Multi-particle system for capture and detection of cellular and molecular analytes

Abstract

Particle-based systems are disclosed that use cavity-containing particles that include respective cavities therein and a plurality of capping particles that dock with the cavity-containing particles to create a compartment for cell confinement as well as a region for various molecular reactions. These systems are advantageous in that cells or other reagents can be loaded into cavity-containing particles and then compartments are produced by the stable interaction or association of each cavity-containing particle with one or more 'capping' particles induced by simple mixing, centrifugation, or other associating operations. In this way thousands to millions of capping events between cavity-containing particles and capping particles lead to the rapid formation of thousands to millions of compartments in parallel that include what is termed "capped particles."

Description

2025-267-2MULTI-PARTICLE SYSTEM FOR CAPTURE AND DETECTION OF CELLULAR AND MOLECULAR ANALYTESRelated Application

[0001] This Application claims priority to U.S. Provisional Patent Application No.63 / 779,083 filed on March 27, 2025, which is hereby incorporated by reference in its entirety7. Priority is claimed pursuant to 35 U.S.C. § 119 and any other applicable statute.Technical Field

[0002] The technical field generally relates to systems and methods to selectively confine cells and / or molecular reactions within multi-particle-based systems using interacting or nesting particles that form a cavity or void that act as a compartment. Specifically, the field of the invention relates to systems and methods which employ cavity-containing particles that associate or interact with capping particles to form an assembly of capped particles in which the cavity / void or compartment is at least partially sealed or occluded from the external environment of the capped particles.Statement Regarding Federally SponsoredResearch and Development

[0003] This invention was made with government support under CA256084 awarded by the National Institutes of Health, and 1648451 awarded by the National Science Foundation. The government has certain rights in the invention.Background

[0004] Screening and selection of cells based on desirable phenotypes and functions has become an indispensable process for leveraging cell-derived bioproducts for biotechnological applications and life science research. Examples encompass screening for hybridoma or primary7B cells that rapidly produce functional antibodies, screening for highly proliferative lipid-producing microalgae, screening for activated T cells based on cytokine secretion profiles, and screening for therapeutically potent mesenchymal stromal cells based on extracellular vesicle secretion. Screening at the single-cell level enables sorting through the vast heterogeneity of cell phenotypes to select out and expand cells yy ith desirable properties. Considering the vast phenotypic heterogeneity7that cells possess even among isogenic cells, it2025-267-2is crucial to screen large populations of cells at the single-cell level to ultimately enrich cells with desirable properties. Screening of libraries of genetically or epigenetically modified or knocked out cells in a pooled fashion also benefits from functional screening tools. Similarly, analysis of single cells to uncover transcriptomic, genomic, epigenomic, or proteomic markers and heterogeneity across cell populations can drive understanding of cell differences that give rise to different functions or therapeutic response. Furthermore, analysis of molecules (proteins, nucleic acids, etc.) is critical for life science research and diagnostics.

[0005] One of the key requirements for analyzing single cells, amplifying the detection of molecules, and screening single cells based on desirable properties is the compartmentalization of cells or molecular reactions into discrete nanoliter to picoliter scale volumes. Compartmentalization is advantageous for limiting transport of produced or released molecules between compartments, thereby ensuring that the signal generated is derived from and representative of the cells, molecules, and other samples that are encapsulated within each separated compartment. This ultimately leads to a higher purify of the functional phenotype of cells that can be identified and sorted out, well-defined transcriptomic, genomic, epigenomic, and proteomic signatures from individual cells, and / or more sensitive detection of molecules. Furthermore, detectable levels of analyte or a reaction product indicative of analyte presence can be accumulated more rapidly in picoliter to nanoliter scale compartments, decreasing the incubation time necessary. This enables cells to maintain a higher viability and overall cell health in downstream analysis such as sorting and culture expansion. A shorter time also increases the throughput for molecular assays and the turnaround time for diagnostics, important for rapidly detecting disease or improving the life science research workflow.

[0006] Currently, microfabricated geometrical structures, microdroplets, and other shaped-microparticles are commonly used for compartmentalizing single cells and molecular reactions. Combined with various methods for readout and downstream analysis such as fluorescence, optofluidics, and imaging, microfabricated arrays, wells, and valves have been used for confinement and analysis of single cells. However, these methods are typically hampered by low throughput and high cost for operation. Droplet microfluidics overcomes the issue of throughput as they enable millions of droplets, though the technology is not widely adaptable as it requires complex and specialized instruments to generate, analyze, and sort droplets. More recently, lab-on-a-particle technology, which carries out microscale reactions and analysis on or in microparticles, have been reported such as cavity -containing2025-267-2particles or hollow core-shell particles. Cavity-containing particles can capture and analyze tens of thousands of single cells and their secretions using standard laboratory equipment such as fluorescence-activated cell sorters or sequencers, though their open-faced system can cause crosstalk and are less compatible with motile or fast-growing cells. Although hollow core-shell particles can readily encapsulate cells, including motile or fast-growing cells such as yeast and bacteria, as well as their products (e.g., proliferated cells and secreted antibody), the particle formation and cell loading steps need to occur simultaneously in a microfluidic droplet generator device that requires expertise and microfluidic laboratory instruments.Siimniai'v

[0007] As disclosed herein, capped particle technology is provided as a new tool for scalable and high-throughput compartmentalization and analysis of cells and their products (or molecular reactions) in a microfluidics-free manner only using standard laboratory equipment. The capped particle technology further enhances the accessibility and applicability of single-cell and molecular assay compartmentalization workflows towards a wider range of biomedical, environmental, chemical, and clinical applications. The technique also introduces other unique advantages which include time dependent introduction of a solid phase, potential for spatial barcoding, and ability to have tunable semi-permeable particle assemblies, compartments, or capsules for selective transport and long-term growth. Capped particle systems create compartments without the use of oils or other separate immiscible liquid phases, so cells or biomolecules can remain in aqueous phases throughout processing, incubation, and analysis steps, leading to higher biocompatibility, improved ease of use, and compatibility with other downstream instruments.

[0008] Generally, the invention relates to systems and methods to selectively confine cells and / or molecular reactions within particle-based systems using interacting or nesting particles that form a cavity or void that function as a compartment for cell confinement as well as a region for various molecular reactions and / or cell proliferation. These systems are advantageous in that cells or other reagents can be loaded into cavity-containing particles and then compartments are produced by the stable interaction or association of each cavitycontaining particle with one or more ‘capping’ particles induced by simple mixing, centrifugation, or other associating operations that can be performed in bulk vessels, tubes, petri dishes, well plates, syringes, or the like. In this way thousands to millions of capping events between cavity-containing particles and capping particles lead to the rapid formation2025-267-2of thousands to millions of compartments in parallel that include was is termed “capped particles.” In some embodiments these compartments are sealed from the external environment of the capped particles to prevent cells from being released or molecules with molecular weights above a cutoff from being released. Similarly, the sealed compartments also prevent cells in the external environment from entering the compartments and / or molecules above a cutoff from entering the compartments. Capped particle-based compartments are advantageous in that no additional oils, or other immiscible phases are required to form compartments, and compartments can be opened without laborious and difficult steps of removing oil or a second immiscible phase.

[0009] In some embodiments, the capped particles are “de-capped” to remove the capping particle from interacting with the cavity -containing particles following a reaction or a period of time. In various embodiments, the cavity-containing particles and / or capping particles may be functionalized with reactive or binding moieties. These reactive or binding moieties may bind cells and / or molecular analytes. Complementary reactive or binding moieties may aid in the formation of a capped particle by creating non-covalent or covalent interactions between a cavity-containing particle and capping particle, increasing binding strength between the two particle types with complementary binding moieties. For example, in one embodiment, capping particles are functionalized with streptavidin and interact with biotinylated cavitycontaining particles (or capping particles are functionalized with biotin and interact with streptavidin coated cavity -containing particles).

[0010] In some embodiments the cavity-containing particles and / or capping particles include unique molecular and / or physical barcodes, such as oligonucleotide barcodes, fluorescent barcodes, magnetic barcodes, or other optical / scatter barcodes. In some embodiments cellular assays are conducted in the capped particle compartments, such as growth assays, colony forming assays, secretion assays, killing assays, gene expression assays, fluorescent reporter assays, single-cell sequencing assays, or the like. In other embodiments molecular assays are conducted in the capped particle compartments, including immunoassays, nucleic acid amplification assays, enzymatic assays, affinity or binding assays and the like. In preferred embodiments, analysis of the cellular assays or molecular assays in the capped particles is conducted using standard laboratory equipment, such as flow cytometers, single-cell sequencing droplet generators, microscopes, mass spectrometers, and other instruments suitable for analyzing single cells. In some embodiments individual capped2025-267-2particles are sorted (e.g., using fluorescence-activated cell sorting, or image-activated cell sorting) and a downstream growth or analysis step is performed on one or more sorted events.

[0011] In a preferred embodiment, the cavity -containing particles and the capping particles are suspendable in an aqueous solution. Cells or other reagents or molecular analytes can be loaded into the compartments with this or another aqueous solution. According to some embodiments, a plurality of cavity-containing particles including respective cavities therein that open to an external environment of the plurality of cavity -containing particles via respective openings are associated with a plurality of capping particles that are configured to interact or associate with the openings of the plurality' of cavity-containing particles to at least partially seal or occlude the respective cavities from the external environment, wherein the associating results in a mixture. Mixing or agitating the formed mixture is performed to generate a plurality’ of capped particles. A variety of techniques can be used for mixing or agitating including centrifugation, pipetting, vortexing, and shaking.

[0012] In one embodiment, a multi-particle system for capture and detection of cellular and molecular analytes includes a plurality of cavity-containing particles including respective cavities therein that open to an external environment of the plurality of cavity -containing particles via respective openings and a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity -containing particles to at least partially seal or occlude the respective cavities from the external environment.

[0013] In another embodiment, a method of forming a multi-particle system for capture and detection of cellular and molecular analytes using capped particles includes associating (1) a plurality of cavity -containing particles including respective cavities therein that open to an external environment of the plurality of cavity-containing particles via respective openings and (2) a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity-containing particles to at least partially seal or occlude the respective cavities from the external environment, wherein the associating results in a mixture. The mixture is then mixed, centrifuged, or agitated to form a plurality of capped particles.

[0014] In another embodiment, a method of performing cellular analysis including: generating a mixture of (1) cells, (2) a plurality of cavity-containing particles including respective cavities therein that open to an external environment of the plurality of cavitycontaining particles via respective openings, and (3) a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity-containing2025-267-2particles to seal or occlude the respective cavities from the external environment. The mixture is then mixed or agitated to form a plurality of capped particles with cells contained in the plurality of capped particles. The plurality of capped particles containing the cells are incubated. The cells or a secretion from the cells is labelled with a dye or fluorescent reporter. The capped particles containing the labelled cells or labelled secretion from the cells are flowed through a cell sorter to sort and enrich a sub-population of the capped particles containing the labelled cells or labelled secretion from the cells based on one or more signals from the cell sorter.

[0015] In another embodiment, a method of performing a cell secretome assay includes generating a mixture of (1) cells, (2) a plurality of cavity -containing particles including respective cavities therein that open to an external environment of the plurality of cavitycontaining particles via respective openings, and (3) a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity -containing particles to seal or occlude the respective cavities from the external environment, wherein the plurality of capping particles are functionalized with non-specific binding elements for all or substantially all secreted proteins from the cells. The mixture is then mixed or agitated to form a plurality of capped particles with one or more cells contained in the plurality of capped particles. The plurality7of capped particles containing the one or more cells are incubated. The plurality of capped particles are de-capped to generate a plurality of capping particles having accumulated secretions thereon. The plurality of capping particles having accumulated secretions thereon are analyzed with a mass spectrometer.

[0016] In another embodiment, a method of performing an enzy me assay includes generating a mixture of (1) enzymes, (2) a plurality of cavity -containing particles including respective cavities therein that open to an external environment of the plurality of cavitycontaining particles via respective openings, and (3) a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity -containing particles to seal or occlude the respective cavities from the external environment, wherein the plurality of cavity-containing particles and / or the plurality7of capping particles contain enzyme substrates. The mixture is mixed or agitated to form a plurality of capped particles with the enzy mes located in the plurality7of capped particles. The plurality of capped particles are incubated, wherein the enz mes interact with the enzyme substrate to generate a colorimetric, fluorescent, or chemiluminescent signal. The signal level for each of the2025-267-2plurality of capped particles is determined and the plurality of capped particles are sorted based at least in part on the determined signal level.

