or a subunit thereof, a characteristic of the structure of the effector or a subunit thereof, and any combination thereof. In some cases, the subunit of the effector comprises a building block (BB) of the effector, and wherein one or more building block(s) of the effector comprise or make up a chemical motif. In some cases, the method further comprises feeding the empirical dataset into the computational program. In some cases, the method further comprises training the computational program using the empirical dataset. In some cases, the training comprises using Artificial Intelligence (Al), Machine Learning (ML), Neural Networks, Predictive Models, or any combination thereof. In some cases, the method further comprises providing or obtaining a secondary dataset and feeding it into the computational program. In some cases, the method further comprises merging the empirical dataset with the secondary dataset, prior to feeding it into computational program. In some cases, the method further comprises processing or cleaning the empirical dataset, the secondary dataset, or both, prior to feeding it into the computational program. In some cases, the secondary dataset is extracted from a database. In some cases, the training comprises increasing the accuracy or efficiency of the computational program in calculating the one or more parameters. In some cases, the method further comprises providing a plurality of computational representations of a plurality of effectors, calculating the one or more parameters for at least a first subset of the plurality of the effectors, selecting a second subset of the plurality of the effectors based on the one or more parameters. In some cases, the calculating the one or more parameters for at least the first subset of the plurality of the effectors informs the selection of the second subset of the plurality of the effectors. In some cases, an algorithm is applied on the one or more parameters to facilitate or guide the selection of the second subset of the plurality of the effectors. In some cases, the interaction comprises binding of the effector to the target, the one or more parameters comprise binding free energy, and the high-performing effectors comprise relatively low binding free energies among the plurality of effectors. In some cases, the computational representation of the target or the computational representation of the effector is imported from a database. In some cases, the computational representation of the target is imported from a database and the computational representation of the effector is designed.

[0004] In some aspects, provided herein is a method comprising: (a) providing or obtaining a computational representation of a target and a plurality of computational representations of a plurality of effectors; (b) using a computational model to calculate one or more parameters indicative of a predicted interaction of the computational representation of the target with each computational representation of the plurality of the computational representations of the plurality of effectors; (c) based on the one or more parameters calculated in (b), ranking the plurality of computational representations of the plurality of effectors; (d) based on the ranking in (c), identifying a plurality of high-performing effectors, wherein performance is based on the predicted interaction; and (e) synthesizing the plurality of high-performing effectors on a plurality of scaffolds, wherein each scaffold comprises an effector of the plurality of high-performing effectors and a barcode corresponding to and identifying the effector, wherein the effector is bound to the scaffold via a cleavable linker and is releasable from the scaffold upon cleavage of the cleavable linker. In some cases, the method further comprises performing an empirical screen of the plurality of scaffolds synthesized in (e), against the target. In some cases, the method further comprises recording a dataset from the empirical screen. In some cases, the method further comprises feeding the dataset into the computational model of (b). In some cases, the method further comprises performing two or more iterative steps to refine the high-performing effectors, wherein each iterative step comprises running or modifying the computational model, performing the empirical screen, or both. In some cases, the effector comprises a plurality of subunits of the effector, the barcode comprises a plurality of subunits of the barcode, and the plurality of subunits of the barcode correspond to and identify the plurality of subunits of the effector. In some cases, the effector is a small molecule. In some cases, the barcode is a nucleic acid molecule. In some cases, the method comprises running the computational model, wherein running the computational model comprises virtual screening. In some cases, the virtual screening comprises molecular docking. In some cases, the interaction comprises binding of the target to the effector, and the parameter comprises binding free energy. In some cases, the effector comprises one or more subunits, and wherein the method comprises indicating a performance metric or identification for at least a subset of the one or more subunits. In some cases, the method further comprises designing the plurality of computational representations of a plurality of effectors. In some cases, designing the plurality of computational representations of the plurality of effectors comprises selecting a subset of the one or more subunits based on the performance metric or identification. In some cases, the predicted interaction comprises a position of the effector relative to the target, an orientation of the effector relative to the target, or a binding free energy of the effector in binding to the target. In some cases, the target comprises one or more binding pocket(s). In some cases, the dataset comprises structure-activity relationships (SARs) between the target and the effector. In some cases, SARs are obtained for a plurality of effectors empirically identified as hits.

[0005] In yet another aspect, provided herein is a method comprising: performing two or more sequential screens, wherein each screen among the two or more sequential screens is informed by or informs another screen among the two or more sequential screens, wherein at least one screen of the two or more sequential screens comprises an empirical screen of a plurality of encoded effectors, wherein each encoded effector comprises: (i) an effector bound to a scaffold via a cleavable linker and releasable upon cleavage of the cleavable linker, and; (ii) a barcode corresponding to and identifying the effector. In some cases, at least one screen of the two or more sequential screens comprises an in-silico screen. In some cases, the in-silico screen comprises: (a) providing or obtaining a computational representation of a target and a plurality of computational representations of the effectors of the plurality of encoded effectors; and (b) using a computational program and running it to calculate one or more parameters predictive of one or more interactions between the computational representation of the target and the computational representation of each effector. In some cases, the in-silico screen is the first screen among the two or more sequential screens to be performed. In some cases, the in-silico screen comprises performing predictive analysis using Artificial Intelligence (Al), Machine Learning (ML), or Neural Networks. In some cases, the method further comprises providing a map of Structure Activity Relationships (SARs) among a target and a plurality of effectors. In some cases, the in-silico screen informs a second screen, and wherein the second screen comprises empirically screening a first library of bead -bound encoded effectors, the second screen informs a third screen, and the third screen comprises empirically screening a second library of bead-bound encoded effector. In some cases, the first library is a diverse library and the second library is a focused library. In some cases, the number of effectors screened in the first screen is larger than the number of the effectors screened in the second screen, and the number of the effectors screened in the second screen is larger than the number of the effectors screened in the third screen. In some cases, at least one screen of the two or more sequential screens comprises an empirical screen of a plurality of bead-bound encoded effectors on cells. In some cases, at least one screen of the two or more sequential screens comprises providing or obtaining a miniaturized screening platform comprising a plurality of compartments, wherein at least a subset of the plurality of compartments comprises at least one bead-bound encoded effector trapped therein, and wherein the bead-bound encoded effector interacts with the target, thereby generating a signal indicative of the activity of the target in presence of the bead-bound encoded effector. In some cases, the compartment is a droplet, a well, a nanopen, or a miniaturized channel or feature. In some cases, the compartment is a droplet formed with the aid of a microfluidic device. In some cases, the microfluidic device comprises an integrated circuit, and wherein the integrated circuit comprises a droplet generated followed by an incubation region, and wherein the incubation region allows for incubating the droplet for at least 12 minutes with a dispersion ratio of less than 9%.

[0006] Further provided herein are methods and systems for miniaturized high- throughput screening of encoded effector libraries using miniaturized compartmentalized systems. Presented are methods and systems for miniaturized high-throughput screening. In some embodiments, 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.

[0007] In an aspect, provided herein is 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.

[0008] In some embodiments, the bead does not comprise a solid core made of a material different from the substantially homogeneous bead polymer material. In some embodiments, the bead does not comprise a core-shell structure. In some embodiments 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. In some embodiments, the inner core is a polymer material. In some embodiments, the bead does not comprise a polystyrene core. In some embodiments, the inner core is a substantially soft material. In some embodiments, the inner core comprises functional groups. In some embodiments, the bead comprises functional groups for synthesizing effectors. In some embodiments, the substantially homogenous polymer material comprises functional groups for synthesizing effectors.

[0009] In some embodiments, the inner core comprises functional groups for synthesizing effectors. In some embodiments, the functional groups for synthesizing effectors comprise amines. In some embodiments, amines may be protected by a protecting group. Examples of protecting groups may comprise F-moc, BOC, Azide, or Carbamide. In some embodiments, the bead further comprises functional groups for oligonucleotide synthesis. In some embodiments, the bead further comprises functional groups for oligonucleotide synthesis. In some embodiments, the functional groups for oligonucleotide synthesis comprise at least about 10 ⁵ molecules per bead. In some embodiments, the functional groups for oligonucleotide synthesis comprise Azide.

[0010] In some embodiments, 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. In some embodiments, the barcode comprises one or more optical barcoding particles on the surface of the bead or inside the bead. In some embodiments, the optical barcoding particles comprise spectral properties in the short-wave infrared (IR) range.

[0011] In some embodiments, the optical barcoding particles comprise a surface. In some embodiments, the optical barcoding particles comprise amphiphilic surfaces. In some embodiments, the surfaces of the optical barcoding particles comprise a surface coating. In some embodiments, the surface coating comprises Si.

[0012] In some embodiments, 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 pm. In some embodiments, the diameter of the cylindrical optical barcoding particle is at most about 5 pm. In some embodiments, the diameter of the cylindrical optical barcoding particle is at most about 2 pm. In some embodiments, the optical barcoding particle is detectable by fluorescence or luminescence

[0013] In some embodiments, the optical barcoding particle comprises an excitation wavelength of from about 1000 to about 1100 nanometers (nm). In some embodiments, 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).

[0014] 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.

[0015] In some embodiments, 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.

[0016] In some embodiments, the volume of the optical barcode is at most about 0.05% of the volume of the bead. In some embodiments, the optical barcode comprises at least 1, 2, 3, 4, 5, or 6 optical barcoding particles. In some embodiments, the barcode comprises a nucleic acid molecule covalently bound to the bead or trapped inside the bead. In some embodiments, the barcode comprises a DNA, an RNA, a peptide, or a peptide nucleic acid (PNA). In some embodiments, the bead further comprises an optical barcode thereon or therein.

[0017] In some embodiments, 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. In some embodiments, the bead diameter is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 micrometers (pm) 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. [0018] In an aspect, provided herein is 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. In some embodiments, the compartment is a droplet or a well. In some embodiments, the effector is bound to the scaffold via a cleavable linker and is releasable upon cleavage of the cleavable linker, and the method comprises exposing the bead to a stimulus to cleave the cleavable linker and release the effector into the compartment. In some embodiments, the compartment further comprises an assay reagent and a target, and the signal is indicative of the activity of the target in presence of the effector, as measured using the assay reagent.

[0019] In an aspect, provided herein is 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.

[0020] In some embodiments, the encoding particles comprises a spectral emission bandwidth of at most about 100 nm. In some embodiments, the solid support comprises a particle. In some embodiments, the particle comprises a diameter of at most about 30 micrometers (pm). In some embodiments, the solid support comprises or is a bead. In some embodiments, solid support is encapsulated in a compartment. In some embodiments, the compartment comprises or is a droplet, a well, a nanopen, or a miniaturized channel.

[0021] In some embodiments, the compartment is a droplet surrounded by an immiscible oil. In some embodiments, the droplet is generated with the aid of a droplet microfluidic device, and the system further comprises the droplet microfluidic device. In some embodiments, the compartment is a well in a miniaturized array platform. In some embodiments, the compartment is a microfluidic or miniaturized compartment comprising four sides, wherein the four sides comprise three closed sides and one open side.

[0022] In an aspect, provided herein is 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.

[0023] In some embodiments, the encoding particles comprise a spectral emission bandwidth of at most about 3 nm. In some embodiments, the first signal and the second signal are spectrally independent. In some embodiments, the first signal is in the visible range. In some embodiments, the second signal is not in the visible range. In some embodiments, the second signal is in the short-wave infrared range.

[0024] In an aspect, provided herein is 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. [0025] In an aspect, provided herein is a screening method comprising: (a) providing or obtaining a computational representation of a target and a computational representation of an effector, (b) providing or obtaining a computer program and running it to calculate one or more parameters indicative of a predicted interaction between the computational representation of the target and the computational representation of the effector, and (c) synthesizing the effector on a scaffold. In some embodiments, the effector is bound to the scaffold via a cleavable linker and releasable upon cleavage of the cleavable linker. In some embodiments, the scaffold further comprises a barcode corresponding to the effector and identifies it.

[0026] In some embodiments, the barcode comprises a nucleic acid molecule or a molecule with detectable spectral or optical properties. In some embodiments, the method further comprises performing an empirical screen to measure a signal indicative of an interaction between the target and the effector, thereby generating an empirical dataset. [0027] In some embodiments, the interaction, the predicted interaction, or both are influenced by a property of the effector, and the property of the effector comprises a structure of the effector, a characteristic of the structure of the effector, a subunit of the effector, a structure of the subunit of the effector, a characteristic of a structure of a subunit of the effector, or any combination thereof. In some embodiments, the effector comprises a plurality of subunits of the effector, the subunits of the effector comprise a building block (BB) of the effector, and the BB of the effector comprise a chemical motif. In some embodiments, a BB or a characteristic thereof may be driving an effect on the target.

[0028] In some embodiments, the method may further comprise feeding (e.g., providing as an input or a part of an input) the empirical dataset into the computational program. In some embodiments, the method may further comprises training the computational program using the empirical dataset. In some embodiments, training may comprise using Artificial Intelligence (Al), Machine Learning (ML), Neural Networks, Predictive Models, or any combination thereof.

[0029] In some embodiments, the method may further comprise providing or obtaining a secondary dataset and feeding it into the computational program. The method may further comprise merging the empirical dataset with the secondary dataset, at any point during the workflow, for example, prior to feeding it into the computational program. In some embodiments, the method further comprises processing or cleaning the empirical dataset, the secondary dataset, or both, prior to feeding it into the computational program. In some embodiments, the secondary dataset is obtained, extracted, or read from a public database. In some embodiments, training comprises increasing the accuracy or efficiency of the computational program in calculating the one or more parameters.

[0030] In some embodiments, a screening method comprises providing a plurality of computational representations of a plurality of effectors, calculating the one or more parameters for at least a first subset of the plurality of the effectors, selecting a second subset of the plurality of the effectors based on the one or more parameters, and synthesizing the second subset of the plurality of the effectors on a plurality of scaffolds via cleavable linkers. In some cases, the second subset may comprise a promising effectors, high-performing effectors, or effectors with a higher likelihood of being active against a target compared to an average effector in a chemical space. In some cases, a high-performing effector may suspected to have an activity against a target based on observations in a preliminary screen which may in some cases be a predictive analysis using a computer program, in-silico screen, an algorithm. The methods may comprise using Artificial Intelligence (Al), machine learning (ML), and/or Neural Networks.