[0017] In another embodiment, a method of performing single-cell RNA-sequencing assay includes generating a mixture of (1) cells of a first cell type, (2) a plurality of cavitycontaining particles including respective cavities therein that open to an external environment of the plurality of cavity -containing particles via respective openings, and (3) a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity-containing particles to seal or occlude the respective cavities from the external environment, wherein the plurality of capping particles have a unique oligonucleotide barcode and nucleic acid capture moiety. The mixture is mixed or agitated to form a plurality of capped particles with single cells of the first type contained in the plurality of capped particles. The single cells of the first type contained in the plurality’ of capped particles are lysed to release mRNA. The mRNA is captured in the plurality’ of capped particles. cDNA is formed from the captured mRNA and amplified. The amplified cDNA is then sequenced.

[0018] In another embodiment, a kit is disclosed that includes a plurality of cavitycontaining particles including respective cavities therein that open to an external environment of the plurality of cavity -containing particles via respective openings; a plurality of capping particles that are configured to interact or associate w ith the openings of the plurality of cavity-containing particles to at least partially seal or occlude the respective cavities from the external environment; and wherein the plurality of cavity -containing particles and the plurality of capping particles are located in either a common container or vessel or separate containers or vessels.Brief Description of the Drawings

[0019] FIG. 1 illustrates the general morphology and shape of capping particles and cavity-containing particles in cross-section. Capping particles interact with cavity -containing particles to create three-dimensional enclosed compartments for cellular or molecular assays.

[0020] FIG. 2 illustrates (from left to right) brightfield images of the cavity-containing particles, capping particles, and capped particles. Scale bar represents 50 pm.

[0021] FIG. 3 illustrates capped particles as a compartmentalization system. Fluorescein-isothiocyanate-dextran (average molecular weight of 60,000-76,000 Da) is encapsulated within the cavity of a cavity-containing particle and sealed by a capping particle. Scale bar represents 50 pm.2025-267-2

[0022] FIG. 4 illustrates variations of surface chemistries forming capped particles. In some illustrated embodiments capping particles and cavity-containing particles have complementary binding moieties with affinity to drive association and formation of capped particles. In other embodiments capped particles may be formed without the presence of complementary' binding moieties. Capping particles and cavity -containing particles may also be functionalized with binding or reactive moieties, such as biotin and streptavidin groups to conjugate biotinylated antibodies and proteins to the surface. Strained alkynes may also be conjugated to particle surfaces as another binding or reactive moiety to attach azido-modified antibodies via click chemistry reactions.

[0023] FIG. 5 illustrates one method of fabricating spherical capping particles using a microfluidic device. PEG (polyethylene glycol). The schematic highlights the flow focusing method of generating spherical, UV-crosslinked hydrogel particles. Other particle fabrication methods know n in the art can be used in addition to the one demonstrated here.

[0024] FIG. 6 illustrates the flow cytometry' scatter profiles of capping particles, cavitycontaining particles, and capped particles. Plotting the forward scatter (FSC) width and side scatter (SSC) height profiles of the particles can be used to visualize the three distinct populations. Cavity-containing particles and capping particles can be stained or labeled with different fluorescent dyes, such as streptavidin conjugated with Alexa Fluor 647 (SA-647) or Alexa Fluor 568 (SA-568).

[0025] FIG. 7 illustrates the application of using capped particles for capturing secretions from single cells. Secreting cells are loaded into cavity-containing particles that have been functionalized with cell-capture moieties. Capping particles are functionalized with secretioncapture moieties and attached to cavity -containing particles to create cell-loaded capped particles. The capped particle samples are incubated to allow secretions from encapsulated cells to accumulate on the corresponding capping particle. Cell staining and secretion detection reagents are mixed with capped particles to generate fluorescence signals associated with the capped particles and cells. Flow' cytometry analysis can be used to analyze capped particles to obtain single-cell secretion profiles. A method to gate for these events is depicted in the bottom panel. Capped particles are identified by their forward and side scatter profile relative to capping particles and cavity-containing particles. Cell-loaded capped particles are identified by the cell stain signal area from previous staining steps. The distribution of secretions for single cells can be measured by generating a histogram of cell-loaded capped particles based on the fluorescence of the secretion detection reagent. (Bottom) Example2025-267-2images of cell-loaded capped particles in brightfield and fluorescence images. Cell stain and secretion detection fluorescence are observed in separate fluorescence channels. Scale bar equal to 40 microns.

[0026] FIG. 8 illustrates an application of using a capped particle system for antimicrobial susceptibility testing (AST). The capping particles and / or cavity-containing particles are functionalized with drugs and / or compounds and corresponding barcodes (e.g.. unique fluorophores or combinations of fluorophores). Microbial cells from a patient sample are mixed with cavity-containing particles, and then are capped using capping particles, such as through vortexing and then centrifugation. The capped samples are incubated to grow clonal microbial populations in the presence of eluted and bound drug. The analysis of the colonies in the capped particles is conducted through FACS, flow cytometry or imaging. A method for gating is illustrated depicting the selection of the capped population with cells growing in the presence of antimicrobial agents through forward scatter (FSC) w idth and side scatter (SSC) height profiles of the particles along with the presence of a viability dye. A subpopulation of resistant clones may be sorted for further downstream regrowth and / or sequencing. The drugs or varying drug concentrations can be then linked to the colony based on the barcodes embedded or conjugated to the capped particles.

[0027] FIG. 9 illustrates the embodiment of using capped particles that include oligobarcoded capping particles for performing barcoded single-cell sequencing. The capping particles restrict transport of released nucleic acids (such as RNA) from cells loaded within cavity-containing particles and lysed therein. The approach may be applied for whole analysis of gene expression, sequencing of specific genes (BCRs, TCRs), CITE-seq / SEC-seq, and / or transcriptome sequencing and analysis.

[0028] FIG. 10 illustrates the embodiment of using capped particles for colony -based selection of yeast cells. A suspension of yeast cells is mixed with a plurality of cavitycontaining particles and a plurality7of capping particles. The mixture is agitated and then capped particles encapsulating single yeast cells are formed. The growth of yeast cells can be analyzed and sorted via fluorescence-activated cell sorting based on forward scatter (FSC) width and side scatter (SSC) height profiles. Alternatively, yeast cells can be sorted based on plasmid-based fluorescence or production of intracellular proteins. Genetic screens as well as other functional screens may be performed by suspending yeast cells in selection media.

[0029] FIG. 11 illustrates an embodiment of using capped particles including fluorescence-barcoded capping particles and cavity-containing particles for performing2025-267-2simultaneous biomarker and patient multiplexed ELISA. The cavity-containing particles are barcoded to distinguish between different biomarkers, while the capping particles are barcoded to indicate which sample the cavity-containing particles were incubated with.

[0030] FIG. 12 illustrates capped particles as a compartmentalization system to monitor colony growth. Mammalian, bacterial, fungal, and other cell ty pes can be encapsulated and grown within capped particles. Introduction of a single cell leads to a stable clonal colony within a capped particle. Scale bar equal to 50 micrometers.

[0031] FIG. 13 illustrates an application of capped particles for measuring and screening genetically-encoded biosensors. E. coli expressing the calcium-sensitive biosensor, GCaMP, were introduced into capped particles and cultured. Varying concentrations of calcium were introduced into the surrounding solution and fluorescence signal was measured, showing an increase in fluorescence intensity with increasing calcium (Ca2+) concentration. A clonal colony has much higher overall fluorescence than a single bacteria expressing GCaMP. Scale bar equal to 50 micrometers.

[0032] FIG. 14 illustrates an application of capped particles for containing fluorescent signal from an ELISA enzyme, horseradish peroxidase (HRP), bound to streptavidin.Cavity-containing particles were incubated with decreasing concentrations of a signal generating enz me (HRP) ty pically used in ELISAs, capped, and then incubated with a signal generating substrate (ADHP). The substrate is converted to a fluorescent product that localized to the capped particle. The localized signal intensity increases with increasing enzyme concentration. Scale bar equal to 50 microns.

[0033] FIG. 15 illustrates an application of capped particles for containing precipitate or polymer generated by an enzyme commonly used for both ELISA and immunohistochemistry staining, horseradish peroxidase (HRP), bound to streptavidin. Cavitycontaining particles were incubated with decreasing concentrations of a signal generating enzyme (HRP), capped, and then incubated with a signal generating substrate (DAB). The substrate is converted from a small colorless molecule to a dark brown polymer product that remains localized in the capped particle. The localized signal intensity increases with increasing enzyme concentration. Scale bar equal to 50 microns.

[0034] FIG. 16 illustrates an embodiment of capped particles used to measure antibody accumulation for different incubation periods after antibody-secreting cells are loaded and capped within capped particles. Left shows flow cytometry profiles of antibody accumulation signal over different incubation times. Right shows brightfield and fluorescence images of2025-267-2cells loaded into capped particles and detection of their secreted antibodies with anti-IgG antibodies following incubation of cells to allow secretion over different times.

[0035] FIGS. 17A-17D illustrate capped particle workflow for single-cell assays. FIG. 17A illustrates capped particles are likened to sealable, microscopic test tubes composed of two multi-functional hydrogel particles that can be decorated with proteins and other chemical moi eties. FIG. 17B illustrates representative brightfield image of capped particles (listed as capped nanovials) loaded with mammalian cells. FIG 17V illustrates how cells are loaded into cavity-containing particles, followed by centrifugation with spherical capping particles in order to create sealed, solid-phase reaction vessels (i.e., capped particles) for downstream applications such as grow th studies, secretion accumulation and detection, and multi-cell interaction assays. FIG. 17D illustrates cavity -containing particles and capping particles are fabricated using an aqueous two-phase system composed of a gelatin stream and a polyethylene glycol (PEG) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) stream. Reducing PEG and gelatin concentrations limits phase separation, resulting in production of capping particles rather than nanovials.

[0036] FIGS. 18A-18K illustrate capped particle formation characterization, molecular retention, and How cytometry profiling. FIG. 18 A show s the effect of numerical capping particle to nanovial (NV) ratio on capping efficiency. FIG. 18B shows the effect of multiple centrifugation steps on capping efficiency. FIG. 18C shows the effect of streptavidin (SA) conjugation of cavity -containing particles (i.e., nanovials) and capping particles on capping efficiency. FIG. 18D shows the effect of streptavidin (SA) conjugation of 40 pm cavitycontaining particles and 30 pm capping particles on capping efficiency effect of numerical capping particle to nanovial (NV) ratio on capping efficiency. FIG. 18E shows the effect of numerical capping particle to nanovial (NV) ratio on capping efficiency for the mixed size population. FIG. 18F shows the effect of multiple centrifugation steps on capping efficiency. FIG. 18G illustrates the effect of particle size (40 pm and 70 pm) and capping solution (WB - wash buffer, YM - yeast media) on capping efficiency. FIG. 18H illustrates flow cytometry7and fluorescence microscopy showing retention of varying molecular weight FITC-dextran molecules. FIG. 181 illustrates how hydrogel particles can be independently stained with fluorescently-bound streptavidin to barcode and detect capped nanovial events. FIG. 18J shows capped particles (i.e., capped nanovials) and constitutive particles can be identified on flow cytometry based on fluorescence. FIG. 18K illustrates that each particle ty pe has a unique forward and side scatter profile. FIG. 18L shows brightfield and fluorescence2025-267-2microscopy images of streptavidin-fluorophore-conjugated nanovials, capping particles, and capped nanovials following gating and sorting. FIG. 18M illustrates bar graphs of capping efficiency achieved over three batches that used ~40 pm cavity-containing particles and ~30 pm capping particles, streptavidin functionalization on capping particles, three centrifugation steps, and a 10:1 numerical ratio of capping particles to cavity -containing particles that resulted in high capping efficiencies ranging from 73.3% to 92.4% across different batches.

[0037] FIGS. 19A-19E illustrate a capped particle growth assay. FIG. I9A shows the flow cytometry profile of cell-loaded, capped particles (i.e., capped nanovials). Forward scatter width and high calcein AM area signals are used to identity' HyHEL5-loaded capped nanovials. FIG. 19B shows that following sorting, HyHEL5 colonies proliferate within the particle compartment. After 96 hours the cell colony erupts from the enclosure. FIG. 19C illustrates that following 72 hours of growth, HyHEL5-loaded capped nanovials are gated for Calcein AM area signal from flow- cytometry and sorted. The top 20% of samples have on average four cells per compartment and the bottom 10% have on average one cell per compartment. FIG. 19D shows the gating strategy for enrichment of yeast-loaded capped nanovials based on SSC height signal and images of S', cerevisiae colonies gated based on SSC post-sort. FIG. 19E illustrates timelapse images of E. coll growth over time in capped particles.

[0038] FIGS. 20A-20D illustrate a capped particle antibody secretion assay. FIG. 20A illustrates how hybridomas are loaded into capped and uncapped nanovials with biotinylated HEL protein conjugated to the capping particle and the cavity-containing particle (i.e., nanovial), respectively. Secreted antibodies are detected with fluorescent anti-mouse IgG. FIG. 20B illustrates a flow cytometry' histogram comparison of fluorescence secretion signal for cell-loaded and unloaded nanovials and cell-loaded and unloaded capped nanovials. FIG.20C illustrates fluorescence and brightfield microscopy images of the single-cell secretion assay7results for ab assay on nanovials and capped nanovials. FIG. 20D illustrates MFI of signal for unloaded nanovials and capped nanovials (left) and cell-loaded uncapped and capped particles (right). Capped nanovial workflow led to an increased signal-to-noise ratio compared to nanovials alone.

[0039] FIGS. 21A-21D illustrate a two-cell interaction assay. FIG. 21 A shows a schematic of co-loading OKT3 hybridomas (CellTracker Blue) with Jurkat NFAT-GFP cells (CellTracker Deep Red) into capped particles. FIG. 2 IB shows a gating strategy to identity' Jurkat-loaded capped particles with OKT3 hybridoma (Q2) and without OKT3 hybridoma2025-267-2(QI). NFAT-GFP area profiles for Jurkat+ capped particles with (histogram, Q2) and without (histogram, QI) 0KT3 cells shows distinct activation profiles. FIG. 21C shows mean fluorescence intensity of events in Q2 versus QI shows a significant increase, * = p < 0.05, n = 3 replicates. FIG. 21D shows fluorescence microscopy of sorted OKT3 positive and Jurkat positive capped particles (Q2).

[0040] FIGS. 22A-22D illustrate a two-cell spiked interaction assay in capped particles. FIG. 22A show's a schematic of hybridoma spiking and co-loading with Jurkat NFAT-GFP cell line into capped particles. FIG. 22B show's flow cytometry analysis of capped particles loaded with Jurkat cells and multiple hybridoma populations. After gating for Jurkat reporter cell presence via CellTracker Deep Red. events are gated for single positive hybridoma populations and histograms of NFAT-GFP activation fluorescence are shown for OKT3+HyHEL5- (-) and OKT3-HyHEL5+ events (+). A >30-fold increase in GFP MFI is observed for on-target OKT3 hybridoma co-loaded with Jurkat reporter cells. FIG. 22C shows a comparison in OKT3 enrichment when gating based on NFAT-GFP signal area alone at different stringencies for capped particles and uncapped particles. FIG. 22D shows fluorescent microscopy images of representative capped particles containing a single Jurkat NFAT-GFP reporter cell and either one OKT3 or HyHEL5 hybridoma.

[0041] FIGS. 23A-23D illustrate capping particle and cavity-containing particle (nanovials - NV) size measurements. Particle size and CVs of (FIG. 23A) 40 pmnanovials, (FIG. 23B) 70 pm nanovials, (FIG. 23C) 30 pm capping particles, and (FIG. 23D) 40 pm capping particles.

[0042] FIG. 24 illustrates biotin and fluorescent streptavidin conjugation to particles. Fluorescent streptavidin conjugation to capping particles and nanovials.

[0043] FIGS. 25A and 25B illustrate capping centrifugation speed, tube size, and capping efficiency. Effect of centrifugation speed (FIG. 25 A) and duration and (FIG. 25B) tube size on capped particle formation efficiency.

[0044] FIGS. 26 A and 26B illustrate flow' cytometry sort yield and purity. FIG. 26A illustrates calculated capped particle sort yield and purity for different Sony SH800S sort delay settings using 40 pm nanovials with 30 pm capping particles. FIG. 26B illustrates capped particle sort yield and purity measurements for 70 pm nanovials with 40 pm capping particles. Yield is defined as fraction of capped particle sorted events that w'ere visible by microscopy. Purity is defined as capped particle sorted events divided by all particles (capped nanovials, capping particles, and uncapped nanovials) imaged by microscopy.2025-267-2

[0045] FIGS. 27A-27B illustrate enrichment of GFP-producing bacterial colonies. FIG.27A shows the gating strategy for enriching bacteria-loaded capped particles following different growth times. FIG. 27B shows fluorescence and brightfield microscopy of GFP-expressing E. coli following flow sorting following varying incubation times.

[0046] FIGS. 28A-28B illustrate a simulation of antibody accumulation within cavitycontaining particles and capped particles. COMSOL simulation results of secreted antibody accumulation in (FIG. 28A) a cavity-containing particle and (FIG. 28B) a capped particle.

[0047] FIG. 29 illustrates the dynamic range of the capping particle and cavity-containing particles. Average capping particle and nanovial fluorescence intensity when incubated with varying dilutions of antibody -laden HyHEL5 supernatant.

[0048] FIG. 30 illustrates single-cell secretion time-course. Average secretion signal on capped particles with increasing secretion incubation times. Brightfield / fluorescence overlaid images for selected time points.Detailed Description of Illustrated Embodiments

[0049] With reference to FIG. 1, cavity-containing particles 10 may have a spherical envelope that encompasses the outer surface and are preferably sub-millimeter in diameter, more preferably < 100 micrometers with respect to an outer diameter, and even more preferably < 60 micrometers in diameter (outer diameter) and sized to not clog flow cytometry and single-cell sequencing instruments. Cavity -containing particles 10 may have a spheroidal or non-spherical envelope. Cavity-containing particles 10 preferably are suspendable in an aqueous solution. The mass density' of cavity -containing particles 10 is preferably similar to water (within ±10% of the density of water). A cavity-containing particle 10 may have one or more cavities 12 which are open to the environment via an opening 14, preferably a single cavity 12 open to the environment via the single opening 14. The cavity 12 opening at the exterior surface of the cavity -containing particle 10 preferably has a circular shape to match and seal with a capping particle 16. In one preferred embodiment, the capping particle 16 is spherical or ellipsoidal, although other cavity-opening shapes and matching capping particles 16 may be used. The cavity 14 defines a volume preferably that is sub-nanoliter. The shape of the cavity 14 is preferably spherical, hemispherical, or another three-dimensional shape containing circular cross-sections, to facilitate capping by spherical capping particles 16.2025-267-2

[0050] The cavity-containing particles 10 preferably are made from a hydrogel material. In some embodiments the hydrogel material includes polyethylene glycol (PEG). In some embodiments the hydrogel material may be permeable to small molecules, peptides, nutrients and the like, but not permeable to larger proteins, oligonucleotides, chromosomes, mRNA, and related biomacromolecules. In some embodiments the hydrogel material may have a molecular weight cutoff above which molecules may not transport through the hydrogel material or the transport through the hydrogel material is severely limited. The molecular weight cutoff may be above 20 kD, 50 kD, 100 kD, 150 kD, 200 kD, or 500 kD, depending on the hydrogel material composition and manufacturing.

[0051] Manufacture of cavity-containing particles using aqueous two-phase systems and microfluidics. Cavity-containing particles 10 may be manufactured using micro fluidic droplet generators 20 as illustrated in FIG. 5 to create droplets of aqueous two-phase systems including a first phase prepolymer and second phase separating polymer. In one embodiment, the droplets of precursor polyethylene gly col (PEG) and gelatin are used for manufacture of cavity-containing particles 10, as described in Lee et al. ACS Nano. 16. 1, 38-49, 2021 and Udani et al. Nature Biotechnology 19, : 354-363, 2024, incorporated herein by reference. Generally, a polymerizable PEG precursor solution containing a photoinitiator is co-flowed with a sacrificial polymer (e.g., gelatin) solution and an oil phase containing surfactant in a flow-focusing droplet microfluidic generator 20 to form an aqueous two-phase system (ATPS) in water-in-oil droplets. See FIG. 5. The PEG and gelatin phases separate into two distinct components within droplets (PEG-rich outer region and gelatin-rich inner region) which are then subj ect to UV exposure to crosslink the PEG component. After deemulsifying the crosslinked particles and washing away the uncrosslinked gelatin phase, cavity-containing PEG particles 10 are formed. More specifically, PEG solution has 27.5% w / v 4-arm 5000 Da PEG-acrylate and 4% w / v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) with phosphate buffered saline (PBS, without Ca and Mg) as the solvent, 20% w / v gelatin from cold water fish skin dissolved in DI water, and 3M™ Novec 7500 fluorinated oil including fluoro-surfactant (e.g., 0.5% w / w Pico-Surf or 2% w / w RAN008) are used as the polymerizable PEG precursor phase, sacrificial polymer phase, and continuous phase, respectively, for the ATPS water-in-oil droplet generation. More specifically, the precursor reagents are flowed through a flow-focusing microfluidic channel at flow rates of 1.2 pL / min, 1.5 pL / min, and 13 pL / min for the gelatin phase, PEG phase, and oil phase, respectively. The size of the generated cavity-containing particle 10 is2025-267-2primarily dictated by the height of the microchannel of the droplet generator 20, specifically the height of the co-flo wing junction area of the microfluidic device. Cavity -containing particles 10 with a diameter of ~35 pm, ~55 pm, and ~90 pm are formed when the height of the microchannel are ~18 pm, ~30 pm, and ~70 pm, respectively.

[0052] A number of other manufacturing approaches known in the art can also be used to manufacture cavity-containing particles 10. For example, Sahin et al. describe a number of flow lithography-based approaches (Lab Chip Oct 25:22(21):4007-4042. doi:10.1039 / d21c00421f. 2022) which is incorporated herein by reference. Approaches to manufacture cavity-containing particles 10 include droplet microfluidics lithography, non-spherical lithography, aqueous two phase lithography, continuous flow lithography, stop flow lithography, mask-less flow lithography, hydrodynamic focusing lithography, lock-n-release lithography, two step lithography, structured flow lithography, non-uniform UV flow lithography, inertial flow lithography, co-flow lithography, two-photon flow lithography, vertical flow lithography, and soft membrane flow' lithography. Additional approaches for manufacturing cavity -containing particles 10, especially with non-spheri cal envelopes, include 3D panting technologies, including stereolithography and roll-to-roll continuous liquid interface production.

[0053] Functionalization of cavity-containing particles with binding moieties. Cavity-containing particles 10 may be optionally functionalized throughout the polymer volume or functionalized locally within the cavity 12 with binding moieties. Cavitycontaining particles 10 may contain an immobilized layer of gelatin in the cavity 12 to which different binding moieties may be attached. In one embodiment, the cavity 12 may be functionalized with biotin. In a preferred embodiment, the cavity 12 may be functionalized with biotin and streptavidin to attach biotinylated molecules including antibodies, antibody fragments, aptamers, peptide-maj or histocompatibility complex (peptide-MHC) molecules, MHC-like molecules with their presented antigens (like MR1 and CDla,b,c,d), antigens, peptides, lectins, nucleic acids, or oligonucleotides. Binding moieties based on proteins or peptides may include AviTag sequences for site-specific biotinylation, or other tag sequences for specific binding including His tag or flag tag, or a variety of other tag sequences known in the art. In another embodiment, the cavity 12 may be functionalized with strained alkynes and further functionalized with azide-conjugated molecules, including antibodies, antibody fragments, nucleic acids, or oligonucleotides. In some embodiments the cavity 12 may be functionalized with more than one binding moiety, such as both biotin and strained alkynes.2025-267-2In other embodiments, the cavity-containing particle 10 may be functionalized with biotin, strained alkynes, or other orthogonally -reactive groups during manufacture by including functional group-containing molecules during the formation and manufacturing of the cavitycontaining particles 10. For example, for acrylate-based polymerization of the cavitycontaining particles 10, biotin-PEG acrylate groups may be added at a weight / weight ratio of 1:10 with other polymer precursors to incorporate into the polymer backbone of the cavitycontaining particle 10.