[0031] In some embodiments, running the computer program may informs the selection of the second subset of the plurality of the effectors (e.g., promising effectors or effectors suspected of having a high likelihood of being active against a target or having an intended effect). In some embodiments, an optimization algorithm is applied on the one or more parameters to facilitate or guide the selection of the second subset of the plurality of the effectors (e.g., after performing a preliminary screen or a first screen, which in some cases is a virtual, computational, or in-silico screen).

[0032] In some embodiments, the screen may model or measure an interaction between a target and an effector, or conduct/facilitate any combination of both, such as to provide information about an effect of an effector. In some embodiments, the interaction may be a binding of the effector to the target. Computational screening may calculate the one or more parameters which in some cases may comprise binding free energy. The promi sing/high- performing effectors may comprise relatively low binding free energies among the plurality of effectors. For example, a threshold may be set or defined for the parameter (e.g., binding free energy), and effectors comprising a binding free energy below the set threshold may be identified as high-performing or promising effectors, and may proceed to the next screen. For example, the high-performing effectors may be synthesized on beads according to any embodiment in the present disclosure and screened using any screening system provided in the present disclosure. The combination of steps of the methods performed may lead to mapping structure activity relationships (SARs), provide information about a plurality of effectors in a chemical space, lead to identification of hits and new drug candidates. The methods may be used for hit-to-lead drug discovery.

[0033] In an aspect, provided herein is a method of screening comprising: (a) providing or obtaining a computational representation of a target and a plurality of computational representations of a plurality of effectors; (b) providing or obtaining a computational model and calculating one or more parameters indicative of a predicted interaction of the computational representation of the target with each computational representation of the plurality of the computational representations of the plurality of effectors, using the computational model; (c) based on the one or more parameters calculated in (b), ranking the plurality of computational representations of the plurality of effectors; (d) based on the ranking in (c), identifying a plurality of high-performing effectors, wherein performance is based on the predicted interaction; and (e ) synthesizing the plurality of high-performing effectors on a plurality of scaffolds, wherein each scaffold comprises an effector of the plurality of high-performing effectors and a barcode corresponding to the effector and encoding it, wherein the effector is bound to the scaffold via a cleavable linker and is releasable upon cleavage of the cleavable linker.

[0034] In some embodiments, the method further comprises performing an empirical screen of the plurality of scaffolds synthesized in (e), against the target. In some embodiments, the method further comprises recording a dataset from the empirical screen. In some embodiments, the method further comprises feeding the dataset into the computational model of (b). In some embodiments, the method further comprises performing two or more iterative steps to refine the high-performing effectors, wherein each iterative step comprises running or modifying the computational model and performing the empirical screen.

[0035] In some embodiments, the effector comprises a plurality of subunits of the effector, the barcode comprises a plurality of subunits of the barcode, and the plurality of subunits of the barcode correspond to and identify the plurality of subunits of the effector. In some embodiments, the effector is a small molecule. In some embodiments, the subunit of the effector is a building block (BB) of a small molecule. In some embodiments, the methods comprise small molecule drug discovery using a combination of in-silico and empirical screening, and screening may comprise using miniaturized compartmentalized high- throughput screening systems such as droplet microfluidics and miniaturized arrays.

[0036] In some embodiments, the barcode is a nucleic acid molecule. In some embodiments, the barcode comprises one or more optical barcoding particles. In some embodiments, the method comprises running the computational model, wherein running the computational model comprises performing a virtual screen (e.g., virtual drug screen). In some embodiments, virtual screening comprises molecular docking. In some embodiments, the interaction between a target and an effector comprises binding and the parameter comprises binding free energy.

[0037] In some embodiments, the effector comprises one or more subunits, and the method comprises indicating a performance metric or identification for at least a subset of the one or more subunits. In some embodiments, the method further comprises designing the plurality of computational representations of a plurality of effectors. In some embodiments, designing the plurality of computational representations of the plurality of effectors comprises selecting a subset of the one or more subunits based on the performance metric or identification. The method may comprise selecting BB for designing and/or synthesizing small molecule drugs.

[0038] In some embodiments, the predicted interaction comprises a position of the effector relative to the target, an orientation of the effector relative to the target, or a binding free energy of the effector in binding to the target. In some embodiments, the dataset (e.g., generated through a virtual screen, an empirical screen, or any combination thereof) comprises structure-activity relationships (SAR) between the target and the effector or a plurality of effectors. In some cases, a subset of the effectors screened may be identified as hits.

[0039] In an aspect, provided herein is a method comprising: performing two or more sequential screens, wherein each screen among the two or more sequential screens is informed by or informs another screen among the two or more sequential screens, or both. In some embodiments, at least one screen of the two or more sequential screens comprises an empirical screen of a plurality of bead-bound encoded effectors, wherein each bead-bound encoded effector comprises: (i) an effector bound to a bead via a cleavable linker and releasable upon cleavage of the cleavable linker, and (ii) a barcode corresponding to the effector that identifies it.

[0040] In some embodiments, at least one screen of the two or more sequential screens comprises an in-silico screen. In some embodiments, the in-silico screen comprises: (a) providing or obtaining a computational representation of a target and a plurality of computational representations of the effectors of the plurality of bead -bound encoded effectors; and (b) providing or obtaining a computational program and running it to calculate one or more parameters indicative of a predicted interaction between the computational representation of the target and the computational representation of each effector. [0041] In some embodiments, the in-silico screen is the first screen to be performed. The in-silico screen may comprise predictive analysis to inform one or more screens subsequent to it. The in-silico screen may comprise a model which may be trained and improved by empirical data, data from a public database or any suitable source, or any combination thereof. In some embodiments, the in-silico screen comprises performing predictive analysis using Artificial Intelligence (Al), Machine Learning (ML), Neural Networks, any other suitable in-silico screening approach, or any combination thereof.

[0042] In some embodiments, the method further comprising providing a map of Structure Activity Relationships (SARs) among a target and a plurality of effectors, in some cases, using a combination of in-silico and empirical screening which may inform each other. In some embodiments, the in-silico screen may be the first screen to be performed, and/or may inform a second screen. The second screen may comprises empirically screening a first library of encoded effectors (e.g., bead-bound encoded effector library or one bead one compound (OBOC) library). In some embodiments, the second screen may inform a third screen. The third screen may comprise empirically screening a second library of bead -bound encoded effector. In some embodiments, the method may be used for screening an encoded effector that is not bound to a bead/scaffold. Any empirical screen performed may comprise generating a training dataset. The training dataset may be used to train the model used as part of the in-silico screen. The empirical screens may further generate a test dataset for testing the quality of the computational model. Any appropriate Machine Learning (ML) workflow may be used.

[0043] In some embodiments, the number of effectors screened in the first screen may be larger than the number of the effectors screened in the second screen. In some embodiments, the number of the effectors screened in the second screen may be larger than the number of the effectors screened in the third screen. As such, the effectors screened may get refined as the workflow proceeds and/or advances. In some embodiments, at least one screen of the two or more sequential screens comprises an empirical screen of a plurality of encoded effectors on cells.

[0044] In some embodiments, at least one screen of the two or more sequential screens comprises providing or obtaining a miniaturized screening platform comprising a plurality of compartments, wherein at least a subset of the plurality of compartments comprise at least one bead-bound encoded effector, and wherein the bead-bound encoded effector interacts with a target in the compartment, thereby generating a signal indicative of the activity of the target in presence of the bead-bound encoded effector. In some embodiments, the same or similar methods may be performed on an encoded effector that is not bound to a bead.

[0045] In some embodiments, the compartment is a droplet, a well, a nanopen, or a miniaturized channel or feature. In some embodiments, the compartment is a droplet formed with the aid of a microfluidic device. In some embodiments, the microfluidic device comprises an integrated circuit. The integrated circuit may comprise a droplet generated followed by an incubation region, and the incubation region may allow for incubating the droplet for at least 5, 10, 15, 20 minutes, or above, with a dispersion ratio of less than about 20%, 10%, 9%, 3%, 2% or less.

INCORPORATION BY REFERENCE

[0046] U.S. Patent Application Nos. 17/067,534, filed October 9, 2020; 63/380,709, filed October 24, 2022; 63/418,915, filed October 24, 2022; and 63/419,623, filed October 26, 2022, are incorporated by reference herein in their entirety and for all purposes. In addition, all publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety and for all purposes.

[0047] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0049] 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. [0050] 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.

[0051] 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.

[0052] FIG. 2C provides a depiction of the released encoded effector-fluorophore from FIG. 2B.

[0053] 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.

[0054] FIGs. 2E and 2F provide exemplary chemical reactions for activating molecules for photocleavage.

[0055] 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. Each type of functional group may be configured to be attached to a barcode and/or an effector.

[0056] 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.

[0057] FIG. 4 schematically illustrates an exemplary droplet microfluidic device for screening.

[0058] FIG. 5 provides an exemplary workflow for encoded effector screening using a droplet microfluidic platform.

[0059] 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. [0060] 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.

[0061] 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).

[0062] FIG. 9 illustrates an exemplary workflow of a split-and-pool method for generating spectrally/optically encoded effector libraries.

[0063] FIG. 10 illustrates an exemplary workflow of using a Bead Index Registry and Dispensing System (BIRDS) for generating beads pre-encoded with optical barcodes. Optically pre-encoded beads can be later used for effector synthesis and creation of One Bead One Compound Spectrally/Optically Encoded Libraries (OBOC-SEL). The term compound can be generalized to any kind of effector.

[0064] 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.

[0065] 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.

[0066] 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. In addition, 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.

[0067] 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.

[0068] 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.

[0069] 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. [0070] 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.

[0071] 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.

[0072] FIG. 18 illustrates an exemplary method for amplifying a primer to maximize cellular nucleic acid capture.

[0073] FIG. 19 depicts a computer system suitable for performing the methods disclosed herein.

DETAILED DESCRIPTION

[0074] Provided herein are methods and systems for sample screening. In some cases, 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. In some examples, 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. In some examples, screening platforms may comprise droplet microfluidic devices. In some examples, 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.

[0075] In some cases, 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. In some cases, screening may comprise high-throughput screening wherein large numbers of effectors are screened in a shorter period compared to preceding technologies.

[0076] In some embodiments, 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 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.

[0077] 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. Alternatively, 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.

[0078] In some examples, a scaffold (e.g., a bead) may comprise on or more effectors bound thereto. The one or more effectors may be similar or different. For example, a scaffold may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different effectors attached thereto. In some cases, the synergistic effects of more than one effector can be screened on the sample in the same compartment. For example, a scaffold (e.g., a bead) may comprise effector A and effector B attached thereto via a cleavable linker. Effector A and effector may be attached to one another. Alternatively, effector A and effector B may not be attached to one another. 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”.

[0079] 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 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. In some cases, 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.

[0080] Screening encoded effector libraries against targets present in live cells may be challenging. Many conventional cell-based high-throughput assays are limited in the number of compounds that can be economically screened due to the time required to process samples as well as the ability to store and manage a large library of effectors. Additionally, combining encoded effector libraries with conventional microtiter plate-based high-throughput screening (HTS) platforms may be challenging. The volume of reagent required to fill a microtiter plate can place an upper limit on the effective concentration of effector released from a bead. Also, sorting individual beads into a microtiter plate is technically challenging and time consuming. [0081] In some embodiments, provided herein are methods and systems for performing high-throughput cell screening and perturbation analysis at a scale greater than existing/conventional high-throughput screening (HTS) systems. This may be facilitated by miniaturizing the screening system. 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). In some cases, by miniaturizing the compartments, 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.

[0082] In some cases, a miniaturized droplet-based microfluidic platform may be used to perform the screens. Alternatively or in addition, platforms such as array -based platforms, well-based platforms, micro-raft arrays, and other compartmentalized screening platforms may be used. Some cell types (e.g., some adherent cells) may be more suitably screened while adhered 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. Alternatively or in addition, 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 microenvironment for the cell.

[0083] In some cases, a hydrogel matrix may provide support for cell adhesion in a screening platform. In some cases, an artificial tumor spheroid may be generated. For example, one or more cells (e.g., suspension cells or adherent cells) may be suspended in a polymerizable monomer. The polymerizable monomer may be subjected to polymerization and gelati on/solidificati on. 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. In some cases, 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. In some cases, more than one cell type may be co-cultured using the described methods and systems and may be perturbed and/or screened.

[0084] 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). In some examples, 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. Stated differently, 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. For example, 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.

[0085] 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 of a cell (e.g., cell lysates or intracellular components, elements, organelles, molecules, or beyond). In some cases, based on the detected signal, 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. Alternatively or in addition, in some cases, 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. For example, 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. Decisions about 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) may be performed during the screen or at a later stage. Stated in other words, 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.

Screening encoded effector libraries in compartmentalized systems

[0086] Provided herein are methods and systems for screening a library of encoded effectors, such as a plurality of different/unique encoded effectors using the screening platform provided anywhere herein on any sample or target provided anywhere herein. In some examples, a plurality of scaffolds (e.g., beads or any other kind of scaffold or solid support mentioned anywhere herein) 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).

[0087] 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. In some examples, the library may comprise 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, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 10000 or more unique encoded effectors. In some examples, 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. In some examples, 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. In some examples, 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.

[0088] In some examples, 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. In some examples, 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. In some examples, 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. In some examples, 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. In some examples, the library may comprise 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, or a smaller number of unique encoded effectors.

[0089] 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. In some examples, 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). [0090] 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). In this example, 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 (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).

[0091] FIG. IB 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. In some cases, the correspondence and identification may be facilitated during the synthesis of the encoded effector. For example, the barcodes (e.g., the nucleic acid barcode, the optical barcode or both) 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. In case of nucleic acid barcodes, subunits may be sequences included in the nucleic acid barcode. In case of optical barcodes, 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. In some cases, the bead may only comprise the optical barcoding particles as barcode(s) and not the nucleic acid barcode. For example, in some cases, 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.

[0092] Provided herein are methods and systems comprising providing, obtaining, and using optical/spectral/photonic encoding particles. 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. 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. 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).

[0093] 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. In some cases, the optical barcode may get excited. In some cases, the particle may resonate to generate a signal. In some cases, the particle may be stimulated or excited by an energy source (electromagnetic waves, heat, acoustic) and resonate this energy internally. In some cases, the internal resonance is maintained through electron orbital conjugation, or through whispering gallery modes (WGMs). In some cases, the particle may dissipate energy through vibration, mechanical forces, or quantum tunneling effects. In some cases, the particle may emit electromagnetic waves through collapse of resonance, or through a lasing cavity. In some cases, the energy of the emitted or lased electromagnetic waves is less than the energy of the stimulus. In some cases, the direction of photon emission depends on the orientation of the optical particle.

[0094] In some examples, 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. In an example, 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.

[0095] In some examples, 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.

[0096] 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 potentially/optionally other factors. In some examples, 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). In some examples, 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. In some cases, 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.

[0097] A screening device used for screening encoded effector libraries may comprise a sorting module. Alternatively, a screening device used for screening encoded effector libraries may not comprise a sorting module. In some cases, 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 structureactivity relationships (SAR) more comprehensively. The methods are described in more detail throughout the disclosure.

[0098] 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.

[0099] An effector can be any type of molecule or substance whose effect on a sample may be investigated. In examples, the effector may comprise or be a compound, a protein, a peptide, an enzyme, a nucleic acid, a gene, or any other substance. In some instances, the encoding allows a user to determine the structure of the effector by measuring/detecting a property of the encoding. Thus, each encoding moiety has a measurable property that, when measured, can be used to determine the structure of the effector which is encoded. Many different encoding modalities can be used. Encoding modalities may comprise nucleic acids, DNA, RNA, peptides, peptide nucleic acids (PNA). In some examples, encoding modalities may comprise optical barcodes, nanoparticles, luminescent materials, and/or quantum dot particles. In some examples, various combinations of encoding modalities may be used. For example, encoding modalities may comprise both nucleic acid molecules and optical barcodes.

[0100] In some examples, encodings may comprise nucleic acid molecules such as DNA, RNA, or PNA. In some cases, 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. When the encoding modalities are nucleic acids, the sequence of the nucleic acid may provide information about the structure of its corresponding effector. In some instances, 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. [0101] In some instances, 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. In some instances, when encoded effectors are placed into solutions or other environments, 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. In some cases, the scaffold may be a bead.

[0102] 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. This is sometimes referred to as “split and pool synthesis.” For illustrative purposes only, 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. This process is repeated as many times as desired to prepare the desired effectors and corresponding encodings. This process can be repeated on a massively parallel scale in small volumes to prepare vast libraries of compounds at low cost and with low amounts of reagents. In some instances, 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. In some instances, 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.

[0103] In some embodiments, the scaffolds further comprise impurities in the effector and/or its encoding. For example, 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. In some instances, 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. In some embodiments, impurities of the effector and its corresponding encoding are present due to defects in the methodologies used to synthesize the encoded effectors. In some embodiments, scaffolds as described herein can comprise a single encoder, an encoding and its impurities, or combinations thereof. In some embodiments, 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. In some embodiments, 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.

[0104] Cleavable linkers can be used to attach effectors to scaffolds. In some embodiments, the effector is bound to a scaffold by a cleavable linker. In some embodiments, the cleavable linker is cleavable by electromagnetic radiation, an enzyme, a chemical reagent, heat, pH adjustment, sound, or electrochemical reactivity. In some embodiments, the cleavable linker is cleavable by electromagnetic radiation. In some embodiments, the cleavable linker is cleavable by electromagnetic radiation such as UV light. In some embodiments, the cleavable linker is a photocleavable linker. In some embodiments the photocleavable linker is cleavable by electromagnetic radiation. In some embodiments the photocleavable linker is cleavable through exposure to light. In some embodiments, the light comprises UV light. In some embodiments, the cleavable linker is cleavable by a cleaving reagent. In some embodiments, the cleavable linker must first be activated in order to be able to be cleaved. In some embodiments, the cleavable linker is activated through interaction with a reagent.

[0105] In some embodiments, the cleavable linker is a disulfide bond. In some embodiments, the cleavable linker is a disulfide bond, and the cleavable reagent is a reducing agent. In some embodiments, the reducing agent is a disulfide reducing agent. In some embodiments, the disulfide reducing agent is a phosphine. In some embodiments, the reducing agent is 2-mercapto ethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol, a combination thereof, or a derivative thereof.

[0106] In some embodiments, 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. [0107] In some embodiments, 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. In some embodiments, the cleavable linker as the structure

Effector scaffold, wherein X is -C(=O)NR-, -C(=O)O-, -C(=O)- or a bond, and R is H or alkyl. In some embodiments, the cleaving reagent is a tetrazine. In some embodiments, 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.

[0108] In some embodiments, the cleavable linker comprises an azido group attached to the same carbon as an ether linkage. In some embodiments, the cleavable linker has the

Scaffold Effector

Effector Scaffold structure . 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.

[0109] In some embodiments, the cleavable linker is cleaved by a transition metal catalyst. In some embodiments, the cleavage reagent is a transition metal catalyst. In some embodiments, the transition metal catalyst is a ruthenium metal complex. In some embodiments, the cleavable linker is an O-allylic alkene. In some embodiments, the cleavable

Scaffold 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. In some embodiments, the transition metal complex is a palladium complex. In some embodiments, the cleavable linker has the structure

Scaffold Effector . Such cleavable linkers are described in 3’-O- modified nucleotides as reversible terminators for pyrosequencing, PNAS October 16, 2007, 104 (42) 16462-16467, which is hereby incorporated by reference.

[0110] In some embodiments, the number of effectors cleaved from the scaffold is controlled. In some embodiments, the number of effectors cleaved from a scaffold is controlled by controlling the amount of stimulus used to cleave the cleavable linker. In this context, a “stimulus” is any method or chemical used to specifically cleave a cleavable linker. In some embodiments, the stimulus is a chemical reaction with a cleaving reagent. In some embodiments, 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.

[OHl] 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.

[0112] In some embodiments, the number of effectors cleaved from the scaffold is controlled by controlling the concentration of the cleaving reagent. In some embodiments, the concentration of the cleavage reagent is controlled in an encapsulation containing an encoded effector bound to a scaffold. In some embodiments, the concentration of chemical reagent used 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 pM, at least 10 pM, at least 100 pM, at least 1 mM. at least 10 mM, at least 100 mM, or at least 500 mM. In some embodiments, 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 pM, at most 10 pM, at most 100 pM, at most 1 mM, at most 10 mM, at most 100 mM, or at most 500 mM.

[0113] In some embodiments, the cleaving reagent is added to a plurality of encapsulations. In some embodiments, 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. In some embodiments, 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.

[0114] In some embodiments, the cleaving reagent is added to the encapsulation by picoinjection. In some embodiments, the encapsulation is passed through a microfluidic channel comprising a pico-inj ection site. In some embodiments, pico-inj ections are timed such that the rate of pico-inj ection matches the rate at which encapsulation cross the pico-inj ection site. In some embodiments, at least 80%, 85%, 90%, 95%, 98%, or 99% of encapsulations passing a pico-inj ection site receive a pico-inj ection. In some embodiments, the pico-inj ections 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.

[0115] In some embodiments, the cleaving reagent is added from a stock solution to the encapsulation. In some embodiments, 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.

[0116] In some embodiments, methods and systems described herein comprise cleaving a photocleavable linker between an encoded effector and a scaffold. In some embodiments, the methods and systems described herein comprise exposing an encapsulation to electromagnetic radiation comprising an effector bound to a scaffold through a photocleavable linker. In some embodiments, the methods and systems described herein comprise exposing an encapsulation to light (for e.g., UV light) comprising an effector bound to a scaffold through a photocleavable linker. In some embodiments, the encapsulation is exposed to the light using a microfluidic device.

[0117] In some embodiments, the photocleavable linker is cleaved by exposure to light (e.g., UV light). In some embodiments, 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. In some cases, 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.

[0118] In some embodiments, the cleavable linker may be cleaved by electromagnetic radiation. In some embodiments, the concentration of the number of effector molecules released from a scaffold is controlled by controlling the intensity or duration of electromagnetic radiation.

[0119] 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, a-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. In some examples, the cleavable linker is an o-nitrobenzyloxy linker, an o-nitrobenzylamino linker, an a-substituted o-nitrobenzyl linker, an o-nitroveratryl linker, a phenacyl linker, p-alkoxyphenacyl linker, a benzoin linker, or a pivaloyl linker. [0120] In some examples, 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. In some cases, 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 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).

[0121] FIG. 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/j a035616d). As depicted, 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. As depicted in FIG. 2E, 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). As depicted, the thio-phenol ester provides a stable covalent linker to compound (R). Specific oxidation of the thio-phenol (shown in middle molecule) can generate an “activated” linker-moiety. Kinetic control of the oxidation step may allow for quantitative “activation” to prescribe compound release. In some embodiments, base treatment causes linker scission through elimination, thereby generating a free acid compound, or with subsequent decarboxylation generates just a compound.

[0122] 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).

[0123] 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. In some cases, 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. For example, in case the screening system is a droplet microfluidic system, pico-inj ection may be used to inject a chemical into a droplet after its formation to cleave the cleavable linker. In another example, if the screening system is an array, 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. In some examples, the activating reagent for activating the cleavable linker may comprise a disulfide reducing reagent. In an example, the activating reagent comprises tetrazine.

[0124] The activating reagent may be added to the encapsulation by pico-inj ection. In some examples, the encapsulation may pass through a microfluidic channel comprising a pico-inj ection site, in case the encapsulation is a droplet in a microfluidic device. Reagent addition (e.g., pico-inj ections) may be timed such that the rate of reagent addition (e.g., pico- injection) matches the rate at which encapsulation cross the pico-inj ection site. In some examples, least 80%, 85%, 90%, 95%, 98%, or 99% of encapsulations passing a picoinjection site receive a pico-inj ection. In some embodiments, the pico-inj ections 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 activating reagent is added to the encapsulation by droplet merging.

[0125] 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. In some examples, the concentration of activating reagent may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100 micromolar (pM). 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.

[0126] 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. [0127] 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 concentrations of effector being screened against a sample or target may be released. As a result, in some cases, making a determination of the efficacy of individual effectors may be difficult to ascertain. By releasing a uniform amount of effectors from each scaffold in a screen, a uniform dose across the screen may be employed, removing bias from lower potency, higher concentration effectors.

[0128] In some examples, 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. 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 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 pM 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.

[0129] 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 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 pM 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 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. For example, 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.

[0130] 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). In some cases, the incubation time may be at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour(s) (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.

[0131] 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 term “effector” is used broadly to encompass any moiety whose effect on a sample is being interrogated. [0132] In some examples, 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.

[0133] 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 nonpeptide 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.

[0134] In some examples, the effectors may elicit a response from the target being interrogated. The response elicited can take any form and may depend on the sample being interrogated. As an example, when the sample comprises a cell, the response may be a change in expression pattern, apoptosis, expression of a particular molecule, or a morphological change in the cell. As another example, when the sample comprises a protein, the effector may inhibit protein activity, enhance protein activity, alter protein folding, or measure protein activity.

[0135] In some examples, 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. In some examples, the effector may be a peptide, a non-natural peptide, a polymer, or an unnatural amino acid. In some examples, the peptide may comprise a nonpeptide region. In some examples, the peptide may be a cyclic peptide. In some examples, the peptide may comprise a secondary structure that mimics a protein.

[0136] The peptide or polymer may be made of a number of units (e.g., a number of amino acids). In some examples, the peptides may comprise a number of amino acids. For example, 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. In some examples, 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. In some cases, 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.

[0137] 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. In some examples, an effector/compound may be a completed chemical that is synthesized by connecting a plurality of chemical monomers to each other. In some examples, 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).

[0138] In some examples, the effector may be an effector nucleic acid. The effector nucleic acid may comprise a number of nucleotides. In some examples, 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. In some examples, 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. In some examples, 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 10 ⁵ , or more. In some examples, 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

[0139] The effectors provided herein can be linked with encodings. In some embodiments, the effectors are linked with an encoding. In some instances, the encoding allows a user to determine the structure of the effector by determining a property of the encoding. Thus, each encoding moiety has a measurable property that, when measured, can be used to determine the structure of the effector which is encoded.

[0140] In some examples, 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.

[0141] In some examples, the barcode/encoding may comprise or be DNA. An example barcode may comprise double-stranded DNA or single-stranded DNA. In some examples, the barcode/encoding may comprise or be RNA. An example barcode may comprise doublestranded RNA or single-stranded RNA. In some examples, the barcode may comprise or be a peptide or a peptide nucleic acid (PNA).

[0142] The barcode encoding the effector may comprise or be a nucleic acid molecule of any suitable length. For example, 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. In some examples, 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. In some examples, 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. In some examples, 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.

[0143] In some embodiments, 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. In some embodiments, 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. These encoding subunits can be ligated together using many known methods, including enzymatic ligation, template-free synthesis, templated polymerase extension, chemical ligation, recombination, or solid phase nucleic acid synthesis techniques. [0144] In some examples, the encoding/barcode may be a molecular weight barcode. The molecular weight barcode may be a peptide. In some cases, 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. In some examples, 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.

[0145] 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. In some examples, the bead may be a TentaGel bead. Alternatively, 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. In some examples, 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. In some cases, the barcode may comprise a beadspecific barcode (BSB) that identifies the bead/scaffold and may be used to count the scaffold.

Miniaturized screening platforms

[0146] In some examples, 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 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. In some cases, each compartment can act as an independent reaction vessel. In some cases, 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. In some cases, the systems provided herein may comprise or be bench-time instruments which may effectively replace large HTS facilities.

[0147] The systems provided herein may comprise a plurality of parti tions/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. For example, 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)). In case droplets are used as compartments, droplet formation may be performed in a variety ways.

[0148] In some examples, in the methods of the present disclosure, the compartments may comprise or be droplets, wells, or combinations of both (e.g., droplets trapped in wells). Among the applications of such screening platforms are single cell analysis, cell culture, cell perturbation analysis, encoded effector library screening, cell perturbation analysis, phenotypic cell screening, drug screening and drug discovery, diagnostics, personalized medicine, synthetic biology, discovery of biologies or antibodies, stem cell research, cell coculture (e.g., culturing multiple cell types and screening them for a variety of metrics), cell signaling pathway analysis, protein degradation analysis, cellular cross-talk analysis, cell morphology analysis, cell viability assays, tissue engineering, directed evolution (e.g., enzyme evolution), a plethora of genomics and proteomics, secretomics, metabolomics, and other applications.

[0149] 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 the screening systems provided anywhere herein or other screening platforms beyond this disclosure which may be used for screening encoded effector libraries provided herein.

[0150] 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. In some cases, 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. In other cases, 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.