[0054] Barcoding approaches and methods for cavity-containing particles. Cavitycontaining particles 10 can be barcoded both at the interface between the cavity 12 and solution, and / or throughout the volume of the cavity-containing particle 10. In one embodiment, the cavity 12 alone is barcoded with one or more of fluorophores with different emission spectra, oligonucleotides with unique sequences, peptides with unique sequences, other polymers with unique molecular identities, or other sets of antigens. Functionalization methods described herein and in the previous section may be used to attach barcodes to the cavity 12 of the cavity-containing particles 10 through binding moieties, including non-covalent and covalent interactions with the cavity-containing particle 10. In other embodiments, the volume of the cavity-containing particle 10 is barcoded with fluorophores with unique emission spectra, chromogens with unique colors, precipitates that cause unique side scatter patterns in flow, unique oligonucleotide sequences, unique peptide sequences, or other unique antigens. For this embodiment, the various barcoding elements can be introduced throughout the volume of the cavity-containing particle 10 during formation / manufacturing of the cavity -containing particle 10. In some embodiments, both the cavity' 12 and the bulk material of the cavity-containing particle 10 may be barcoded with a combination of the methods listed above.

[0055] Split-pool synthesis. One preferred method for forming cavity-containing particles 10 with numerous unique barcodes is split-pool synthesis. For example, split-pool synthesis may be used to produce a number of cavity -containing particles 10 each with unique oligonucleotide barcode sequences. Particular reaction chemistries for split-pool synthesis are known in the art (e.g., see Macosko et al. Cell 2015.161(5): 1202-1214. doi:10.1016 / j. cell.2015.05.002 which is incorporated herein by reference). In this method a plurality of cavity-containing particles 10 with an initial reactive group is introduced into separate reaction vessels (e.g., four reaction vessels for each of the four DNA bases, A, G, C, T). An initial reaction is performed to add a DNA base with a protected group to the initial2025-267-2reactive group on each of the cavity -containing particles 10. The cavity-containing particles 10 are washed, pooled, and the protected group is removed, revealing a new reactive group. Then the cavity-containing particles 10 are separated back to four new pools for a subsequent reaction adding a subsequent DNA base with a protected group. Following reaction, these samples are washed, pooled, de-protected, and the cycle continues with the next element in the barcode. These split-pool cycles are repeated until a barcode of the desired size is achieved, which may include 5 elements, 10 elements, 15 elements, 20 elements, or more. In a preferred embodiment for oligonucleotide barcodes, the barcode size may have 15 elements. A similar approach may be used to introduce peptide barcodes, fluorophores, or combinations of barcodes that are correlated. For example, in one embodiment particular fluorophores and particular DNA bases are reacted together in each reaction pool such that the oligonucleotide barcode and fluorophore combination are associated with each other enabling mapping of optical signatures from flow cytometry or microscopy to oligonucleotide signatures for downstream sequencing-based readouts.

[0056] Addition of magnetic, iron oxide, particles into cavity-containing or capping particles. In some embodiments cavity-containing particles 10, capping particles 16, or both cavity-containing particles 10 and capping particles 16 including magnetic materials to impart magnetic properties on the respective particles 10, 16. The ability to manipulate the cavity-containing particles 10 or capping particles 16 imparts new functionality such as the ability to coalesce and “pull down7’ and wash particles 10, 16 in parallel without centrifugation using an external magnetic field. In embodiments where only the cavitycontaining particles 10 are magnetic, these particles 10 and capped particles 18 (i.e., a cavitycontaining particles 10 with a capping particle 16) can be separated from free capping particles 16 through magnetic pull down. In embodiments where only the capping particles 16 are magnetic, these particles 16 and capped particles 18 can be separated from free cavitycontaining particles 10 (that are not capped) through magnetic pull down. Then free capping particles 16 may also be separated by size-based fdtration, enriching only the capped particles 18. Magnetic materials may include magnetic nanoparticles or magnetic microparticles including, e.g., iron oxide or other magnetizable material. Magnetic materials may be embedded within cavity -containing particles 10 or capping particles 16 during manufacture by crosslinking the cavity -containing particles or capping particle precursors while magnetic particles are dispersed within the precursors. Alternatively, magnetic particles with binding2025-267-2moieties may be noncovalently or covalently linked to cavity-containing particles 10 or capping particles 16 following manufacture e.g., through binding moieties.

[0057] Description of the capping particles. Capping particles 16 are particles that are preferably larger than the cavity 12 of cavity-containing particles 10 (preferably greater than 30 micrometers in diameter) but smaller than the cavity-containing particles 10 themselves (preferably less than 60 micrometers). Capping particles 16 may be spherical, ellipsoidal, or polyhedral. In some embodiments capping particles 16 contain their own ellipsoidal, hemispherical, or spherical cavities on the particle surface. In a preferred embodiment, capping particles 16 are spherical and are made substantially from a single material, shaped to be able to stably remain attached to and block, occlude, or partially-occlude the opening 14 of the cavity 12 in cavity -containing particles 10. Similar to cavity -containing particles 10. capped particles 18 are preferably suspendable in an aqueous solution. Capping particles 16 are preferably composed of hydrogel materials, although other polymer materials may be used, or inorganic materials like glass, metal, or metal alloy particles. Capping particles 16 more preferably made from a biocompatible hydrogel such as PEG. In some embodiments the hydrogel material may be permeable to small molecules, peptides, nutrients and the like, but not permeable to larger proteins, oligonucleotides, chromosomes, mRNA, and related biomacromolecules. In some embodiments the hydrogel material may have a molecular weight cutoff above which molecules may not transport through the hydrogel material or the transport through the hydrogel material is severely limited. The molecular weight cutoff may be above 20 kD, 50 kD, 100 kD, 150 kD, 200 kD, or 500 KD, depending on the hydrogel material composition and manufacturing. Capping particles 16 may include binding or adhesive moieties to facilitate interaction with and stable capping of cavity -containing particles 10 including complementary binding or adhesive moieties. For example, capping particles 16 may be functionalized with biotin while the cavity-containing particles 10 are functionalized with streptavidin. Alternatively, capping particles 1 may be functionalized with streptavidin while the cavity -containing particles 10 are functionalized with biotin. These are exemplary and other binding chemistries such as click chemistry are contemplated.

[0058] Preferred polymer concentration (and crosslinking density) for capping particles. The polymer concentration used to manufacture the capping particles 16 and cavity-containing particles 10 may affect the rate of which capped particles 18 form w hen combining these tw o particle ty pes, through phy sical and chemical interaction differences. In a preferred embodiment, a plurality of the capping particles 16 with 22.5% w / v 5 kDa 4-arm2025-267-2PEG acry late are combined with a plurality of cavity -containing particles 10 with 27.5% w / v 5 kDa 4-arm PEG acrylate, resulting in 55% of the cavity -containing particles 10 becoming capped in a single cycle of mixing and centrifugation. In a more preferred embodiment, a plurality of the capping particles 16 with a 15% w / v 5 kDa 4-arm PEG acrylate are combined with a plurality of cavity -containing particles 10 with 27.5% w / v 5 kDa 4-arm PEG acrylate, resulting in 68% of the cavity-containing particles 10 becoming capped. In another embodiment, a plurality of the capping particles 16 with a 7.5% w / v 5 kDa 4-arm PEG acrylate are combined with a plurality of cavity-containing particles 10 with 27.5% w / v 5 kDa 4-arm PEG acry late, resulting in 45% of the cavity -containing particles 10 becoming capped.

[0059] Manufacture of capping particles using microfluidic devices. Capping particles 16 can be fabricated using microfluidic droplet generators 20 to produce spherical capping particles 16. Spherical capping particles 16 can be produced using any droplet generating microfluidic device 20, including flow focusing microfluidic droplet generators 20 (e.g., FIG. 5), step emulsification devices, or co-flow droplet generators 20. In these embodiments, the disperse phase of the fluids introduced into the droplet generators 20 includes an aqueous hydrogel precursor (polymeric, biomolecular, etc.) combined with a continuous phase of an immiscible fluid (oil w ith surfactant). The aqueous hydrogel precursor is crosslinked to form a spherical capping particle 16. In a preferred embodiment, a flow focusing microfluidic droplet generator 20 is used with a disperse phase of four-arm PEG-acrylate (which can range in size from 2kDa arm length to 40kDa arm length and can range in weight percent in solution from 5% to 50%, but in a most preferred embodiment is in the range of 7.5% to 20%) combined with a photoinitiator such as LAP (which can range in weight percent in solution from 0.5% to 50%, but in its most preferred embodiment is at 2.5%) flowed at a rate of 2pl / min combined with a continuous phase of Novec 7500 with 0.5% v / v Pico-Surf flowed at a rate of 18pl / min and crosslinked using UV. In some embodiments the crosslinking is UV-initiated, in others it is pH-initiated or otherwise chemically -initiated and proceeds with a change in temperature and / or over time. In embodiments where the capping particles 16 are crosslinked using UV-initiated crosslinking, they can be produced by methods other than droplet based microfluidics such as contact molding, continuous How its lithography, stop How lithography, mask-less How lithography, hydrodynamic focusing flow lithography, lock and release flow lithography, structured flow lithography, multi-step flow lithography, non-uniform UV flow lithography, inertial flow2025-267-2lithography, coaxial flow lithography, soft membrane flow lithography, vertical flow lithography, two photon flow lithography, layer-by-layer particle printing, roll-to-roll particle printing, imprint lithography, soft photolithography, among other methods. These methods are able to produce more complex, non-spherical capping particle shapes.

[0060] Methods for manufacturing degradable capping particles. Capping particles 16 can be fabricated to be degradable by including known degradable moieties or materials within the particle manufacture process (such as peptide crosslinkers or dithiol linkages). For peptide-based degradable moieties the addition of proteases, peptidases, matrix metalloproteinases, TEV protease, gelatinases, try psin, or the like with a suitable peptide substrate may be applied for a period of time (minutes to hours) to degrade the capping particles 16. For linkages such as disulfide bonds, dithiothreitol (DTT). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), or the like may be added to degrade capping particles 16.

[0061] Functionalization of capping particles with binding moieties. Capping particles 16 can be functionalized with binding moieties both during and after particle fabrication. In one embodiment, functional groups are included in the particle precursor solution and are crosslinked into the capping particle hydrogel network. For example, for embodiments where the bulk material of the hydrogel is made using a PEG-acrylate precursor, then the capping particles 16 can be biotin functionalized by including biotin-acrylate in the particle precursor solution. Biotin groups, thiol groups and amine groups among others can be incorporated into the capping particles 16 using this method. In a most preferred embodiment, 7.5 weight percent four- arm 5kDa PEG-acrylate with 2.5 weight percent LAP and 1.25 weight percent biotin-PEG-acrylate makes up the particle precursor solution. In another embodiment, materials with functional groups can be physically entangled in the hydrogel network of the capping particle 16. For example, in some embodiments gelatin or other proteins containing functional groups is included in the particle precursor solution, where it is physically entangled in the capping particle 16 during particle formation and is later functionalized with biotin groups, strained alkynes or a combination of the two. In a preferred embodiment, 7.5 weight percent four- arm 5kDa PEG-acrylate with 2.5 weight percent LAP and 1 - 2 weight percent gelatin makes up the particle precursor solution. Gelatin or other proteins may be functionalized at lysine residues using N-hydroxysuccinimide (NHS) or l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistries known in the art. Functionalization of2025-267-2other residues may also be performed according to well-known bioconjugation approaches known in the art.

[0062] Barcoding approaches and methods for capping particles. Capping particles 16 can be barcoded w ith one or more of fluorophores with different emission spectra, oligonucleotides with unique sequences, peptides with unique sequences, other polymers with unique molecular identities, or other sets of antigens. In some embodiments, capping particles 16 can be barcoded during the time of their fabrication by including fluorophores with unique emission spectra, chromogens with unique colors, precipitates that cause unique side scatter patterns in flow , unique oligonucleotide sequences, unique peptide sequences, or other unique antigens into the particle precursor solution during fabrication. In other embodiments, capping particles 16 are barcoded after fabrication by attaching fluorophores with unique emission spectra, chromogens with unique colors, unique oligonucleotide sequences, unique peptide sequences, or other unique antigens via functional groups in the particle (biotinstreptavidin, azide-alkyne, di-thiol, etc.).