[0151] In some examples, encoded effectors may comprise or be molecules whose structures can be measured or identified by measuring a property of the corresponding encoding. For example, in an integrated workflow comprising a screening system, an assay, and an effector library, 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).

[0152] The assay may generate a signal which can be detected by a screening system (e.g., a detector). In case the compartment comprises an effector (e.g., a member of an encoded effector library), 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). For example, the signal may be indicative of an effect of the effector on the assay, the target, or the sample.

[0153] In some cases, based on the signal, 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. The terms compartment and encapsulation may be used interchangeably. In some instances, 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 effector library. The encoded effector library may comprise a plurality of unique encoded effectors described in further detail elsewhere herein. In some cases, a plurality of unique and/or different encoded effectors (e.g., a large, encoded effector library) 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. In some cases, the miniaturized systems may comprise automated benchtop instruments.

[0154] In some cases, 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. In some cases, the effects of a multi-member library (e.g., a one-millionmember library of encoded effectors) may be screened against a sample/target. 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. For example, a rule may be defined on a software of the system which is in communication with the miniaturized screening chip through a computer. In some cases, the rule may comprise defining a threshold for the signal. Based on the rule, 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

[0155] 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. In some embodiments, 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 some embodiments, the methods and systems provided herein measure genomic, metabolomic, and/or proteomic data from cells screened against the encoded effectors. In some embodiments, 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.

[0156] 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. In some examples, the scaffold may be a bead, a fiber, nanofibrous scaffold, a molecular cage, a dendrimer, or a multi-valent molecular assembly.

[0157] In some examples, 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. In some examples, the bead may be a polymer bead. In some examples, the bead may comprise a polystyrene core. In some examples, the beads may be derivatized with polyethylene glycol. In some examples, the beads may be grafted with polyethylene glycol (e.g., a polystyrene core grafted with polyethylene glycol). In some examples, the polyethylene glycol may contain reactive groups for the attachment of other functionalities, such as effectors or barcodes. In some examples, the reactive groups may comprise or be an amino or carboxylate group. In some cases, the reactive group may be at the terminal end of the polyethylene glycol chain. In some examples, the bead is a TentaGel® bead.

[0158] The polyethylene glycol (PEG) attached to the beads may be any size. In some embodiments, the PEG is up to 20 kDa. In some embodiments, 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.

[0159] In some embodiments, 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.

[0160] In some embodiments, the bead comprises a PEG attached to a bead through an alkyl linkage and the bead comprises two bifunctional species. In some embodiments, the beads comprise surface modification on the outer surface of the beads that are orthogonally protected to reactive sites in the internal section of the beads. In some embodiments the beads comprise both cleavable and non-cleavable ligands. In some embodiments, the bead is a TentaGel® B resin. [0161] 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.

[0162] In some examples, the bead diameter may be at least about 1 nm, 10 nm, 100 nm, 200 nm, 500 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 12 pm, 14 pm, 16 pm, 20 pm, 25 pm, 30 pm, 50 pm, 80 pm, 100 pm, 90 pm, 100 pm, 120 pm, 140 pm, 160 pm, 180 pm, 200 pm, 300 pm, or larger. The bead size may differ based on the application. In some examples, the bead size may be at most about 300 pm, 200 pm, 160 pm, 100 pm, 80 pm, 50 pm, 40 pm, 30 pm, 20 pm, 10 pm, 8 pm, 6 pm, 4 pm, 3 pm 2 pm, 1 pm, 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 (pm) in water.

[0163] In some examples, 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. In some cases, the bead mean diameter may be at least about 1 nm, 10 nm, 100 nm, 200 nm, 500 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 12 pm, 14 pm, 16 pm, 20 pm, 25 pm, 30 pm, 50 pm, 80 pm, 100 pm, 90 pm, 100 pm, 120 pm, 140 pm, 160 pm, 180 pm, 200 pm, 300 pm, or larger. The bead size may differ based on the application. In some examples, the bead size may be at most about 300 pm, 200 pm, 160 pm, 100 pm, 80 pm, 50 pm, 40 pm, 30 pm, 20 pm, 10 pm, 8 pm, 6 pm, 4 pm, 3 pm 2 pm, 1 pm, 500 nm, 200 nm, 100 nm, 10 nm, 1 nm, or less.

[0164] In some examples, the bead mean diameter may be at least about 1 nm, 10 nm, 100 nm, 200 nm, 500 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 12 pm, 14 pm, 16 pm, 20 pm, 25 pm, 30 pm, 50 pm, 80 pm, 100 pm, 90 pm, 100 pm, 120 pm, 140 pm, 160 pm, 180 pm, 200 pm, 300 pm, or larger. The bead size may differ based on the application. In some examples, the bead size may be at most about 300 pm, 200 pm, 160 pm, 100 pm, 80 pm, 50 pm, 40 pm, 30 pm, 20 pm, 10 pm, 8 pm, 6 pm, 4 pm, 3 pm 2 pm, 1 pm, 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 (pm) in water.

[0165] Provided herein is a population of beads. 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. In some cases, the beads may be used for synthesizing encoded effector libraries. In some examples, 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. This quality may be referred to as monodispersity. 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 %). In some cases, 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.

[0166] In some examples, provided herein is 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.

[0167] 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.

[0168] 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). The effector may be a chemical compound synthesized on the bead and/or inside the bead. For example, in some cases, 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. In some examples, the effector molecules may be synthesized in the internal volume but not on the surface. In some examples, 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. In some examples, 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 reactive sites. In other examples, the bead may be polymerized with selective functional groups between the outer and inner layers of the bead matrix. [0169] In some examples, 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. For example, 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). 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. In some examples, the hydrogel may comprise or be PEG.

[0170] In some cases, 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. Tolerance may include being compatible with a condition. For example, 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. For example, 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. [0171] The bead may comprise swelling properties suitable for chemical synthesis according to the methods of the present disclosure. For example, 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. Swelling may be quantified as the percentage of change in the diameter of the bead once the bead is suspended in a solvent, as compared to its diameter when dry. For example, 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. For example, 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% larger than its dry diameter. In some cases, 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.

[0172] In some examples, 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 deswelled or shrunk in a solvent such as water, DMA, DMF, ACN, DCE, Methanol, Dioxane, Diethylether, Ethanol, Isopropanol, DCM, NMP, DMSO, Ethyl Acetate, and Acetone. For example, in some cases, 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. In some cases, 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.

[0173] The soft material of the bead/bead resin (e.g., the polymer/hydrogel material) may comprise functional groups for effector synthesis and the bead may be compatible with such chemical synthesis. In some cases, the polymer material may be made of monomers comprising functional groups for effector/barcode synthesis. Alternatively or in addition, the polymer resin may be made of monomers which do not comprise functional groups. For example, 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.

[0174] The bead may be compatible with peptide synthesis (e.g., solid phase peptide synthesis (SPPS)). The bead may remain substantially morphologically intact and/or unresolved during solid phase peptide synthesis. For example, 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.

[0175] In some examples, 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. For example, the thickness of the peripheral 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. In some cases, 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.

[0176] In some cases, the inner core may comprise or be a hard core (e.g., a core harder than the peripheral section of the bead). In some cases, 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. In some cases, the inner core may be a solid core. TentaGel beads comprise a polystyrene core. In some cases, 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). In some examples, 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.

[0177] In some examples, the inner core of the bead may comprise functional groups. The bead may comprise functional groups for synthesizing effectors. In some cases, 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. In some examples, the functional groups for synthesizing effectors may comprise Amine groups, hydroxyl groups, Alkyne groups. The inner core may be functional. In some cases, the inner core may comprise effectors covalently bound thereto or therein. The covalent bond may comprise a cleavable linker described anywhere herein.

[0178] The bead may further comprise functional groups for oligonucleotide synthesis or conjugation. In some examples, 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. In some cases, 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). In some cases, 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. [0179] In some examples, the bead comprises an inner portion, an external surface, and a peripheral section. In some cases, 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. For example, 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. In some cases, 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.

[0180] 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. In this example, the functional groups are shown to be mostly on the surface of the bead. In FIG. 3A, 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. In this example, 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)

[0181] 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 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. In some cases, the functional groups are comprised in the monomer building blocks. Alternatively or in addition, 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. For example, 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.

[0182] 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. Alternatively or in addition, 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. In some cases, the barcode may be a nucleic acid molecule. In other examples, 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. For example, 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. In some examples, 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).

[0183] In some examples, 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. Microfluidic methods and systems for bead generation

[0184] The present disclosure provides methods and systems for generating beads (e.g., polymer/hydrogel beads) using a microfluidic droplet generator. 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) may react and polymerize the contents of the monomer droplets to form solid or semi-solid polymer droplets or beads.

[0185] 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. In some examples, 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. In some cases, the energy may be light (e.g., UV light). For example, 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. In some examples, the polymerization reaction may be initiated by a chemical or a change in PH. In some examples, the polymerization reaction may comprise a redox reaction. Upon application of the stimulus of any kind, 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. Upon polymerization the emulsions/droplets may be broken such as by washing the beads with a proper solvent. [0186] In some examples, 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. In some examples, the UV exposure region may be similar to or the same as the cleavage region 206 shown in FIG. 4. In some cases, 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 (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. Upon post-processing and/or washing, 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. In some cases, such material may be PEG [0187] In some examples, the UV exposure may not be performed in an integrated microfluidic circuit (e.g., such as a microfluidic device shown in FIG. 4). Alternatively, in some cases, 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. In some cases, the stimulus may be light (e.g., UV light). UV light may be exposed to the droplets in bulk. For example, in case the monomer droplets are formed in a microfluidic droplet generator, 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). For example, the container (e.g., as a whole or in part may) be exposed to the stimulus (e.g., light). The stimulus may initiate the polymerization reaction. [0188] In some examples, the stimulus for initiating polymerization may comprise a chemical. In some examples, such chemical may act as a catalyst. In some examples, the catalyst may comprise cupper (Cu). In some examples, 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. Alternatively or in addition, the droplets may be collected from the microfluidic device and exposed to the stimulus after collection. In some 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). For example, 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). In some examples, the outlet tubing may pass through a light region and be exposed to light.

[0189] In some examples, 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. For example, 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. In some examples, 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. In some examples, 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.

[0190] 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. Alternatively or in addition, the bead may be compatible with conditions used for screening. Chemical synthesis may comprise using solvents (e.g., organic and/or mineral solvents). For example, 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. For example, the bead may remain substantially morphologically intact, solid, uncompromised, unresolved, integrated, and/or swelled during SPPS and/or in organic solvents. In some examples, 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., 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. [0191] 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. In some cases, the beads may be compatible with enzymatic transformation reactions. Examples of 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). [0192] The bead may comprise functional groups or functional sites capable of synthesis of chemical compounds (e.g., effector compounds and/or barcodes). In some cases, functional groups may comprise or be amine groups. In some examples, 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. In some cases, 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.

Bulk emulsification and particle-templated emulsification

[0193] In some examples, 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.

[0194] In some examples, 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.

[0195] Provided herein is a method of bulk emulsification or vortex -based emulsification. In some examples, a plurality of beads may be provided in an aqueous solution in a container or compartment. In some cases, the container or compartment may be a tube of any size or 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). In some cases, the compartment or plurality of compartments may be a plurality of wells. In some cases, 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. In a compartment (e.g., in a compartment of the plurality of compartments), 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. In some cases, 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.

[0196] In some examples, 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. In some cases, 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.

[0197] In some cases, the beads may act as a template for the droplets. For example, 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). Such method may be referred to as particle-templated emulsification (PTE). The droplets containing beads may be used as compartments for performing screens using the methods and systems provided anywhere herein.

[0198] In some examples, the compartments used for screening may comprise droplets. In some examples, 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.

[0199] An example 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. In this example, the second 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. In other examples of a microfluidic device, there may only be a single inlet stream. 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. 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. In the example of FIG. 4, 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. In some embodiments, 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)). In some embodiments, 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. In some embodiments, the height of the chamber is about 80 pm and the height of the connecting channel is about 50 pm. As an additional design feature to aid in prevention of trapping of bubbles within the device, 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. If fluid is diverted out of the main microfluidic channel at bypass shunt 208a, the material will not pass through 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. 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. Alternatively, the objective for the excitation light may be configured to also collect the emission. In some embodiments, 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. In some embodiments, the objective or fiber aligned at detection position 216 is not coupled to the device. When 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. When not coupled to the device, 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 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. In some embodiments, the Optical Glue helps to minimize scattering of the light from the fiberoptic wave guide.

[0200] In some embodiments, for any microfluidic device described herein (e.g., FIG. 4), beads may be provided with an aqueous fluid via an inlet (e.g., inlet 201, inlet 203, or inlet 218). In some embodiments, the beads are suspended in the aqueous fluid, and provided from a bead source. In some embodiments, a tubing or other channel (“bead tubing”) provides fluidic communication between the bead source and a microfluidic device described herein. In some embodiments, said bead tubing comprises a rigid material. In some embodiments, 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. For example, without such vibration, in instances, the beads may settle along the walls of the bead tubing. In some instances, 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. In some instances, such settling of beads along the walls of a bead tubing and/or formation of “clumps” of beads results in a reduced amount or lack of beads that enter a microfluidic chip. In some embodiments, 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).

[0201] In some embodiments, the vibration motor is configured to deliver high frequency vibration and/or low power (i.e., low amplitude of vibration of frequency). In some embodiments, 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. In some embodiments, 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. [0202] In some embodiments, beads settling out in the bead tubing (for example along the walls of the bead tubing) results in an accumulation of beads within the bead tubing and may prevent beads from being disposed on the microfluidic chip. In some embodiments, 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. In some embodiments, a feedback controller is provided and configured to modify the operation mode of a vibration motor based on no beads being detected. For example, if no beads are detected by the microfluidic device (for example at the detection and/or sorting regions), 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. In some embodiments, upon detecting beads (for e.g., by the detection region or sorting region), the feedback controller may turn off the vibration motor or revert the vibration frequency to a predetermined value.

[0203] The droplet formation junction 204 comprises a microfluidic channel that continues down the flow path towards cleavage region 206. Near cleavage region 206 is a UV waveguide 205 configured to deliver light into the microfluidic channel of the cleavage region 206. In some embodiments, 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. In some embodiments, the UV waveguide comprises a parabolic lens at an end closest to the cleavage region. In some embodiments, the parabolic lens is configured to columnate light inside the cleavage region. In some embodiments, the parabolic lens, or a curved lens, minimizes the tendency for the light from the UV waveguide to be scattered. In some embodiments, 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. In some embodiments, an Optical Glue 217 is provided with the UV waveguide. In some embodiments, the Optical Glue 217 helps to minimize light being scattered by UV waveguide. Also, 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.