[0063] One preferred method for forming capping particles 16 with numerous unique barcodes is split-pool synthesis. For example, split-pool synthesis may be used to produce a number of capping particles 16 each with unique oligonucleotide barcode sequences.Particular reaction chemistries for split-pool synthesis are known in the art (e.g., see Macosko el al. Cell 2015.161(5): 1202-1214. doi: 10.1016 / j. cell.2015.05.002 which is incorporated herein by reference). In this method, a plurality of capping particles 16 with an initial reactive group or binding moiety introduced throughout the capping particle polymer volume is introduced into separate reaction vessels (e.g., four reaction vessels for each of the four DNA bases, A, G, C, T). An initial reaction is performed to add a DNA base with a protected group to the initial reactive group on each of the capping particles 16. The capping particles 16 are w ashed, pooled, and the protected group is removed, revealing a new reactive group. Then the capping particles 16 are separated back to four new pools for a subsequent reaction adding a subsequent DNA base with a protected group. Following reaction, these samples are washed, pooled, de-protected, and the cycle continues with the next element in the barcode. These split-pool cycles are repeated until a barcode of the desired size is achieved, which may include 5 elements, 10 elements, 15 elements, 20 elements, or more. In a preferred embodiment for oligonucleotide barcodes, the barcode size may have 15 elements. A similar approach may be used to introduce peptide barcodes, fluorophores, or combinations of barcodes that are correlated. For example, in one embodiment particular fluorophores and2025-267-2particular DNA bases are reacted together in each reaction pool such that the oligonucleotide barcode and fluorophore combination are associated with each other enabling mapping of optical signatures from flow cytometry or microscopy to oligonucleotide signatures for downstream sequencing-based readouts.

[0064] Processes and methods for forming capped particles. Capped particles 18 are those particles that include a cavity-containing particle 10 and capping particle 16 that combine to form an occluded or sealed cavity 12, preferably using physical and / or chemical methods of facilitating contact between cavity-containing particles 10 and capping particles 16 in a bulk solution. Physical methods include agitation, centrifugation, and fluid shearing of bulk suspensions of cavity -containing particles 10 and capping particles 16 to force contact between capping particles 16 and cavity-containing particles 10. Other methods include applying magnetic forces to capping particles 16 and / or cavity-containing particles 10 that include magnetic-responsive material to facilitate interactions and contact, such as with an external magnetic field, permanent magnet, or electromagnet. Chemical methods include incorporating complementary functional groups on cavity -containing particles 10 and capping particles 16 that form non-covalent or covalent bonds upon interaction of a cavitycontaining particle 10 and capping particle 16. In a preferred embodiment, capped particles 18 are formed by centrifugation in a centrifuge by combining capping particles 16 and cavitycontaining particles 10 in an aqueous solution within a 0.5 mL lube. Centrifugation for a period of time (e.g., 3 minutes, 5 minutes. 10 minutes) may be used to force contact between capping particles 1 and cavity-containing particles 10. In a preferred embodiment, capping particles 16 and cavity-containing particles 10 are centrifuged at 350 RCF. In other embodiments the capping particles 16 and cavity-containing particles 10 may be centrifuged at 100 RCF, 200 RCF, 400 RCF, or 500 RCF. In another embodiment, capped particles 18 are formed via centrifugation in a swing bucket centrifuge by combining capping particles 16 and cavity-containing particles 10 in an aqueous solution within a 1.5 mL tube. In yet another embodiment, capped particles 18 are formed via centrifugation in a fixed rotor centrifuge by¬ combining capping particles 16 and cavity -containing particles 10 in an aqueous solution within a 0.5 mL tube. In preferred embodiments the ratio of the number of capping particles 1 to cavity-containing particles 10 mixed in the aqueous suspension to achieve capped particle formation is greater than 1 (e.g., ratios of 2:1, 3:1, 4:1, 5:1, or even 10:1). Following formation of capped particles 18 (e.g., through centrifugation), unbound capping particles 16 may be removed through filtration or other separation processes. In other embodiments2025-267-2unbound capping particles 16 are not removed and remain as part of the mixed suspension containing capped particles 18 and excess capping particles 16. FIG. 2 illustrates (from left to right) brightfield images of the cavity -containing particles 10, capping particles 16, and capped particles 18.

[0065] Preferred numbers and number ratios. In general, to achieve a high number of capping events, the number of capping particles 16 should exceed the number of cavitycontaining particles 10 when in a mixture together. In a preferred embodiment, when mixed in a 0.5 mL Eppendorf tube 500,000 capping particles 16 are combined with 100,000 cavitycontaining particles 10. In other embodiments, a 5:1 numerical ratio of capping particles 16 to cavity-containing particles 10 is used with 1,000,000 capping particles 16 and 200,000 cavity-containing particles 10 or 100,000 capping particles 16 and 20,000 cavity-containing particles 10. In other embodiments, a 10:1 or 2:1 numerical ratio of capping particles 16 to cavity-containing particles 10 may be used.

[0066] Preferred methods of mixing or agitating cavity-containing particles and capping particles to maximize capping events. In general, capped particle events are generated by the interaction of a plurality of capping particles 16 and a plurality of cavitycontaining particles 10. Methods for creating the interaction of the two types of particles may include, but not limited to, the following: adding a plurality of capping particles 16 and a plurality of cavity-containing particles 10 into a container and subjecting the mixture to centrifugation, upon which the two types of particles physically and / or chemically interact to create capped particle events. FIG. 4 illustrates that chemical functionalization may optionally be used to promote capping events. Adding affinity functional groups is optional as capping may still be accomplished with unmodified capping particles 16 as seen in FIG. 4. As yet another alternative as seen in FIG. 4, another approach is to add a plurality of capping particles 16 having biotin and streptavidin and a plurality of cavity-containing particles 10 having biotin and streptavidin into a container and subjecting the mixture to centrifugation, upon which the two types of particles physically and / or chemically interact to create capped particle events. Alternatively, a plurality of capping particles 16 functionalized with biotin and streptavidin may be added to a plurality of cavity-containing particles 10 functionalized with biotin and streptavidin and then incubating with agitation in a vessel, well, tube, or plate, upon which the two types of particles chemically interact to create capped particle events. The plurality of capping particles 16 and a plurality of cavity -containing particles 10 may be capped by incubating with agitation on a shaker, mixer, vortexer or the like, upon which the2025-267-2two ty pes of particles physically interact to create capped particle events. In yet another alternative, a plurality of capping particles 16 functionalized with one of biotin or streptavidin are added to a plurality of cavity -containing particles 10 functionalized with the other of biotin or streptavidin (e.g., capping particle 16 with biotin and cavity-containing particle 10 with streptavidin or vice-versa) and then incubating with agitation in a vessel, well, tube, or plate, upon which the two types of particles chemically interact to create capped particle events.

[0067] In another embodiment, a plurality of cavity-containing particles 10 are disposed on a surface and a plurality of capping particles 16 are added that settle under gravity or another body force (e.g., magnetic, electrical), upon which the two types of particles physically and / or chemically interact to create capped particle events. Strained alkynes may also be conjugated to particle surfaces as another binding or reactive moiety to attach azidomodified antibodies via click chemistry reactions as seen in FIG. 4.

[0068] Preferred materials for cavity-containing particles and capping particles to achieve a high fraction of capping. In some embodiments, the capping particles 16 and cavity-containing particles 10 include complementary binding moieties which enable a more stable formation of capped particles 18 and maintenance of a bound state over hours, days, or weeks upon interaction. In these embodiments, the capping particles 16 include binding moieties that interact with and lead to non-covalent or covalent binding with binding moieties present on the cavity -containing particles 10. For example, biotin and / or streptavidin on the capping particles 16 may interact with streptavidin and / or biotin on the cavity-containing particles 10 to form a noncovalent binding between the capping particles 16 and cavitycontaining particles 10 and strong affixation of the capping particle 16. Other complementary' binding or reactive moieties may be used for enhancing capped particle formation, such as antibody-antigen, azide-strained alkyne (click chemistry), complementary oligonucleotides, aptamer-antigen, thiol-thiol condensation reactions, or other covalent chemical interactions.

[0069] Preferred crosslinking ratios for cavity-containing particles and capping particles. In a preferred embodiment, cavity-containing particles 10 are crosslinked using 27.5% w / v PEG and 5% w / v photosensitive crosslinker and capping particles are crosslinked using 10% w / v PEG and 1.25% w / v photosensitive crosslinker. In another embodiment, capping particles 16 are crosslinked using 7.5%, 15%, or 22.5% w / v PEG with 1.25% w / v photosensitive crosslinker.2025-267-2

[0070] Preferred sizes, cavity-sizes and size ratios to form compartments. In general, to achieve open cavities 12. capping particles 16 with a diameter greater than the cavity diameter of the cavity -containing particle 10 are used. This functionality creates a fluid-filled space not containing highly crosslinked polymer within the capped particle 18 forming a cavity 12 where molecular assays can be compartmentalized, cells can be grown and incubated, and where fluorescent probes can be used to generate detectable signals for microscopy and flow cytometry. In a preferred embodiment, capping particles 16 with a diameter of 37 microns are used with cavity-containing particles 10 of a total diameter of 60 microns and a cavity diameter of 32 microns. It should be appreciated that other dimensions of the capping particles 16, cavity -containing particles 10 and cavity diameter are contemplated.

[0071] Properties of capped particles and transport of species into or out of capped particles. Capped particles 18 may act as sub-nanoliter-scale compartments for cell growth, cell-cell interactions, chemical reactions, accumulation of reaction products, and / or accumulation of secretions as seen in FIG. 7. In some embodiments capped particles 18 may be completely sealed from the external environment such that only water and small molecules and ions below- a molecular weight cutoff can easily transport through the hydrogel materials encapsulating the inner compartment formed within the capped particle 18. FIG. 3, for example, illustrates how capping particles 16 are able to seal the cavity 12 of the cavitycontaining particle 10 from the external environment and prevent transport of larger molecules out of the cavity'.

[0072] In these embodiments transport through the hydrogel is achieved through diffusive transport. Molecules and reagents can be exchanged and transported into the inner compartment by exchanging the surrounding fluid around the capped particles 18, e.g.. using centrifugation, resuspension and solution exchange operations, or using dilution operations where a new fluid is added to the current fluid in which the capped particles 18 are suspended. These operations of solution exchange and transport into the inner compartment occur while other cells, biopolymers, or other larger molecules remain isolated and compartmentalized within each capped particle 18. In other embodiments capped particles 18 are partially sealed from the external environment such that transport of larger biomolecules and biopolymers may occur either through small gaps betw een the capping particle 16 and cavity-containing particle 10 that form at the interface, or through the hydrogel matrix of the cavity-containing particle 10 and / or capping particle 16. In these embodiments convective2025-267-2transport is minimized because there is little or no flow within the inner compartment of the capped particle 18 and diffusive transport of molecules into or out of the capped particles 18 is achieved. In these embodiments, biomolecules that are not bound to the cavity-containing particle 10 or capping particle 16 can freely diffuse into the surrounding solution or be diluted out by addition of new solutions. Additionally, large biopolymers or other molecules may transport into the inner compartment through diffusive transport to e.g., initiate reactions, label or stain molecules (e.g., using antibodies or oligonucleotides), or start enzymatic processes, e.g., in vitro transcription and translation. Note that for either embodiment transport into and out of the inner compartment or cavity 12 is restricted and reduced (e.g., 10-fold reduced, or 100-fold slower transport) compared to a cavity -containing particle 10 without a capping particle 16 bound.

[0073] Methods for washing capped particles. Washing of capped particles 18 generally may follow approaches used to wash cells using tools and techniques known in the art. These include a series of steps starting with centrifugation, followed by aspiration of supernatant, and refilling with additional solution. Additionally, cell strainers or other membranes can be employed to remove supernatants from capped particles 18 and exchange capped particles 18 into an elution solution.