[0204] 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. In some embodiments, 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. In some embodiments, 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)). In some embodiments, the height of the chamber is about 30 pm to about 1,000 pm. In some embodiments, 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. If fluid is diverted out of the main microfluidic channel at bypass shunt 208a, the material will not pass through the 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. 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. In some embodiments, 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. Alternatively, the objective for the excitation light may be configured to also collect the emission. In some embodiments, 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. In some embodiments, the objective or fiber aligned at detection position 216 is not coupled to the device. When 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. When not coupled to the device, 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 is determined to lack a desired signal. The discrimination junction electrode 212 is connected to a discrimination junction ground circuit, which is connected to the device at circuit connection points 213a and 213b.

Droplet characteristics

[0205] In some examples, 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.

[0206] In some examples, the droplets are placed in an oil emulsion. In some examples, 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. In some examples, the oil comprises a silicone oil. In some examples, the oil comprises a fluorosilicone oil. In some examples, the oil comprises a hydrocarbon oil. In some examples, the oil comprises a mineral oil. In some examples, the oil comprises a paraffin oil. In some examples, the oil comprises a halogenated oil. In some examples, the oil comprises a fluorocarbon oil.

[0207] In some examples a population of substantially monodispersed droplets may be formed. In some embodiments, 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.

[0208] 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. In some examples, 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. In some examples, 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.

[0209] In some examples, a single encoded effector is present in a compartment of encapsulation of the present disclosure (e.g., a droplet or a well). In some examples, a single scaffold comprising an encoded effector and its encoding are present in an encapsulation. In some examples, a plurality of scaffolds, each scaffold comprising a different encoded effector and its respective encoding, are present in a compartment.

[0210] In some examples, encapsulations comprise biological samples. In some embodiments, encapsulations comprise single cells. In some embodiments, encapsulations comprise one or more cells. In some embodiments, the encapsulations comprise nucleic acids. In some embodiments, the encapsulations comprise proteins.

[0211] In some examples, 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.

[0212] In some examples, 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. I some examples, this percentage may be at most about 50%, 40%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, or less of the total number of droplets formed per unit time.

Droplet screening workflows

[0213] FIG. 5 shows an exemplary workflow of an effector screen performed in a microfluidic device. In the exemplary workflow shown, 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 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. As the encapsulations travel through the incubation region, 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). In some embodiments, the 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. Following completion of the screen, 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)). The sequenced encodings can then be decoded to reveal the effectors which had the desired activity. In some embodiments, 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.

[0214] Provided herein are methods and systems for screening encoded effectors on samples using encapsulations, wherein the sample and an encoded effector are encapsulated or co-localized in a compartment (e.g., a droplet or a well). In some cases, 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. [0215] In some examples, 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. In some cases, the microfluidic device may comprise one or more droplet generation junction, such as 1, 2, 3, 4, 5, 6, or more droplet generation junctions integrated in the droplet microfluidic platform.

[0216] 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. In a particular example, 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. As long as the materials of the first stream and second stream are held in separate reservoirs, the assay does not start. 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. Alternatively, 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.

[0217] 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 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). In some cases, 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. For example, the percentage of droplets containing 1, 2, or more beads may be calculated according to the Poisson distribution using the size of the droplet.

[0218] In some examples, the barcode encoding the effector may comprise one or more optical barcoding particles on the surface of the scaffold/bead or inside it. In some cases, the optical barcoding particles may comprise spectral properties in the short-wave infrared (IR) range. In some examples, the optical barcoding particle may be detectable by fluorescence or luminescence. In some examples, the optical barcodes may be detected by line scan cameras or line scan spectrometers. In some examples, the optical barcoding particles may comprise an excitation wavelength of from about 1000 to about 1100 nanometers (nm). In some examples, 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). [0219] 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. In some examples, 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. In some examples, 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, 30 nm, 50 nm or greater/broader. In an example, the optical barcoding particles may comprise an emission bandwidth of at most about 0.5 nm.

[0220] 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. For example, 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).

[0221] 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. In some examples, 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. [0222] 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 ⁶ ,

9 x 10 ⁶ , 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 , 10 ⁷ , 2 x

10 ¹⁰ , 3 x 10 ¹⁰ , 4 x 10 ¹⁰ , 5 x 10 ¹⁰ , 6 x 10 ¹⁰ , 7 x 10 ¹⁰ , 8 x 10 ¹⁰ , 9 x 10 ¹⁰ , or more unique optical signatures.

[0223] In some examples, the one or more optical barcoding particles, together or in combination, 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. [0224] The optical barcoding particles may be made of any suitable material. In some examples, 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.

[0225] 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. In some examples, the optical barcoding particle may comprise or be a cylindrical optical barcoding particle. In some examples, the height of the cylindrical optical barcoding particle is at most about 20 pm, 15 pm, 10 pm, 9 pm, 8 pm, 7 pm, 6 pm, 5 pm, 4 pm, 3 pm, 2 pm, 1 pm, 0.9 pm, 0.8 pm, 0.7 pm, 0.6 pm, 0.5 pm, 0.4 pm, 0.3 pm, 0.2 pm, 0.1 pm, or less. In some examples, diameter of the cylindrical optical barcoding particle is at most about 20 pm, 15 pm, 10 pm, 9 pm, 8 pm, 7 pm, 6 pm, 5 pm, 4 pm, 3 pm, 2 pm, 1 pm, or less. In some examples, the diameter of the cylindrical optical barcoding particle may be at least about 0.1 pm, 0.2 pm, 0.3 pm, 0.4 pm, 0.5 pm, 0.6 pm, 0.7 pm, 0.8 pm, 0.9 pm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, or larger. In some examples, the volume of the optical barcode is at most about 0.05% of the volume of the bead.

[0226] In some examples, the barcode may comprise a nucleic acid molecule covalently bound to the bead or trapped inside the bead. In some examples, the barcode may comprise a Deoxyribonucleic acid (DNA), a Ribonucleic acid (RNA), a peptide, or a peptide nucleic acid (PNA). In some examples, a bead comprising a nucleic acid barcode may further comprise an optical barcode thereon or therein.

[0227] The bead of the present disclosure may remain substantially structurally intact during solid phase peptide synthesis (SPPS). For example, 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, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 24 hours, 72 hours or longer (e.g., under conditions required for library synthesis). In some examples, the bead remains substantially structurally intact during and after suspension in an organic solvent. [0228] In some examples, 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 (pm) 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. In some examples, 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).

[0229] 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.

[0230] Provided herein are methods and systems for capturing Structure-Activity Relationships (SAR). For example, 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. As such, 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. In some examples, this method may replace or eliminate sorting and/or post-processing steps such as sequencing. As such, in some cases, the DOTF method may not comprise a physical sorting step. In some cases, a physical sorting step may also be implemented. As such, in some cases, DOTF and sequencing may both be performed. For example, a scaffold (e.g., bead) may comprise one or more barcoding modalities. For example, 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. For example, a bead may comprise an optical barcode, the optical barcode may be decoded on the fly by detecting the second signal described above. In addition, the bead may comprise a nucleic acid molecule which can be sequenced. The barcoding modality may be chosen or optimized based on the application. [0231] Provided herein is 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, 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.

[0232] In some examples, 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. In some examples, 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.

[0233] Provided herein is a system comprising a solid support comprising an effector bound to the solid support via a cleavable linker. 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 (pm). The solid support comprises or be a bead. The bead may be any bead mentioned/described anywhere herein.

[0234] 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. In some examples, the compartment may be a droplet surrounded by an immiscible oil.

[0235] In some examples, 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. In some examples, the compartment may be a well in a miniaturized array platform. In some examples, 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.

[0236] In some examples, provided herein is 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 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.

[0237] 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.

[0238] The encoding particles may comprise a spectral emission bandwidth of at most about 3 nm. In some examples, the first signal and the second signal may be spectrally independent. In some examples, the first signal may be in the visible range. In some examples, the second signal may not be in the visible range. In some examples, the second signal may be in the short-wave infrared (IR) range. The bead, barcode, encoding particles, the effector, and the rest of the elements used may be according to any embodiments described anywhere herein.

[0239] In some examples, provided herein is 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. The bead, barcode, encoding particles, the effector, and the rest of the elements used may be according to any embodiments described anywhere herein.

[0240] 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.

[0241] 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.

[0242] 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).

[0243] FIG. 9 illustrates an exemplary workflow of a split-and-pool method for generating spectrally/optically encoded effector libraires.

[0244] FIG. 10 illustrates an exemplary workflow of using a Bead Index Registry and Dispensing System (BIRDS) for generating beads pre-encoded with optical barcodes.

Optically pre-encoded beads can be later used for effector synthesis and creation of One Bead One Compound Spectrally/Optically Encoded Libraries (OBOC-SEL). The term compound can be generalized to any kind of effector.

[0245] 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. In this example, the optical barcoding particles are randomly distributed in the beads. In some cases, 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. For example, 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).

[0246] 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. In some cases, the surfaces may be modified in terms of any intended physical or chemical property. In an example, 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 Tween, Span, PEGylated di or triblock copolymer surfactants. In some cases, the surfaces are made hydrophobic or hydrophilic. Generally, 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. In an example, the surface of the optical barcoding particles may be coated with Si. Si may make the surface of the optical barcoding particle hydrophobic. In some cases, 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. In some cases, 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.

[0247] 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. In addition, 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. In an example, the inner core of the bead may comprise Dextran, and the peripheral section of the bead may comprise PEG. The bead may be similar to any bead embodiment described anywhere herein. [0248] In some examples, the methods may comprise performing multiplexed doseresponse studies simultaneously. In some cases, optical barcoding and DOTF may be used to perform dose-response studies. For example, an encoded effector library according to any embodiment described herein may be prepared. The encoded effector library may comprise any unique number of effectors. Alternatively or in addition, the effector loading (e.g., the amount or concentration of an effector loaded on an individual bead) may be variable among the population of the beads (among the library). Alternatively or in addition, 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 same stimulus. For example, in a library, 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. For example, 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) may encode effector structure, effector load/concentration and/or both, and may be decoded after the screen or during the screen (DOTF). 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

[0249] The screens of the present disclosure may be performed in any suitable screening platform, in some cases, in miniaturized compartmentalized screening platforms. Any beadbound encoded effector embodiment may be combined with any screening system disclosed. In some cases, screens may be performed in arrays.

[0250] 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). In some cases, 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.

[0251] A variety of array platforms with any shape, size, geometry, and materials can be used for performing the methods of the present disclosure. In an example, 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. In an example, a Globe Scientific 1324 Glass Microscope Slide was 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.

[0252] 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. In a particular example, the diameter of the fabricated wells may be from about 200 to about 300 micrometers/microns (pm). The height or depth of the well walls (e.g., the thickness of the material in which the wells are imprinted) may be from about 50 to about 200 microns. In some examples, the spacing between the wells may be from about 50 to about 100 microns. In some examples, the total internal volume of the wells may be from about 20 picoliters to about 100 nanoliters. [0253] In a particular example, the well diameter may be about 200 pm and the well height (thickness of the well wall) may be about 50 pm. Accordingly, the internal volume of the well may be about 1.57 nanoliters (nL). In another example, the well diameter may be about 200 pm and the well height may be about 100 pm. Accordingly, the internal volume of the well may be about 3.14 nL. In another example, the well diameter may be about 200 pm and the well height may be about 200 pm. Accordingly, the internal volume of the well may be about 6.28 nL. In other examples, the diameter of the well may be about 300 pm, and the height of the well may be about 50 pm, 100 pm, or 200 pm. Accordingly, the internal volume of the well may be about 3.53 nL, 7.065 nL, or 14.13 nL, respectively. [0254] In some cases, the diameter of the well may be at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400 pm, or above. In some cases, the diameter of the well may be at most about 1 cm, 500 pm, 400 pm, 300 pm, 200 pm, 150 pm, 100 pm, 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 pm or greater. In some cases, the height of the well wall may be at most about 500 pm, 400 pm, 300 pm, 200 pm, 100 pm, 50 pm, 30 pm, 20 pm or smaller. The volume of the well may be at least about 0.1, 0.2, 0.3 0.4, 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. In some cases, 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.

[0255] In some examples, 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. In some cases, the plurality of compartments may comprise at least 2, 10, 20, 30, 40, 50, 60, 100, 200, 300, 400, 500, 1000, 5000, 10,000, 20,000, 40,000, 60,000, 100,000, 1000,000, 10,000,000, 100,000,000 or more compartments.

[0256] 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. In this example, 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. In some cases, the bottom substrate may be glass or plastic. The walls may be made of any material. In some cases, the wall material may be a hydrogel or polymer.

[0257] The compartments or wells of a miniaturized well -based platform can be made of any suitable material. In some examples, 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. In case of using a porous material for fabricating the wells, the mesh size can be small enough to prevent material diffusion therein, or such material transfer may be otherwise substantially blocked. In some cases, the wells (e.g., microwells) seek to isolate individual populations of cells and any compounds the effects of which on the cells are to be examined. As such, 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.

[0258] In some examples, array platform/device material may be resistant to extracellular protein adsorption and/or cell growth. In some examples, the methods and systems of the present disclosure may be used to screen ion channels in cells in the array-based system. In some cases, 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. If the array platform material is resistant to ECM protein adsorption and cell growth, this may minimize the chance for cells growing between two wells.

[0259] In some examples, preferred well wall materials may be malleable into a predetermined shape or feature at the correct resolution (e.g., a high resolution). In some cases, 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. In some cases, 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. In some cases, the material of the compartments may have low autofluorescence.

[0260] The materials of the well walls and the bottom substrate can be the same or different. In a particular example, 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. 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. In another example, 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. In some cases, 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. In some examples, any Zwitterionic polymers such as (poly(phosphorylcholine), poly(carboxybetaine), poly(sulfobetaine), poly(trimethylamine A-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. In such examples both the bottom and walls of the wells could be made of the same material (e.g., glass or plastic).

[0261] The bottom surface and/or walls may be made of any material. In some cases, glass, plastic, silicon, silica, polymer, polydimethylsiloxane (PDMS). In some examples, the bottom of the compartments/wells (e.g., the solid surface in the bottom of the platform) may be treated with a cell adhesive material to render the surface adhesive to cells and/or ECM. This will improve the surface properties for cell seeding. Any suitable cell adhesive material or chemical can be used. In some examples, 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). [0262] In some cases, 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. In some examples, the material of the walls may comprise any level of wettability or hydrophilicity. In some cases, the material may be hydrophobic. In some examples, the well walls may be made of a material (e.g., hydrogel) with a molecular weight of from about 10 to about 2000 Daltons. [0263] The well walls comprise a top surface. In some cases, the top surface of the well walls may be or may be rendered substantially non-adherent to cells. In some examples, the well walls may be made of a material that by itself is substantially non-adherent to cells. In some examples, the material of the wells may comprise or be glass through-holes or plastics (e.g., Cyclic Olefin Copolymer COC). In case of using COC, it can be made such that only the bottom surface of the well interior is tissue-culture treated (plasma treated) while the surfaces of the well walls (e.g., a material such as COC) have inherent low-specific protein and cell binding properties. In some examples, 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. Some examples of the coating materials coating the top surface(s) of the well walls may comprise PEG-DA, PDMS-Pluronic F127, PDMS- PDMS-PEG-BCP, PDMS-501W, fluoropolymers such as Novec 1720/1702/1700/2202 and Cytop, and zwitterionic polymers (including poly(phosphorylcholine), poly(carboxybetaine), poly(sulfobetaine), poly(trimethylamine A-oxide). In other examples, materials such as organogels and/or fluorogels may be used coat the well surfaces.

[0264] 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. In this example, 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 (PEGDA). Prior to fabricating the wells on the glass slide, 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. After fabricating the array platform, 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. Initially, 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. 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). Next, the water was removed from the wells (e.g., aspirated), and a charged surface treatment (e.g., poly-D Lysine) was applied on the entire micro-array. 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. Next, 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.

[0265] 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) facilitated improving surface properties for cell seeding and growth. 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.

[0266] 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.

[0267] 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. Based on the measured signal, 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.

[0268] After the assay is completed, post-processing may be performed on the hits. In some cases, post-processing may comprise selective polymerization as described elsewhere herein. For example, 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).

[0269] 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. [0270] 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. For example, effectors that are found to be active against an intended target (e.g., based on a threshold of a signal detected from the assay in presence of such effector in the compartment after release) 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. For example, 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).

[0271] 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. In some cases, 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-vali dated hit is a false positive. In some cases, hits may comprise or be high-interest events of unknown veracity. In some cases, hits may not be treated as bona-fide until validated in replicate tests afterwards.

[0272] Upon performing an assay in the screening platform and/or once the signals are measured form the compartments, 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. In some cases, cleaving the barcode from the bead may be performed or catalyzed by an enzyme.

[0273] In some examples, provided herein are methods for bead sorting, recovery, and barcode decoding to elucidate the identity of hits. An example workflow may comprise 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.

[0274] In some cases, 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. For example, the scaffolds may be introduced into the compartments before or after the cells. In some cases, the cells may be suspension cells floating in the compartments and not necessarily pre-seeded in the compartments or adhered thereto.

[0275] 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.

[0276] 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). In some cases, a positive compartment may comprise more than one scaffold. For a compartment to be called as a positive compartment, 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.

Nucleic acid barcode decoding

[0277] In some examples, in case 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. In some examples, 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. In many examples, 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).

[0278] In some examples, 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). 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).

[0279] 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. Any of the cleavable linkers and cleaving reagents described herein may work for this purpose. In some examples, a nicking enzyme or a restriction enzyme can be used to cleave. In some examples, enzymatic, chemical reagent, photocleavage, or other methods can be used to cleave the encodings.

[0280] In some examples, the nucleic acid encoding may comprise a sequencing primer. The sequencing primer may allow for facile amplification of the nucleic acid encoding. In some examples, 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. [0281] 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. In some embodiments, the libraries comprise at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , IO ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , or 10 ¹⁶ unique effectors. In some embodiments, the libraries comprise at least about 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , IO ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , or 10 ¹⁶ unique effectors.

[0282] In some embodiments, 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. In some embodiments, 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. [0283] Any of the methods or systems described herein for a single encoded effector may be utilized by a library of encoded effectors. In some embodiments, provided herein, is a method of screening a library of encoded effectors, the method comprising using any of the methods previously described herein with a library of encoded effectors. In some embodiments, libraries of encoded effectors comprise a plurality of different encoded effectors. In some embodiments, libraries comprise multiple copies of substantially identical effectors or scaffold encoded effectors.

[0284] 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.

Methods and systems for in-silico screening and sequential screening

[0285] Provided herein are screening methods. Screening methods of the present disclosure may comprise vast applications in drug discovery. Screening in drug discovery may comprise exploration of a chemical space. A chemical space may comprise an ensemble of a plurality of molecules or structures (e.g., effectors). A chemical space may be explored to find hits. A chemical space may be explored to identify hits. Hits may comprise an effect on a target. The effect may be any effect mentioned anywhere herein (e.g., inhibition, antagonism, or other effects). The effect may comprise an effect on an activity of a target. For example, a target may be an enzyme, and an effector may be an enzyme inhibitor.

[0286] A screen may be performed to gather information indicative of the effect of an effector on a target or an interaction between them. The effect may be measured or predicted. The screen may be an empirical screen, a virtual screen, a computational screen, an in-silico screen using any suitable approach (e.g., described anywhere herein), and/or any combination thereof. In some cases, a plurality of screens may be performed to explore a chemical space. The plurality of screens may inform each other. The plurality of screens may refine the findings in a series of steps (e.g., sequential steps).

[0287] In an aspect, provided herein is a screening method comprising: (a) providing or obtaining a computational representation (e.g., computational model) of a target and a computational representation (e.g., computational model) of an effector. A computational representation may in some cases comprise a digital file comprising information which can be used by a computer program to do a set of calculations. In some examples, a computational representation of an effector may comprise information about the effector. A computational representation of a target may comprise information about the target. Information may comprise any reasonable information about the properties, characteristics, features, compositions, structures, chemical properties, physical properties, molecular properties, biological properties, and beyond.

[0288] In some examples, a computational representation or otherwise information provided to a computer program (e.g., as input) may comprise one or more equations, one or more letters, one or more symbols, one or more numbers, one or more strings, a text, or information in any suitable format which may at least partially describe a target or an effector (e.g., theoretically or mathematically).

[0289] In some examples, simplified molecular-input line-entry system (SMILES) strings may be provided to a computer program, is a specification in the form of a line notation for describing the structure of chemical species using short ASCII strings.

[0290] Computational representations, models, or the strings used for modeling a molecule may comprise information about atoms, bonds, rings, aromaticity, branching, stereochemistry, isotopes, structures (e.g., 3D structure), orientation of a molecule in space, and more properties of any molecule (e.g., an effector or a target).

[0291] The methods presented herein may comprise providing or obtaining a computer program and running it to calculate one or more parameters indicative of a predicted interaction between the computational representation of the target and the computational representation of the effector. The method may be applied on any number of effectors and any number of targets in any intended sequence or order, in parallel, or in a multiplexed manner. For example, interactions of any number of effectors may be tested on one target. Alternatively, interactions of any number of effectors may be tested on any number of targets. In some examples, interactions of one effector may be tested on any number of targets.

[0292] Interaction may comprise any reasonable kind of interaction any two molecules (e.g., a target and an effector or any number of targets and/or any number of effectors) may have with each other under a set of experimental conditions. Experimental conditions may affect the interactions. Experimental conditions may comprise solvents, temperature, pressure, PH, ionic strength, and other parameters.

[0293] An interaction may comprise any molecular, electronic, atomic valence, or orbital. In some cases, an interaction may be more likely to happen if it plays a role in stabilizing or equilibrating a system, such as by minimizing thermodynamic enthalpy and/or increasing the entropy of the system. In some cases, an interaction may be more likely to happen if a Gibbs free energy of the system decreases as a result of the interaction. In this context, the system may comprise the molecules interacting and their environment/surroundings considering the experimental conditions. For example, the system may comprise the target(s), the effector(s), the salts and solvent (e.g., water) and other milieu of the solution, assay reagents, and additives, considering the experimental conditions such as PH, temperature, pressure, and other parameters. If an effector is to bind, it must displace what is there. In some examples, an interaction may occur without energy input, if the Gibbs free energy of the system decreases as a result of the interaction

[0294] In some examples, an interaction between an effector and a target may comprise binding, where the loss of hydrogen doner-accepter enthalpy due to desolvation may be compensated by free-energy minimizing van der Waals forces and pi-orbital overlap between the effector and target residues, where the equilibrium promotes effector residing within molecular proximity to the target. In another example, an interaction between an effector and a target, may comprise forming a covalent bond between a latent reactive moiety on the effector and a reactive residue on the target. In some examples, an interaction may comprise a transient interaction between two or more molecules. In some examples, an interaction may comprise a non-transient interaction between two or more molecules.

[0295] An interaction may lead to an effect. In some cases, an interaction may comprise an effect. An interaction may in some cases be a prerequisite for an effect. An effect can be any effect mentioned anywhere herein. In some cases, an effect may be inhibition, agonism, catalysis, or another interaction. As an example, an interaction may comprise the binding of an effector (e.g., a compound, ligand, small molecule, biologic, drug candidate, or another effector) to a target (e.g., at a binding pocket of the target which in some examples may comprise or be a protein or enzyme). The binding may play a role in or may cause an activity of a target to be affected (e.g., the effector has an effect on the target). For example, the binding of a small molecule to an enzyme may inhibit the enzyme.

[0296] The method may further comprise synthesizing the effector on a scaffold. In some examples, the effector may be bound to the scaffold via a cleavable linker and releasable upon cleavage of the cleavable linker. In some examples, the scaffold further comprises a barcode corresponding to the effector and identifies it. Bead-bound encoded effectors or One Bead One Compound Libraries (OBOC libraries) may comprise various embodiments described in great detail throughout the present disclosure. Any encoded effector embodiment may be used in any of the screens. In some cases, synthesizing the effector may comprise combinatorial synthesis, solid phase peptide synthesis, split-and-pool synthesis or any other suitable synthesis method (e.g., any suitable synthesis method mentioned anywhere herein). In some examples, the barcode comprises a nucleic acid molecule or a molecule with detectable spectral or optical properties as described anywhere herein.

[0297] In some examples, the method further comprises performing an empirical screen to measure a signal indicative of an interaction between the target and the effector, thereby generating an empirical dataset. The empirical screen may comprise providing a target and an effector in a compartment of a compartmentalized system described in the present disclosure, allow them to interact, detect a signal, and in some cases, optionally proceed to postprocessing the bead-bound encoded effector such as by sorting it, reading the barcode, and/or identifying the effector using the barcode. Such methods are detailed throughout the present disclosure.

[0298] The interaction, the predicted interaction, or both may be influenced by a property of the effector. The property of the effector may comprise a structure of the effector, a characteristic of the structure of the effector, a subunit of the effector, a structure of the subunit of the effector, a characteristic of a structure of a subunit of the effector, or any combination thereof. In some examples, the effector comprises a plurality of subunits of the effector, the subunits of the effector may comprise a building block (BB) of the effector, and the BB of the effector may comprise a chemical motif. In some examples, a BB or a characteristic thereof may be driving an effect on the target. In some cases, a subunit of the effector may comprise more than one BB (e.g., two or more BBs and the bonds between them. Any subsection of the effector, individually, or in combination with the rest of it, independently, or under given experimental conditions, may at least partially contribute to an interaction and/or an effect which may in some cases be predicted, in some cases be empirically measured, and in some cases, both.

[0299] In some examples, the method may further comprise feeding (e.g., providing as an input or a part of an input) the empirical dataset into the computational program. In some examples, the method may further comprise training the computational program using the empirical dataset. In some examples, training may comprise using Artificial Intelligence (Al), Machine Learning (ML), Neural Networks, Predictive Models, or any combination thereof. Any suitable Al or ML technique may be used.

[0300] In some examples, the method may further comprise providing or obtaining a secondary dataset and feeding it into the computational program. The method may further comprise merging the empirical dataset with the secondary dataset, at any point during the workflow, for example, prior to feeding it into the computational program. In some examples, the method further comprises processing or cleaning the empirical dataset, the secondary dataset, or both, prior to feeding it into the computational program. Feeding may comprise providing as an input or a portion of an input using any suitable method.

[0301] In some examples, the secondary dataset may be obtained, extracted, striped, or read from a public database. In some examples, training comprises increasing the accuracy or efficiency of the computational program in calculating the one or more parameters. In some examples, a screening method may comprise providing a plurality of computational representations of a plurality of effectors, calculating the one or more parameters for at least a first subset of the plurality of the effectors, selecting a second subset of the plurality of the effectors based on the one or more parameters, and synthesizing the second subset of the plurality of the effectors on a plurality of scaffolds via cleavable linkers. In some cases, the second subset may comprise a promising effectors, high-performing effectors, or effectors with a higher likelihood of being active against a target compared to an average effector in a chemical space. In some cases, a high-performing effector may suspected to have an activity against a target based on observations in a preliminary screen which may in some cases be a predictive analysis using a computer program, in-silico screen, an algorithm. The methods may comprise using Artificial Intelligence (Al), machine learning (ML), and/or Neural Networks.

[0302] In some examples, running the computer program may inform the selection of the second subset of the plurality of the effectors (e.g., promising effectors or effectors suspected of having a high likelihood of being active against a target or having an intended effect). The selection may be conducted in a variety of ways, by a person, an algorithm, or both. In some examples, an algorithm (e.g., a selection algorithm or optimization algorithm) may be applied on the one or more parameters to facilitate or guide the selection of the second subset of the plurality of the effectors (e.g., after performing a preliminary screen or a first screen, which in some cases is a virtual, computational, or in-silico screen).

[0303] In some examples, the screen may model or measure an interaction between a target and an effector, or conduct/facilitate any combination of both, such as to provide information about an effect of an effector. In some examples, the interaction may be a binding of the effector to the target. Computational screening may calculate the one or more parameters which in some cases may comprise binding free energy. The promi sing/high- performing effectors may comprise relatively low binding free energies among the plurality of effectors. For example, a threshold may be set or defined for the parameter (e.g., binding free energy), and effectors comprising a binding free energy below the set threshold may be identified as high-performing or promising effectors, and may proceed to the next screen. For example, the high-performing effectors may be synthesized on beads according to any embodiment in the present disclosure and screened using any screening system provided in the present disclosure. The combination of steps of the methods performed may lead to mapping structure activity relationships (SARs), provide information about a plurality of effectors in a chemical space, lead to identification of hits and new drug candidates. The methods may be used for hit-to-lead drug discovery.