[0074] Methods of de-capping of capped particles. In various embodiments described herein it may be advantageous to remove the capping particles 16 following an assay or reaction occurring within a capped particle 18. By removing the capping particle, the contents of the cavity-containing particle 10 become open to free transport of reagents of all sizes, or for cells to regrow and form a new colony within a vessel in which the now open-capped particle 18 is seeded. The capping particle 16 may also have acquired data (e.g., bound secreted, amplified, or released molecules, including proteins, nucleic acids, lipids, and extracellular vesicles) from the reaction within the capped particle 18 environment and removal of a capping particle 16 may facilitate further downstream analysis, such as input into a system for flow cytometry, sequencing, mass spectrometry or the like. Although generally it is advantageous to have stable sealed capping of capped particles 18 that is robust to manipulation and agitation, various methods for de-capping or removal of capped particles 18 may be employed. In one embodiment, cells compartmentalized within the space between the cavity -containing particle 10 and capping particle 16 can de-cap the capping particle 16. Specifically, when cell-containing capped particles 18 are incubated, the compartmentalized cells can proliferate, fill the volume of the compartment, and eventually apply force to2025-267-2dislodge the capping particle 16. In another embodiment, mixing, sonication, agitation or other physical methods may be used to remove capping particles 16. While normal pipetting and mixing in a tube is not expected to remove capping particles 16, vigorous mixing with rapid uptake and withdrawal through narrow lumen pipettes, pipetting over a longer time period, or sonication may be applied to remove a fraction of the capping particles 16, by applying high levels of fluid shear stress to break the interaction between capping particles 16 and cavity-containing particles 10. In some embodiments, the process of removal of the capping particle 16 can also be facilitated through the use of chemical agents that interfere with complementary chemical binding moieties present on cavity-containing particles 10 and capping particles 16. For example, addition of excess concentrations of streptavidin and / or biotin or a higher affinity version to interfere with biotin / streptavidin linkages between capping particles 16 and cavity-containing particles 10 and disrupt binding. For all of these physical and chemical processes, they may be done in a dilute suspension, and after sorting and / or filtration to enrich for capped particles 18 such that the large volume ensures that free capping particles 16 do not easily find anew cavity -containing particle 10 to form anew capped particle 18. Alternatively, or in addition, chemical degradation of capped particles 18 may be used to remove either the capping particle 16 or cavity-containing particle 10.Chemical moieties, such as peptides, disulfide linkages, and the like, known in the art may be used during initial crosslinking of particles to impart degradability. For peptide-based degradable moieties the addition of proteases, peptidases, matrix metalloproteases. TEV protease, gelatinases, trypsin, or the like with a suitable peptide substrate may be applied for a period of time (minutes to hours) to degrade particles. For linkages such as disulfide bonds, dithiothreitol (DTT), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), or the like may be added to degrade particles.

[0075] Capped particle solutions and kits. Capped particles 18 can be formed in various aqueous solutions or other single-phase liquid suspensions. In a preferred embodiment, capped particles 18 may be formed by mixing an aqueous suspension containing cavitycontaining particles 10 with a second aqueous suspension containing capping particles 16. The formation of capped particles 18 with sealed or occluded compartments occurs in an aqueous phase without the use of oil or a second immiscible phase. The aqueous suspensions may include DI w ater, phosphate buffered saline (PBS), body fluids such as blood, serum, urine, cerebral spinal fluid, wound exudate, tears, saliva, perfusate, dialysate, or the like. In other embodiments, focused on molecular assays, the capped particles 18 may be formed in2025-267-2aqueous solutions or other single-phase liquids, like ethanol, DMSO, or the like. In some embodiments cavity -containing particles 10 and / or capping particles 16 are in different but miscible solutions, such as an aqueous suspension for cavity-containing particles 10 and DMSO for capping particles 16. In some embodiments one or both of cavity-containing particles 10 or capping particles 16 are in an aqueous suspension with a higher concentration of salts or ions, and capped particles 18 are formed in a suspension with high salt concentration (e.g., > 1 M or > 300 mM).

[0076] Following formation of capped particles 18, the particles may be washed and resuspended into another solution with lower salt or ionic strength (e.g., < 300 mM, or < 150 mM), which may selectively expand the capping particle diameter due to particle swelling and enhance the sealing of the capped particles 18. In some embodiments the cavitycontaining particles 10 and / or capped particles 18 are provided in a kit in separate containers or vessels as an aqueous suspension with a defined number of particles per volume such that when the entire suspensions from the two vessels are mixed the cavity-containing particles 10 and capping particles 16 are mixed at an optimum ratio as described herein. The kit may contain other reagents in aqueous solutions stored in separate labeled vessels that may be added to the capped particles 18 for performing the assays described herein. In some embodiments the cavity-containing and / or capping particles 16 are provided in a dried or lyophilized state in separate containers and are rehydrated by a user in an aqueous solution or buffer prior to performing an assay. In other embodiments the cavity -containing and capping particles 16 are provided as a dried or lyophilized mixed powder containing an optimum ratio of cavity-containing particles 10 to capping particles 16 and the mixture is rehydrated together by an end user prior to performing an assay. In some embodiments the kit may contain one container including cavity-containing particles 10, and a number of separate containers containing capping particles 16 with different barcodes, which may be mixed with separate samples and introduced to cavity -containing particles 10 to form a set of barcoded capped particles 18 associated with different samples.

[0077] Cellular assays in capped particles.

[0078] Types of compatible cell populations or mixtures / multicell assays. Various ty pes of cells may be loaded into cavity-containing particles 10 and sealed by capping particles 16 for cellular assays, including bacteria, yeast, fungi, microalgae, parasites, and mammalian cells. In some preferred embodiments single cells are loaded into cavitycontaining particles 10. In some embodiments single cells are bound to the cavity-containing2025-267-2particles 10 through binding-to-binding moieties or antigens on the cell surface. In other embodiments single cells are loaded into the cavity 12 of the cavity-containing particle 10 and not bound but sealed and maintained in the capped particle 18 through sealing by the capping particle 16. In other preferred embodiments multiple cell types are loaded into cavity -containing particles 10 such as tyvo mammalian cells of different types, a mammalian cell and bacteria, a mammalian cell and yeast cell, or a microalgae and bacteria. Following loading in cavity-containing particles 10, and formation of compartments with capping particles 16, cells may be cultured to grow single-cell-derived colonies within capped particles 18. In some embodiments libraries of cells are loaded into capped particles 18, such as gene knockout libraries (CRISPR / Cas9 knockout libraries), gene mutant libraries, libraries of cells producing different recombinant proteins or biosensors, libraries of cells producing different chimeric antigen receptors, cells containing libraries of synthetic receptor proteins (like synNotch), libraries of cells producing therapeutic molecules, like therapeutic antibodies, bispecific engager proteins, multispecific proteins, cytokine fusions, synthekines, degrader proteins such as genetically-encoded lysosomal targeting chimera proteins, or libraries of yeast displaying different scFv, libraries of cells containing different constructs encoding Al-designed proteins, and the like.

[0079] General approaches to label cells in capped particles. Cells may be stained with different fluorescence-based dyes for visualization and characterization. In some embodiments cells may be stained prior to loading into capped particles 18. In some embodiments cells may be stained after loading into capped particles 18. Cells may be stained yvith live / dead stains such as calcein AM or propidium iodide. In some embodiments cells may be stained with fluorescent antibodies or other molecules specific to surface markers on the cell. In some embodiments cells may be stained with CellTracker dyes. In embodiments yvith one or more different cell types, different cell types may be pre-stained yvith different markers or dyes yvith different fluorophores to determine the presence of one or more cell ty pes in the capped particles 18.

[0080] Cell growth I colony formation assay. One embodiment includes a mammalian cell growth assay using capped particles 18. See FIG. 12. Mammalian cells may include a heterogenous population from a tissue, a cell line, a mutant library, a knockout library, a production library, or the like. A plurality of cavity -containing particles 10 is loaded yvith antibodies specific to surface markers on one or more target cell types. Following binding of mammalian cells with antibody-specific surface markers into the cavity-containing particles2025-267-210, the cavity-containing particles 10 are capped using a capping particle 16 to form capped particles 18 as described herein. The capped particles 18 containing mammalian cells are introduced into a growth medium and incubated to grow and expand within the capped particles 18. In some embodiments the cells are stained with a viability dye, fluorescent surface marker, CellTracker dye, or other fluorescent probe specific to the cell type. Capped particles 18 are then introduced into a flow sorter and capped particles 18 containing a growing colony of mammalian cells are sorted to enrich cell populations that are proliferating based on a threshold or gate on forw ard scatter, side / back scatter, and / or fluorescence intensity. Higher scatter and / or fluorescence intensity7can be used to identity7and sort faster growing colonies for further downstream analysis, regrow th, or sequencing of mRNA or DNA. In some embodiments, slower growing colonies (e.g., colonies susceptible to a drug or growth-inhibitor) are desired, and these can be selected based on gating for lower side scatter, forward scatter, and / or fluorescence intensity of the events. Gated and sorted events may be pooled or sorted into individual compartments e.g., of a multiwell plate, such as 96-well or 384-well plate for further growth, dislodging of the capping particle 16 and spread throughout the well and / or analysis.

[0081] In another embodiment, bacterial cell growth assays are performed using a similar approach. See FIG. 12. Bacterial cells may be diluted to an appropriate concentration for Poisson loading and mixed with cavity-containing particles 10 and capping particles 16. Bacterial cells may include a heterogenous population, a clinical isolate, an infected clinical sample, a mutant library, a knockout library, or the like. In some embodiments binding or adhesive moieties for bacteria are conjugated to the cavity-containing particles 10, such as lectins, antigens, or antibodies. The bacterial cells are captured within the cavity-containing particles 10 through specific chemical interactions or by physical trapping within the cavities. The cavity-containing particles 10 are subsequently capped w ith capping particles 16 to create capped particles 18 containing bacterial cells following methods described herein. These capped particles 18 are then incubated under conditions optimized for bacterial growth in growth medium and at growth temperatures. In some embodiments the growth medium contains antibiotics and a subset of the bacterial cells contain plasmids providing antibiotic resistance. In other embodiments the growth medium is lacking essential nutrients and a subset of the bacterial cells contain plasmids that enable production of the essential nutrient. In some embodiments, the bacterial cells may contain fluorescent protein encoding plasmids or be labeled with a viability dye, metabolic activity dye, or other fluorescent markers to2025-267-2enable downstream analysis. After incubation, capped particles 18 containing growing bacterial colonies may be processed through a flow sorter to isolate and enrich bacterial populations demonstrating desired growth characteristics. Capped particles 18 containing a growing colony of bacterial cells are sorted to enrich cell populations that are proliferating based on a threshold or gate on forward scatter, side / back scatter, and / or fluorescence intensity. Higher scatter and / or fluorescence intensity can be used to identify and sort faster growing colonies for further downstream analysis, regrowth, or sequencing of mRNA or DNA. In some embodiments, slower growing colonies (e.g., colonies susceptible to a drug or growth-inhibitor) are desired, and these can be selected based on gating for lower side scatter, forward scatter, and / or fluorescence intensity of the events. Gated and sorted events may be pooled or sorted into individual compartments e.g., of a multiwell plate, such as 96-well or 384-well plate for further growth, dislodging of the capping particle 16 and spread throughout the well and / or analysis.

[0082] In a similar embodiment, yeast growth assays (see FIG. 12 showing yeast cell growth) can be performed using the capped particle system. FIG. 10 illustrates an illustrative workflow for a yeast growth assay. Yeast cells are co-loaded with a plurality of cavity -containing particles 10 and a plurality of capping particles 16 into a tube. Following agitation by pipetting or vortex, the mixture is centrifuged to form capped particles 18 with a single or multiple budding yeast cells encapsulated within the cavity 12 of the cavity-containing particles 10 and capped by the capping particle 16. The cell-encapsulating capped particles 18 can be incubated in cell media at grow th temperatures for cell growth. Following an incubation period, capped particles 18 containing a growing colony of yeast cells can be analyzed / sorted using commercially available flow cytometers, including fluorescence-activated cells sorters and imagine flow cytometers. Upon enrichment of yeast cells expressing desirable phenotype (i. e. , slow-growing or fast-growing cells), the cells can be recovered by de-capping the capping particles 16 for further dow nstream analysis dow nstream analysis, regrow th. or sequencing, and the like. Yeast assays that are compatible with the capped particle system includes, but not limited to. the following: drug discovery, where candidate antifungal therapeutics can be tested by using the yeast growth as a readout for the efficacy; functional genomics, where a cell sample of gene knockout or overexpression cell samples can be cultivated on varying liquid selection media for an auxotrophic screening workflow; industrial applications, where budding yeast cells that produce desirable products (lipids, ethanol) and have high proliferative potential are2025-267-2screened: cell cycle and aging, where cells with altered budding patterns (e.g., using budding scars as readout), cell cycle, replicative lifespan can be screened.

[0083] Functional yeast display assay. One embodiment includes a yeast display assay using capped particles 18. A plurality of cavity -containing particles 10 loaded with target antigen or antigen-expressing cells are exposed to a yeast display library7presenting affinity7molecules (e.g., scFvs) to pan for yeast with binding affinity to the antigen. Following binding of yeast displaying molecules with affinity to antigen the cavity-containing particles 10 are capped using a capping particle 16 to form capped particles 18. The capped particles 18 containing yeast are incubated to grow7and expand within the capped particles 18. Capped particles 18 are introduced into a flow sorter and capped particles 18 containing a grow ing colony of yeast are sorted based on a gate for forward scatter, side / back scatter, and / or fluorescence intensity above a threshold of control non-growing cells to accumulate specific yeast with binding affinity7. Yeast with binding affinity to antigen are prepared for downstream sequencing or regrowth and subsequent rounds of screening. The compartmentalized growth within capped particles 18 is advantageous in that it prevents competition for growth / nutrients between different single-cell-derived clones in bulk panning processes in standard yeast display workflows. In a related embodiment, an antigenexpressing cell is loaded into the cavity-containing particle 10 which also expresses a fluorescent reporter for gene expression. The binding yeast displaying molecules that act as an agonist / antagonist or other function on the target cell is assayed through downstream modulation of gene expression and corresponding fluorescence signal change on the antigenexpressing cell. For example, the reporter antigen-expressing cell may be a Jurkat cell expressing CD3 and a green fluorescent protein reporter for nuclear factor of activated T cells (NF AT). A single yeast cell or multiple yeast cells, following growth, interacting over time with CD3 of the target reporter Jurkat cell(s) can activate NF AT and increase GFP levels. Sorting of capped particle events with high GFP levels can enrich not only yeast with scFv binders to antigen, but also functional binders that lead to functional responses (e.g., agonism or antagonism). This capability7is not available with standard yeast display panning.

[0084] Biosensor screening assay. In one preferred embodiment, a biosensor selection and evolution assay is implemented wherein bacteria / yeast producing a library of biosensor constructs are encapsulated within a capped particle system, allowed to grow7to a colony, as described herein, to amplify biosensor signal, and induced to produce the biosensor at higher levels than visible with a single cell. Following biosensor production by the colony within the2025-267-2capped particles 18 the capped particles 18 are introduced into a solution containing a target analyte of the biosensor at a first concentration (e.g., high concentration) and analyzed by flow cytometry or sorted using FACS or flow sorters based on a gate or threshold of fluorescence intensity of the event. This process can be repeated after introducing the sorted events into a solution containing the analyte target of the biosensor at a second concentration (e.g., low concentration). This process can be used to isolate biosensor constructs that have a high dynamic range of fluorescence across a range of concentrations from low to high. In some embodiments the colonies within the capped particles 18 are lysed following biosensor production to release the biosensors into the cavity 12 of the capped particle 18 and enable access to a target analyte that does not transport into the cells. In some embodiments binding moieties on the cavity -containing particle 10 and / or capping particle 16 bind to the released biosensor to capture and retain it (e.g., through an antibody to a His tag, FLAG tag, or other affinity epitopes on the biosensor). Biosensors are proteins that respond to different concentrations of a target analyte to produce a different output signal, such as fluorescence signal. Example biosensors include sensors for calcium, glucose, glutamate, dopamine, or other small molecules. For calcium sensors, a calcium binding domain may be fused to a fluorescent reporter protein, such as for GCaMP. The biosensor construct may encode a fluorescent protein (e.g., GFP, mCherry), a luminescent enzy me (e.g., luciferase), a colorimetric marker (e.g., [3-galactosidase), or an electrochemical signal generator, enabling real-time detection and quantification of the target analyte.

[0085] FIG. 13 illustrates an application of capped particles 18 for measuring and screening genetically-encoded biosensors. E. coll expressing the calcium-sensitive biosensor, GCaMP, were introduced into capped particles 18 and cultured. Varying concentrations of calcium were introduced into the surrounding solution and fluorescence signal was measured, showing an increase in fluorescence intensity with increasing calcium (Ca2+) concentration. A clonal colony has much higher overall fluorescence than a single bacteria expressing GCaMP enabling higher fidelity sorting based on biosensor signal and function by flow sorters compared to analysis and sorting of single bacteria.

[0086] Antimicrobial susceptibility testing (AST) using capped particles. In one preferred embodiment, and with reference to FIG. 8, an AST assay is implemented utilizing a barcoded capped particle-based system to assess the impact of different therapeutic agents on cell viability, proliferation, or metabolic activity. The assay includes cavity-containing particles 10 and hydrogel-based capping particles 16, wherein the capping particles 16 are2025-267-2functionalized or loaded with specific therapeutic agents and further encoded with distinct barcodes for downstream identification of the therapeutic agent. The barcodes may include nucleic acids, fluorophores with different spectral signatures, fluorophores with different intensities or other optically distinct markers, enabling multiplexed analysis via microscopy or flow cytometry7. The therapeutic agents may be conjugated or infused into the hydrogelbased capping particles 16, providing a localized and sustained release profile of a therapeutic agent within the encapsulated microenvironment containing loaded cells. Each barcoded capping particle 16 may contain a differing amount or type of therapeutic agent. Upon exposure to the therapeutic agent at a dosed concentration, loaded cells within the cavity containing particles 10 may undergo drug-induced responses, which may be monitored through cell viability markers, metabolic readouts, growth phenotypes, formation of colonies, or phenotypic changes. The barcoded capping particles 16 facilitate parallelized screening of multiple therapeutic agents and therapeutic agents across varying concentrations, allowing for high-throughput dose-response profiling and susceptibility7testing. A similar approach may be used for drug screening or high-throughput drug discovery, enabling cell-based assays to characterize a library of drugs and their effects on various heterogeneous cell types, such as tumor cells, immune cells, fibroblasts, bacteria, yeast, or plant cells.

[0087] In a preferred implementation, a clinical AST assay may be performed using the capped-particle system. A clinical isolate including microbial populations is obtained e.g., from blood, urine, or other body fluid. The microbial populations are prepared and loaded into cavity-containing particles 10 to obtain a loading ratio of approximately 1 microbe per 10 cavity -containing particles 10 or less, to ensure single-cell loading following Poisson statistics. Background solution and cells are washed away and cavity-containing particles 10 are then capped by introduction of a set of capping particles 16. wherein each capping particle 16 is uniquely barcoded and has a unique therapeutic agent loaded therein. The capped particles 18 are then incubated in microbial growth medium and held stationary7in a culture vessel. A release of the therapeutic agent from the capping particle 16 is triggered to modulate growth in the cavity 12 of the capped particle 18. Triggering of the release of therapeutic agent may be achieved using approaches known in the art for controlled drug release. For example, a temperature change, light that photocleaves a covalent coupling reaction betw een the capping particle 16 and therapeutic agent, pH change, or a chemical competitor to displace and release therapeutic agent (e.g., high levels of free biotin to release biotinylated therapeutic agent attached to streptavidin on the capping particle). The amount of2025-267-2growth and corresponding drug can be analyzed by microscopy and / or flow cytometry. For example, growth of microbial populations can be analyzed by the intensity of viability dye or scatter profile of a capped particle, and the corresponding therapeutic agent identified for the same event by linking this information to the fluorescent barcode intensity levels for the capping particle 16. By combining greater than hundreds, thousands, or 10,000 different microbe-containing events covering the range of fluorescent barcodes and the corresponding growth signature obtained by flow cytometry, an AST readout of susceptibility or resistance may be obtained, as well as a minimum inhibitory concentration (MIC) for a microorganism. In some embodiments, rare capped particle events including resistant clones may be sorted out based on a threshold of scatter and / or fluorescent viability dye for further analysis and sequencing.

[0088] Antibody-secreting cell (ASC) screening assay. Capped particle methods and systems may be used to perform screens to identify and sort ASCs secreting antigen-specific antibodies (e.g., IgG) with reduced crosstalk to neighboring cavity -containing particles 10. ASCs can be regrown (e.g., hybridoma, plasmablasts / plasma cells with proper supporting medium) and / or nucleic acids from the ASCs sequenced to isolate matched heavy and light chains of the secreted antigen-specific antibody. In one embodiment of the ASC cell screening assay, cavity-containing particles 10 are functionalized with antibodies or other affinity agents that bind the ASC to the surface of the cavity-containing particle 10, and are also functionalized with target antigen proteins or peptides. In a preferred embodiment, cavity-containing particles 10 are functionalized with antibodies or other affinity agents (e.g., anti-CD27, anti-CD138, anti-CD45) that bind the ASC to the surface of the cavity -containing particle 10, and capping particles 16 are functionalized with target antigen proteins or peptides. This enables loading of ASCs in a high background of other cells without binding of secreted antibodies non-specifically, since antigen-specific capture of secreted antibodies (e.g., IgG) only occurs following the introduction of the functionalized capping particles 16 and the formation of capped particles 18 which have reduced transport out of the capped particle 18. In another embodiment, capping particles 16 are functionalized with immunoglobulin-specific antibodies. ASCs are loaded into cavity -containing particles 10. Cavity-containing particles 10 are then capped with functionalized capping particles 16. Cells are incubated in the capped particle 18 to allow secreted antibodies to accumulate (e.g., for 30 minutes, 1 hr, or 3 hrs) and bind if they have affinity to functionalized sites on the capped particle 18. Fluorescent antibody-binding molecules (Protein A, Protein G, Fc receptor2025-267-2proteins, aptamers, minibodies) or antibodies specific to the secreted antibody (anti-IgG) are introduced to label any bound antibody on the capped particle 18. Capped particles 18 are introduced into a flow sorter or FACS and sorted based on gating for the presence of a fluorescent signal in the antibody channel above a threshold or within a gate indicative of specific antibody binding. Gating for sorting may also include gating based on the presence of both a cavity-containing and capped particle, and the presence of a live ASC, using a viability stain (see FIG. 7). Follow ing sorting of capped particles 18 into tubes or index sorting into wells of a well plate, single ASC clones may be regrown and / or nucleic acids may be extracted and sequenced to identify matched heavy and light chain antibody sequences for antibodies specific to antigen. In some embodiments each capping particle 16 includes a nucleic acid capture sequence and unique barcode. ASCs are lysed within the capped particle, and extracted nucleic acids may be captured on the capping particle 16 for unique barcoding of matched heavy and light chain antibody sequences following reserve transcription and cDNA formation. The intensify of the bound antibody signal may also be used to prioritize antibodies based on potential affinity to antigen. In a related embodiment, ASC-loaded capped particles 18 containing anti-IgG capture molecules instead of antigen are introduced to a flow sorter to enrich single ASC clones for secreted antibody production rate for regrowth and cell line development (e.g., CHO cell line development for IgG production). In some embodiments, following loading of ASCs and capping, ASCs are allowed to grow within capped particles 18 for a period of time (e.g.. 1 day, 2 days, or 3 days) to also ensure both growth and secretion of antibody or antigen-specific antibody, and high-growing ASCs are sorted by FACS based on forward scatter, side / back scatter, and / or fluorescence of a cellspecific dye. In related embodiments, both an ASC and a target-expressing cell are co-loaded into cavity-containing particles 10 prior to formation of capped particles 18 by the introduction of capping particles 16. Capped particles 18 are incubated and binding of secreted antibodies to the target-expressing cell are detected by introduction of antibodies or other antibody-binding molecules that bind and label (e.g., fluorescently) the secreted antibodies. Capped particle events containing high levels of antibody -binding molecules are detected and sorted by FACS based on a threshold or gate on fluorescence intensify as seen in FIG. 7. In some embodiments the FACS gates include additional forward scatter and side scatter gates to detect capped particles 18 containing cells, and / or gates in other fluorescence channels to detect capped particles 18 containing at least one target expressing cell and one ASCs. Target-expressing cells may also be reporter cells that produce a fluorescent reporter2025-267-2molecule when a particular signaling pathway is activated (e.g., NF AT), and sort gates are used based on a threshold in fluorescence of the fluorescent reporter molecule indicating signaling pathway activation. Sorted events are regrown or nucleic acids are extracted from ASCs to sequence and link the matched heavy and light chains for the antigen-specific antibody (or antibody that binds antigen and activates a signaling pathway) in the native conformation in the cell membrane of the target-expressing cell.

[0089] Cell binding and activation assays

[0090] Antigen-specific T cell assays. Capped particle methods and systems may be used to perform screens to identify and sort antigen-specific T cells secreting cytokines (e.g., IFN-gamma) or other effector molecules (e.g., granzyme B) with reduced crosstalk to neighboring cavity-containing particles 10. Antigen-specific T cells can be regrown (e.g., expanded T cell clones with proper supporting medium) and / or nucleic acids from the T cells sequenced to isolate T cell receptor (TCR) sequences corresponding to the antigen-specific response.