[0304] In some aspects, provided herein is a method of screening comprising: (a) providing or obtaining a computational representation of a target (or a plurality of targets) and a plurality of computational representations of a plurality of effectors; (b) providing or obtaining a computational model and calculating one or more parameters indicative of a predicted interaction of the computational representation of the target with each computational representation of the plurality of the computational representations of the plurality of effectors, using the computational model; (c) based on the one or more parameters calculated in (b), ranking the plurality of computational representations of the plurality of effectors; (d) based on the ranking in (c), identifying a plurality of high- performing effectors, wherein performance is based on the predicted interaction; and (e ) synthesizing the plurality of high-performing effectors on a plurality of scaffolds, wherein each scaffold comprises an effector of the plurality of high-performing effectors and a barcode corresponding to the effector and encoding it, wherein the effector is bound to the scaffold via a cleavable linker and is releasable upon cleavage of the cleavable linker. The effector-bound encoded effector may be according to any embodiment mentioned anywhere herein.

[0305] In some examples, the method further comprises performing an empirical screen of the plurality of scaffolds synthesized in (e), against the target or the plurality of targets. In some examples, the method may further comprise recording a dataset from the empirical screen. In some embodiments, the method further comprises feeding the dataset into the computational model of (b). In some examples, the method further comprises performing two or more iterative steps to refine the high-performing effectors, wherein each iterative step comprises running or modifying the computational model and performing the empirical screen. The refinement may be performed by a person (e.g., using medicinal chemistry approaches and know-how) or using a selection algorithm, Al, ML, or any combination thereof.

[0306] In some examples, the effector may comprise a plurality of subunits of the effector, the barcode comprises a plurality of subunits of the barcode, and the plurality of subunits of the barcode correspond to and identify the plurality of subunits of the effector. In some examples, the effector is a small molecule. In some examples, the subunit of the effector is a building block (BB) of a small molecule. In some examples, the methods comprise small molecule drug discovery using a combination of in-silico and empirical screening, and screening may comprise using miniaturized compartmentalized high- throughput screening systems such as droplet microfluidics and miniaturized arrays (any such method or system described anywhere herein).

[0307] In some examples, the barcode is a nucleic acid molecule. In some examples, the barcode comprises one or more optical barcoding particles. In some examples, the method comprises running the computational model, wherein running the computational model comprises performing a virtual screen (e.g., virtual drug screen). In some examples, virtual screening comprises molecular docking. In some examples, the interaction between a target and an effector comprises binding and the parameter comprises binding free energy.

[0308] In some examples, the effector comprises one or more subunits, and the method comprises indicating a performance metric or identification for at least a subset of the one or more subunits. In some examples, the method further comprises designing the plurality of computational representations of a plurality of effectors. In some examples, designing the plurality of computational representations of the plurality of effectors comprises selecting a subset of the one or more subunits based on the performance metric or identification. The method may comprise selecting BB for designing and/or synthesizing small molecule drugs. In some cases, an intended interaction or effect may be driven by a BB. Alternatively or in addition, an intended interaction or effect may be driven by a combination of a two or more BBs and/or the bonds and/or interactions therebetween.

[0309] In some examples, the predicted interaction may comprises a position of the effector relative to the target, an orientation of the effector relative to the target, or a binding free energy of the effector in binding to the target. In some embodiments, the dataset (e.g., generated through a virtual screen, an empirical screen, or any combination thereof) comprises structure-activity relationships (SAR) between the target and the effector or a plurality of effectors. In some cases, a subset of the effectors screened may be identified as hits.

[0310] In an aspect, provided herein is a method comprising: performing two or more sequential screens, wherein each screen among the two or more sequential screens is informed by or informs another screen among the two or more sequential screens, or both. In some examples, at least one screen of the two or more sequential screens comprises an empirical screen of a plurality of bead-bound encoded effectors, wherein each bead-bound encoded effector comprises: (i) an effector bound to a bead via a cleavable linker and releasable upon cleavage of the cleavable linker, and (ii) a barcode corresponding to the effector that identifies it.

[0311] In some examples, at least one screen of the two or more sequential screens comprises an in-silico screen. In some examples, the in-silico screen comprises: (a) providing or obtaining a computational representation of a target and a plurality of computational representations of the effectors of the plurality of bead-bound encoded effectors; and (b) providing or obtaining a computational program and running it to calculate one or more parameters indicative of a predicted interaction between the computational representation of the target and the computational representation of each effector.

[0312] In some examples, the in-silico screen is the first screen to be performed. The in- silico screen may comprise predictive analysis to inform one or more screens subsequent to it. The in-silico screen may comprise a model which may be trained and improved by empirical data, data from a public database or any suitable source, or any combination thereof. In some examples, the in-silico screen comprises performing predictive analysis using Artificial Intelligence (Al), Machine Learning (ML), Neural Networks, any other suitable in-silico screening approach, or any combination thereof.

[0313] In some examples, the method further comprising providing a map of Structure Activity Relationships (SARs) among a target (or a plurality of targets) and a plurality of effectors, in some cases, using a combination of in-silico and empirical screening which may inform each other in any order and/or combination. In some examples, the in-silico screen may be the first screen to be performed, and/or may inform a second screen. The second screen may comprises empirically screening a first library of encoded effectors (e.g., beadbound encoded effector library or one bead one compound (OBOC) library). In some examples, the second screen may inform a third screen. The third screen may comprise empirically screening a second library of bead-bound encoded effector. In some examples, the method may be used for screening an encoded effector that is not bound to a bead/scaffold. Any empirical screen performed may comprise generating a training dataset. The training dataset may be used to train the model used as part of the in-silico screen. The empirical screens may further generate a test dataset for testing the quality of the computational model. Any appropriate Machine Learning (ML) workflow may be used. [0314] In some examples, the number of effectors screened in the first screen may be larger than the number of the effectors screened in the second screen. In some examples, the number of the effectors screened in the second screen may be larger than the number of the effectors screened in the third screen. As such, the effectors screened may get refined as the workflow proceeds and/or advances. In some examples, at least one screen of the two or more sequential screens comprises an empirical screen of a plurality of encoded effectors on cells. The cell screening methods may be according to any cell screening method described anywhere herein.

[0315] In some examples, at least one screen of the two or more sequential screens comprises providing or obtaining a miniaturized screening platform comprising a plurality of compartments, wherein at least a subset of the plurality of compartments comprise at least one bead-bound encoded effector, and wherein the bead-bound encoded effector interacts with a target in the compartment, thereby generating a signal indicative of the activity of the target in presence of the bead-bound encoded effector. In some examples, the same or similar methods may be performed on an encoded effector that is not bound to a bead.

[0316] In some examples, the compartment is a droplet, a well, a nanopen, or a miniaturized channel or feature. In some examples, the compartment is a droplet formed with the aid of a microfluidic device. In some embodiments, the microfluidic device comprises an integrated circuit. The integrated circuit may comprise a droplet generated followed by an incubation region, and the incubation region may allow for incubating the droplet for at least 5, 10, 15, 20 minutes, or above, with a dispersion ratio of less than about 20%, 10%, 9%, 3%, 2% or less.

Applications and samples

Bead capture of nucleic acids

[0317] In addition to measuring activity from detectable signals, additional information can be gathered from a screen by incorporating nucleic acids from the sample onto encodings. In some examples, the methods 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 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.

[0318] In some examples, provided herein is a method for detecting sample nucleic acids in a nucleic acid encoded effector screen. The method 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) may produce a signal. In some cases, 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. In some examples, the expression fingerprint of the cell can be generated in response to treatment with the effector. As described herein, 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.

[0319] In order to release the cellular nucleic acids, the cell may be lysed. In some examples, the method may further comprise the step of lysing the cell. Lysing the cell may comprise adding lysis buffer to the compartment. In some cases, the lysis buffer may be added by pico-inj ection (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. In some examples, 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.

[0320] Any type of cellular nucleic acid can be transferred to the nucleic acid encoding. In some examples, the method may comprise transferring one or more cellular nucleic acids from the sample to the nucleic acid encoding. In some embodiments, the nucleic acids may comprise or be mRNA. In some cases, 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 nucleic acids may proximity extension products. In some examples, a plurality of different cellular nucleic acids may be attached to nucleic acid encodings.

[0321] 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.

[0322] In some cases, 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.

[0323] In some embodiments, the additional reagents to facilitate the transfer of the nucleic acids comprise an enzyme. In some embodiments, the enzyme is a polymerase, a ligase, a restriction enzyme, or a recombinase. In some embodiments, the enzyme is a polymerase. In some embodiments, the additional reagents comprise a chemical cross-linking reagent. In some embodiments, the chemical cross-linking reagent is psoralen.

[0324] FIG. 18 illustrates an exemplary method for amplifying a primer to maximize cellular nucleic acid capture, as described herein. As shown in FIG. 18, in step 1, 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. In some embodiments, 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. In some embodiments, the nicking site corresponds to a specific nucleotide sequence in the nucleic acid encoding. As shown in step 2, the nucleic acid encoding is nicked at the nicking site. As shown, in some embodiments, nicking herein refers to a single strand of the encoding being displaced from the nucleic acid encoded scaffold. As shown in steps 3-4 of FIG. 18, an amplification enzyme may interact with the 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. In some embodiments, the encoded nucleic acid primer labels the cellular encoding. In some embodiments, the capture site of the encoded nucleic acid primer prescribes a targeted cellular nucleic acid. In some embodiments, an enzyme enables such labeling. As shown in step 6, 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.

[0325] The cell may be lysed in order to release the desired nucleic acids and to make the desired nucleic acids available for amplification. In some embodiments, the encapsulation further comprises a cell lysis buffer. In some embodiments, the lysis buffer is added by picoinjection. In some embodiments, 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-inj ection.

[0326] 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. In some embodiments, the isothermal amplification mix comprises reagents for loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicasedependent amplification (HAD), recombinase polymerase amplification (RPA), rolling circle replication (RCA), or nicking enzyme amplification reaction (NEAR). In some embodiments, the encapsulation further comprises reagents for isothermal amplification of the target nucleic acid. In some embodiments, the method comprises adding reagents for isothermal amplification to the encapsulation. In some embodiments, the reagents for isothermal amplification are targeted to the specific nucleic acid sequence. In some embodiments, 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, the amplification mix comprises an amplification enzyme. In some embodiments, the amplification enzyme comprises a polymerase.

[0327] In some embodiments, the specific nucleotide sequence of interest can be amplified within the encapsulation. In some embodiments, the method comprises amplifying the cellular nucleic acid comprising the specific nucleotide sequence to produce amplified cellular nucleic acids. In some embodiments, amplifying the cellular nucleic acids is accomplished by PCR. In some embodiments, amplifying the cellular nucleic acids is accomplished by isothermal amplification. In some embodiments, cellular nucleic acids comprising the specific nucleotide sequence are amplified. In some embodiments, the amplified cellular nucleic acid is barcoded with the nucleic acid encoding the scaffold.

Morphological changes in the sample

[0328] The signal from the sample may be a morphological or visual change in the sample which can be measured by imaging the encapsulation. In some embodiments, 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.

[0329] 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 highspeed shutter, where composite video frames offers multiple full-cell images that can aid in shape determination. 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 from a high frequency pulsed light source, increasing temporal resolution and sharpening the perimeter of the cell. In one manifestation, morphology changes can be detected by fluorescence emission from a cell traversing a laser-light sheet excitation region. In some embodiments, 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.

[0330] In some embodiments, detecting the signal comprises recording images of the sample, wherein the sample is a cell. In some embodiments, 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. In some embodiments, the encapsulation comprises a detection agent. In some embodiments, the detection agent is an intercalation dye. In some embodiments, 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. In some embodiments, the detection agent highlights different regions of the cell. In some embodiments, the detection agent highlights a particular organelle. In some embodiments, 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.

[0331] 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.

Cell types

[0332] In some embodiments, the sample is a cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cancer cell. In some embodiments, 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; 3T3 LI, Mouse Embryo;

HL60, Human Caucasian promyelocytic leukaemia; PC- 12, Rat adrenal phaeochromocytoma; HT29, Human Caucasian colon adenocarcinoma; OE33, Human Caucasian oesophageal carcinoma; OE19, Human Caucasian oesophageal carcinoma; NIH 3T3, Mouse Swiss NIH embryo; MDA-MB-231, Human Caucasian breast adenocarcinoma; K562, Human Caucasian chronic myelogenous leukemia; U-87 MG, Human glioblastoma astrocytoma; MRC-5 (PD 25), Human fetal lung; A2780cis, Human ovarian carcinoma; B9, Mouse B cell hybridoma; CHO-K1, Hamster Chinese ovary; MDCK, Canine Cocker Spaniel kidney; 1321N1, Human brain astrocytoma; A431, Human squamous carcinoma; ATDC5, Mouse 129 teratocarcinoma AT805 derived; RCC4 PLUS VECTOR ALONE, Renal cell carcinoma cell line RCC4 stably transfected with an empty expression vector, pcDNA3, conferring neomycin resistance.; HUVEC (S200-05n), Human Pre-screened Umbilical Vein Endothelial Cells (HUVEC); neonatal; Vero, Monkey African Green kidney; RCC4 PLUS VHL, Renal cell carcinoma cell line RCC4 stably transfected with pcDNA3-VHL; Fao, Rat hepatoma; J774A.1, Mouse BALB/c monocyte macrophage; MC3T3-E1, Mouse C57BL/6 calvaria; J774.2, Mouse BALB/c monocyte macrophage; PNT1 A, Human post pubertal prostate normal, immortalised with SV40; U-2 OS, Human Osteosarcoma; HCT 116, Human colon carcinoma; MA104, Monkey African Green kidney; BEAS-2B, Human bronchial epithelium, normal; NB2-11, Rat lymphoma; BHK 21 (clone 13), Hamster Syrian kidney; NSO, Mouse myeloma; Neuro 2a, Mouse Albino neuroblastoma; SP2/0-Agl4, Mouse x Mouse myeloma, non-producing; T47D, Human breast tumor; 1301, Human T-cell leukemia; MDCK-II, Canine Cocker Spaniel Kidney; PNT2, Human prostate normal, immortalized with SV40; PC-3, Human Caucasian prostate adenocarcinoma; TF1, Human erythroleukaemia; COS-7, Monkey African green kidney, SV40 transformed; MDCK, Canine Cocker Spaniel kidney; HUVEC (200-05n), Human Umbilical Vein Endothelial Cells (HUVEC); neonatal; NCI-H322, Human Caucasian bronchioalveolar carcinoma; SK.N.SH, Human Caucasian neuroblastoma; LNCaPEGC, Human Caucasian prostate carcinoma; OE21, Human Caucasian oesophageal squamous cell carcinoma; PSN1, Human pancreatic adenocarcinoma; ISHIKAWA, Human Asian endometrial adenocarcinoma; MFE-280, Human Caucasian endometrial adenocarcinoma; MG-63, Human osteosarcoma; RK 13, Rabbit kidney, BVDV negative; EoL-1 cell, Human eosinophilic leukemia; VCaP, Human Prostate Cancer Metastasis; tsA201, Human embryonal kidney, SV40 transformed; CHO, Hamster Chinese ovary; HT 1080, Human fibrosarcoma; PANC-1, Human Caucasian pancreas; Saos-2, Human primary osteogenic sarcoma; Fibroblast Growth Medium (116K-500), Fibroblast Growth Medium Kit; ND7/23, Mouse neuroblastoma x Rat neuron hybrid; SK-OV-3, Human Caucasian ovary adenocarcinoma; COV434, Human ovarian granulosa tumor; Hep 3B, Human hepatocyte carcinoma; Vero (WHO), Monkey African Green kidney; Nthy-ori 3-1, Human thyroid follicular epithelial; U373 MG (Uppsala), Human glioblastoma astrocytoma; A375, Human malignant melanoma; AGS, Human Caucasian gastric adenocarcinoma; CAKI 2, Human Caucasian kidney carcinoma; COLO 205, Human Caucasian colon adenocarcinoma; COR- L23, Human Caucasian lung large cell carcinoma; IMR 32, Human Caucasian neuroblastoma; QT 35, Quail Japanese fibrosarcoma; WI 38, Human Caucasian fetal lung; HMVII, Human vaginal malignant melanoma; HT55, Human colon carcinoma; TK6, Human lymphoblast, thymidine kinase heterozygote; SP2/0-AG14 (AC -FREE), Mouse x mouse hybridoma non-secreting, serum-free, animal component (AC) free; AR42J, or Rat exocrine pancreatic tumor, or any combination thereof [0333] The sample may comprise a protein, a recombinant protein, a mutant protein, an enzyme, a mutant enzyme, a protease, a hydrolase, a kinase, a recombinase, a reductase, a dehydrogenase, an isomerase, a synthetase, an oxidoreductase, a transferase, a lyase, a ligase, or any mutant thereof. 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

[0334] 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. For example, 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. In some cases, 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.

[0335] In some cases, 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). Alternatively, the ion channel may be sensitive to stimulation (e.g., optical stimulation) for another reason. In some examples, 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.

[0336] In some examples, 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 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. [0337] 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. In some cases, 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. In some cases, the ion channel may be a protein expressed by a cell.

[0338] 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. Any encoded effector embodiment or screening system embodiment may be used for ion channel screening.

[0339] The set of voltage sensor probes may comprise any suitable probe. For example, the set of voltage sensor probes comprise a FRET pair, a voltage-sensitive oxonol, a fluorescent coumarin, a Di SB AC compound, a coumarin phospholipid, a Di SB AC compound, a coumarin phospholipid, a DiSBAC?, DiSBAC ₄ , DiSBACe, CC1-DMPE, CC2-DMPE, a DiSBAC ₂ (3), DiSBAC ₂ (5), DiSBAC ₄ (3), DiSBAC ₄ (5), DiSBAC ₆ (3), DiSBAC ₆ (5), CC1- DMPE, CC2-DMPE, DiSBACe, CC2-DMPE or any combination or derivative thereof.

[0340] 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 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.

[0341] 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. In an example, 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

[0342] In some examples, 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. In some examples, the observed change may be a redistribution of the signal in space. For example, 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. In some cases, condensate formation may be a measure of protein-protein interactions (ppi) in a sample or in a cell in a sample. For example, an assay may measure one or more protein-protein interactions through manifestation of a phase condensation (formation of condensates) in the sample. In some examples, such assays may be performed to study protein-protein interaction networks or protein-nucleic acid interaction networks (e.g., protein-RNA interaction network). In some examples, 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 terms “condensates” and “stress granules” (SG) may be used interchangeably. In some cases, 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. [0343] Provided herein are method and systems for detecting formation of ribonucleoprotein (RNP) granule assembly. In some cases, 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. In some cases, SG formation may be detected in cells. In some cases, SG formation may be detected in cell-free samples. In some cases, SGs may form or assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. In some examples, 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. In some cases, increasing the levels of RNA in a sample may lead to SG formation or condensate formation. In some cases, 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. Other factors affecting SG assembly may arise through positive or negative cooperativity by extrinsic G3BP1 -binding factors that strengthen or weaken, respectively, the core SG network. SGs may show up as condensates in screening and may be referred to as condensates.

[0344] In some examples, 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.

[0345] In some cases, 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). In this condensed phase, 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 domains (LCDs), undergo concentration dependent LLPS. In some examples, the methods and systems of the present disclosure may facilitate detection, screening, and perturbation of RNA-binding proteins (RBPs). 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. [0346] 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. Therefore, 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. In some cases, a plurality of condensates may form in a sample over time. The plurality of condensates may comprise different sizes and intensities. In some examples, the number of condensates formed in a sample may be an indication of a condition of the sample. In some examples, the intensity of the condensates may be an indication of a condition of the sample. In some examples, 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.

[0347] In some cases, an effect of an effector or effector library on a sample may be screened. In some cases, 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. In a particular example, 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. Alternatively, 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). [0348] 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 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).

[0349] In some examples, condensates may be detected in a droplet-based screening platform provided herein. In such case, 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. As a result of an assay, one or more condensates may be formed inside the droplets. 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. 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).

[0350] In case of condensate detection by imaging, 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. In some cases, 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. In some cases, the intensity of the condensates under different conditions (e.g., presence of effector) may be compared. In some cases, the condensates may aggregate together to form larger and/or brighter aggregated condensates. In some cases, 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. In each case, 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) may be assessed by observation and/or detection of the condition and properties of the condensates, however such properties manifest in the compartment (e.g., droplet or well). In some cases, an effector changes the properties of condensates over time, compared to a control sample not containing an effector. In some cases, the condensates may manifest or appear as small spikes on a droplet digital signal trace detected by a system presented herein.

[0351] 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. For example, 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.

Computer systems

[0352] The methods of the disclosure may involve the use of one or more computer systems. FIG. 19 shows a computer system (also “system” herein) 1901 programmed or otherwise configured to implement the methods of the disclosure. The system 1901 may include a central processing unit (CPU, also “processor” and “computer processor” herein) 1905, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The system 1901 may also include memory 1910 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1915 (e.g., hard disk), communications interface 1920 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1925, such as cache, other memory, data storage and/or electronic display adapters. The memory 1910, storage unit 1915, interface 1920 and peripheral devices 1925 may be in communication with the CPU 1905 through a communications bus (solid lines), such as a motherboard. The storage unit 1915 can be a data storage unit (or data repository) for storing data. The system 1901 may be operatively coupled to a computer network (“network”) 1930 with the aid of the communications interface 1920. The network 1930 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 1930 in some cases is a telecommunication and/or data network. The network 1930 can include one or more computer servers, which can enable distributed computing, such as cloud computing.

The network 1930 in some cases, with the aid of the system 1901, can implement a peer-to- peer network, which may enable devices coupled to the system 1901 to behave as a client or a server.

[0353] The system 1901 may be in communication with a processing system 1940. The processing system 1940 can be configured to implement the methods disclosed herein. The processing system 1940 can be in communication with the system 1901 through the network 1930, or by direct (e.g., wired, wireless) connection. The processing system 1940 can be configured for analysis, such as nucleic acid sequence analysis.

[0354] Methods and systems as described herein can be implemented by way of machine (or computer processor) executable code (or software) stored on an electronic storage location of the system 1901, such as, for example, on the memory 1910 or electronic storage unit 1915. During use, the code can be executed by the processor 1905. In some examples, the code can be retrieved from the storage unit 1915 and stored on the memory 1910 for ready access by the processor 1905. In some situations, the electronic storage unit 1915 can be precluded, and machine-executable instructions are stored on memory 1910.

[0355] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, can be compiled during runtime or can be interpreted during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled, as-compiled or interpreted fashion.

[0356] Aspects of the systems and methods provided herein can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non -transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0357] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer- readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0358] The computer system 1901 can include or be in communication with an electronic display that comprises a user interface (UI) for providing, for example, a customizable menu of genetic variants that can be analyzed by the methods of the disclosure. Examples of UFs include, without limitation, a graphical user interface (GUI) and web-based user interface. [0359] In some embodiments, the system 1901 may include a display to provide visual information to a user. In some embodiments, the display is a cathode ray tube (CRT). In some embodiments, the display is a liquid crystal display (LCD). In further embodiments, the display is a thin film transistor liquid crystal display (TFT-LCD). In some embodiments, the display is an organic light emitting diode (OLED) display. In various further embodiments, on OLED display is a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display. In some embodiments, the display is a plasma display. In other embodiments, the display is a video projector. In still further embodiments, the display is a combination of devices such as those disclosed herein. The display may provide one or more biomedical reports to an end-user as generated by the methods described herein.

[0360] In some embodiments, the system 1901 may include an input device to receive information from a user. In some embodiments, the input device is a keyboard. In some embodiments, the input device is a pointing device including, by way of non-limiting examples, a mouse, trackball, track padjoystick, game controller, or stylus. In some embodiments, the input device is a touch screen or a multi-touch screen. In other embodiments, the input device is a microphone to capture voice or other sound input. In other embodiments, the input device is a video camera to capture motion or visual input. In still further embodiments, the input device is a combination of devices such as those disclosed herein.

[0361] The system 1901 can include or be operably coupled to one or more databases. The databases may comprise genomic, proteomic, pharmacogenomic, biomedical, and scientific databases. The databases may be publicly available databases. Alternatively, or additionally, the databases may comprise proprietary databases. The databases may be commercially available databases.

[0362] Data can be produced and/or transmitted in a geographic location that comprises the same country as the user of the data. Data can be, for example, produced and/or transmitted from a geographic location in one country and a user of the data can be present in a different country. In some cases, the data accessed by a system of the disclosure can be transmitted from one of a plurality of geographic locations to a user. Data can be transmitted back and forth among a plurality of geographic locations, for example, by a network, a secure network, an insecure network, an internet, or an intranet.

Definitions

[0363] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0364] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, 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. This applies regardless of the breadth of the range.

[0365] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0366] The terms “determining,” “measuring,” “detecting,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include 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.

[0367] The term “zzz vivo" is used to describe an event that takes place in a subject’s body.

[0368] The term “ex vivo" is used to describe an event that takes place outside of a subject’s body. An 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 “zzz vitro" assay.

[0369] The term “zzz 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.

[0370] The term “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. In some cases, hits may be high-interest events of unknown veracity. In some cases, 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.

[0371] The term “screen” as used herein refers to performing an assay using a plurality of effectors in order to determine the effect various effectors have on a particular sample.

[0372] The term “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. Nextgeneration 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; 7,21 1,390; 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. US20090061439; US20080087826;

US20060286566; US2006002471 1; US20060024678; US20080213770; and US20080103058; 3) Applied Biosystems (e.g. SOLiD sequencing); 4) Dover Systems (e.g., Polonator G.007 sequencing); 5) Illumina, Inc. as described in US Patent Nos. 5,750,341; 6,306,597; and 5,969,1 19; and 6) Pacific Biosciences as described in US Patent Nos. 7,462,452; 7,476,504; 7,405,281; 7,170,050; 7,462,468; 7,476,503; 7,315,019; 7,302,146; 7,313,308; and US Application Publication Nos. US20090029385; US20090068655;

US20090024331; and US20080206764. Such methods and apparatuses are provided here by way of example and are not intended to be limiting.

[0373] The term “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.

[0374] The term “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. through channels of a microfluidic chip of the disclosure, comprises a flow. This is so, according to the disclosure, whether or not the molecules, cells or virions are carried by a stream of fluid also comprising a flow, or whether the molecules, cells or virions are caused to move by some other direct or indirect force or motivation, and whether or not the nature of any motivating force is known or understood. The application of any force 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.

[0375] 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.

[0376] 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.

[0377] 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 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.

[0378] 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),

- I l l - measurement, or sorting. In a chip designed for 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.

[0379] 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. In an embodiment, 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. For example, 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. In sorting embodiments, the detection region is in fluid communication with a discrimination region and is at, proximate to, or upstream of the discrimination region. [0380] 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. Nonlimiting 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.

[0381] 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 to the flow of fluid in the main channel. In some embodiments, 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.

[0382] 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. Typically, 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. [0383] 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. Alternatively, 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.

[0384] The term “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. In some embodiments, 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.

[0385] 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. This may be useful for more accurate sorting, for example, by allowing for confirmation of a sorting decision, selection of particular branch channel, or to correct an improperly selected channel.

[0386] Different “sorting algorithms” for sorting in the microfluidic device can be implemented by different programs, for example under the control of a personal computer. As an example, consider 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. However, mechanical compliance in the system is likely to cause the fluid switching speed to become rate-limiting with the “forward” sorting program. Since the fluid is at low Reynolds number and is completely reversible, when trying to separate rare molecules, cells or virions, one can implement a sorting algorithm that is not limited by the intrinsic switching speed of the device. The molecules, cells or virions flow at the highest possible static (non-switching) speed from the input to the waste. When an interesting molecule, cell or virion is detected, the flow is stopped. By the time the flow stops, the molecule, cell or virion may be past the junction and part way down the waste channel. 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. At that point, the molecule, cell or virion is “saved” and the device can be run at high speed in the forward direction again. Similarly, 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). Reversible algorithms are particularly useful for collecting rare molecules, cells or virions or making multiple time course measurements of a molecule or single cell.

[0387] The term “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, whereas the second liquid is referred to as the continuous phase or the dispersion medium. In one embodiment, 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). Such an emulsion is referred to here as an oil in water emulsion. In another embodiment, an emulsion may be a water in oil emulsion. In such an embodiment, the discontinuous phase is an aqueous solution 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”. [0388] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value. [0389] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.