[0091] In one embodiment of the antigen-specific T cell screening assay, cavity containing particles 10 are functionalized with antibodies or other affinity agents that bind the T cell to the surface of the cavity-containing particle 10 and / or functionalized with peptide-MHC monomers presenting specific antigenic peptides. In some embodiments the cavitycontaining particles 10 are functionalized with peptide-MHC monomers and a signal-2-activating moiety (e.g., anti-CD28). In one embodiment, cavity -containing particles 10 are functionalized with antibodies or other affinity agents (e.g.. anti-CD45) that bind the T cell to the surface of the cavity-containing particle 10, and capping particles 16 are functionalized with peptide-MHC monomers presenting antigenic peptides. This enables loading of T cells in a high background of other cells without non-specific capture of secreted cytokines, since antigen-specific capture of secreted cytokines (e.g., IFN-gamma) only occurs following the introduction of the functionalized capping particles 16 and the formation of capped particles 18, which have reduced transport out of the capped particle 18.

[0092] In another embodiment, capping particles 16 are functionalized with capture antibodies specific to cytokines or other secreted effector molecules. T cells are loaded into cavity-containing particles 10 based on antigen-specific binding to peptide-MHC through the TCRs. Cavity-containing particles 10 are then capped with functionalized capping particles 16. Cells are incubated in the capped particle 18 to allow secreted cytokines to accumulate (e.g., for 30 minutes, 1 hr, or 3 hrs) and bind if they have affinity to functionalized sites on the capped particle 18. Fluorescent cytokine-detecting molecules (e.g., anti -IFN-gamma. anti-2025-267-2granzyme B, cytokine receptor proteins, aptamers) or antibodies specific to the secreted molecule are introduced to label any bound cytokine or effector molecule on the capped particle 18. Capped particles 18 are introduced into a flow sorter or FACS and sorted based on gating for the presence of a fluorescent signal in the cytokine channel above a threshold or within a gate indicative of specific cytokine binding. Gating for sorting may also include gating based on the presence of both a cavity-containing particle 10 and capping particle 16 and the presence of a live T cell, using a viability stain, anti-CD3 stain, anti-CD4 / 8 stain, or a combination thereof.

[0093] Following sorting of capped particles into tubes or index sorting into wells of a well plate, single T cell clones may be expanded, and / or nucleic acids may be extracted and sequenced to identify matched TCR alpha and beta chains for antigen-specific TCRs. The intensity of the bound cytokine signal may also be used to prioritize T cell clones based on their cytokine secretion levels. In some embodiments each capping particle 16 includes a nucleic acid capture sequence and unique barcode, T cells are lysed within the capped particle, and extracted nucleic acids may be captured on the capping particle 16 for unique barcoding of matched alpha and beta TCR sequences following reverse transcription and cDNA formation.

[0094] In some embodiments, following loading of T cells and capping, T cells are allowed to grow within capped particles for a period of time (e.g., 1 day, 2 days, or 3 days) to also ensure both proliferation and / or secretion of cytokines, and viable or proliferating antigen-specific T cells are sorted by FACS based on forward scatter, side / back scatter, and / or fluorescence of a cell-specific dye. In related embodiments, both a T cell and an antigen-presenting cell (APC) are co-loaded into cavity-containing particles 10 prior to formation of capped particles by the introduction of capping particles 16. Capped particles 18 are incubated, and the binding of secreted cytokines to the capped particles 18 is detected by introduction of fluorescently labeled antibodies or receptor proteins that bind the secreted cytokines. Capped particle events containing high levels of cytokine-binding molecules are detected and sorted by FACS based on a threshold or gate on fluorescence intensity. In a related embodiment for cytotoxic T cells, cell killing of the APC is detected using a live / dead stain and sorting based on a threshold of dead stain and presence of one or more viable T cells is performed to isolate cytotoxic T cells.

[0095] In some embodiments, the FACS gates include additional forward scatter and side scatter gates to detect capped particles 18 containing cells, and / or gates in other fluorescence2025-267-2channels to detect capped particles 18 containing at least one APC and one T cell. APCs may also be engineered reporter cells that produce a fluorescent reporter molecule when a particular signaling pathway is activated (e.g., MAPK, CD40 signaling, NF-kB, or apoptosis), and sort gates are used based on a threshold in fluorescence of the fluorescent reporter molecule indicating signaling pathway activation. Sorted events are regrown, or nucleic acids are extracted from T cells to sequence and link the matched TCR alpha and beta chains for antigen-specific T cells in their native conformation in the cell membrane of the APC. In some embodiments each capping particle 16 includes a nucleic acid capture sequence and unique barcode, T cells are lysed within the capped particle, and extracted nucleic acids may be captured on the capping particle 16 for unique barcoding of matched alpha and beta TCR sequences following reverse transcription and cDNA formation. Assays may be conducted for conventional T cells as well as unconventional T cells (like iNK-T cells, gamma-delta T cells, and mucosal associated invariant T cells) using appropriate MHC-like molecules bound to the capped particle 18 that are recognized by these cells (e.g., CDld, MR1, etc.).

[0096] CAR-T cell functional library screening assay. Capped particle methods and systems may be used to perform screens to identify and sort functional chimeric antigen receptor (CAR) T cells based on their secretion of cytokines (e.g., IFN-gamma) or other effector molecules (e.g., granzyme B) with reduced crosstalk to neighboring cavitycontaining particles 10. Libraries of different CAR gene constructs introduced into T cells may be screened, or CAR-T cells with one construct but different knockout genes (e.g., through CRTSPR / Cas9 and a guide RNA library) may be performed. CAR-T cells can be regrown (e.g., expanded T cell clones with proper supporting medium) and / or nucleic acids from the CAR-T cells sequenced to isolate CAR sequences corresponding to the most functional response.

[0097] In one embodiment of the CAR-T cell screening assay, cavity-containing particles 10 are functionalized with antibodies or other affinity agents that bind the CAR-T cell to the surface of the cavity-containing particle 10 and / or functionalized with antigen molecules that interact with the CAR. In some embodiments, the cavity-containing particles 10 are functionalized with antigen and a co-stimulatory molecule (e.g., anti-CD28 or 4- IBB ligand). In one embodiment, cavity-containing particles 10 are functionalized with antibodies or other affinity agents (e.g., anti-CD45) that bind the CAR-T cell to the surface of the cavitycontaining particle 10, and capping particles 16 are functionalized with the corresponding antigen. This enables loading of CAR-T cells in a high background of other cells without2025-267-2non-specific capture of secreted cytokines, since antigen-specific capture of secreted cytokines (e.g., IFN-gamma) only occurs following the introduction of the functionalized capping particles 16 and the formation of capped particles 18, which have reduced transport out of the capped particle 18.

[0098] In another embodiment, capping particles 16 are functionalized with capture antibodies specific to cytokines or other secreted effector molecules. CAR-T cells are loaded into cavity-containing particles 10 based on antigen-specific binding to the antigen on the cavity-containing particle 10 through the CAR. Cavity-containing particles 10 are then capped with functionalized capping particles 16. Cells are incubated in the capped particle 18 to allow secreted cytokines to accumulate (e.g., for 30 minutes, 1 hr, or 3 hrs) and bind if they have affinity to functionalized sites on the capped particle 18. Fluorescent cytokine-detecting molecules (e.g., anti-IFN-gamma, anti-granzyme B, cytokine receptor proteins, aptamers) or antibodies specific to the secreted molecule are introduced to label any bound cytokine or effector molecule on the capped particle 18. Capped particles 18 are introduced into a flow sorter or FACS and sorted based on gating for the presence of a fluorescent signal in the cytokine channel above a threshold or within a gate indicative of specific cytokine binding. Gating for sorting may also include gating based on the presence of both a cavity -containing particle 10 and capping particle 16 and the presence of a live CAR-T cell, using a viability' stain, anti-CD3 stain, or other cell markers.

[0099] Following sorting of capped particles 18 into tubes or index sorting into wells of a well plate, single or pools of CAR-T cell clones may be expanded, and / or nucleic acids may be extracted and sequenced to identify CAR constructs with optimal functionality7. The intensity of the bound cytokine signal may also be used to prioritize CAR-T cell clones based on their cytokine secretion levels.

[0100] In some embodiments, following loading of CAR-T cells and capping, CAR-T cells are allowed to grow within capped particles 18 for a period of time (e.g., 1 day, 2 days, or 3 days) to also ensure both proliferation and / or secretion of cytokines, and viable or proliferating functional CAR-T cells are sorted by FACS based on forward scatter, side / back scatter, and / or fluorescence of a cell-specific dye. In related embodiments, both a CAR-T cell and a target antigen-expressing cell are co-loaded into cavity -containing particles 10 prior to formation of capped particles 18 by the introduction of capping particles 16. Capped particles 18 are incubated, and the binding of secreted cytokines to the capped particles 18 is detected by introduction of fluorescently labeled antibodies or receptor proteins that bind the secreted2025-267-2cytokines. Capped particle events containing high levels of cytokine-binding molecules are detected and sorted by FACS based on a threshold or gate on fluorescence intensity. In a related embodiment for cytotoxic CAR-T cells, cell killing of the target antigen-expressing cell is detected using a live / dead stain, and sorting based on a threshold of dead stain and presence of one or more viable CAR-T cells is performed to isolate the most cytotoxic CAR-T cells.

[0101] In some embodiments, the FACS gates include additional forward scatter and side scatter gates to detect capped particles 18 containing cells, and / or gates in other fluorescence channels to detect capped particles 18 containing at least one target cell and one CAR-T cell. Sorted events are regrown, or nucleic acids are extracted from CAR-T cells to sequence and optimize CAR constructs for improved functionality. Assays may be conducted for various CAR designs, including those with single-chain variable fragments (scFv), dual-targeting CARs, sideCARS, or armored CAR-T cells expressing additional stimulatory cytokines.

[0102] Multispecific antibody assay. One embodiment includes a functional assay for multispecific molecules that have two or more affinity moieties with binding affinity to different targets. In a particular embodiment, a plurality of cavity-containing particles 10 are loaded with one multispecific-secreting cell and the same or another cell possessing a surface marker that the secreted multispecific molecule has affinity7to. The multispecific molecule may be labeled with one or more additional labeled antigens (e.g., with a fluorophore) or one or more additional cells with affinity to the additional affinity moieties. A specific example of this embodiment is provided for screening of bispecific T-cell engagers. Capped particle methods and systems may be used to perform screens to identify and sort functional cells secreting bispecific T cell engager molecules (BiTEs) using a base embodiment where BiTE-secreting cells, such as CHO cells or HEK cells, are introduced into cavity-containing particles 10. Secreted BiTE molecules are captured by functionalized surfaces on the cavitycontaining particle 10, capping particle, or antigen-expressing target cells within the capped particle 18. T cells are also introduced into the assay, where they can bind to the captured BiTEs and potentially be activated to secrete cytokines, which can be detected on the cavitycontaining particle 10 or capping particle 16. Cells secreting BiTEs can be regrown (e.g., expanded clones with proper supporting medium) and / or nucleic acids from the BiTE-secreting cells sequenced to isolate constructs corresponding to the most functional response.

[0103] In one embodiment of the BiTE screening assay, cavity -containing particles 10 are functionalized with antibodies or other affinity agents that bind BiTE-secreting cells (e.g.,2025-267-2CHO or HEK cells) to the surface of the cavity -containing particle 10 and are functionalized with target antigens or T cell-binding moieties (e.g.. CD3-binding domains). The secreted BiTE molecules can then bind to these surfaces or cells, allowing for interactions with T cells or labeled antigens. Capping particles 16 may also be functionalized with antigen to facilitate BiTE capture and T cell binding. Capped particles 18 enable reduced transport out of the capped particle, leading to higher levels of BiTE capture to enable strong T cell engagement.

[0104] In another embodiment, cavity -containing particles 10 are functionalized with antigen specific to BiTE molecules, and T cells are introduced into the assay. Cells secreting BiTEs are loaded into cavity -containing particles 10, and BiTE molecules secreted by CHO or HEK cells bind to the cavity -containing particle 10, capping particle 16, or target antigenexpressing cells. T cells subsequently introduced into the capped particle 18 can interact with bound BiTEs, leading to activation and cytokine secretion (e.g., IFN-gamma, IL-2, TNF-alpha). T cells are allowed to bind and interact over a period of time to accumulate, such as 3 hrs, 6 hrs, or 12 hrs. The secreted cytokines accumulate and bind to detection sites on the cavity-containing particle 10 or capping particle 16. Fluorescent cytokine-detecting molecules (e.g., anti-IFN-gamma, anti-IL-2, cytokine receptor proteins) or other antibodies specific to the secreted cytokines are introduced to label any bound cytokine. Capped particles 18 are introduced into a flow sorter or FACS and sorted based on gating for the presence of a fluorescent signal associated with the BiTE antigen or cytokine channels above a threshold or within a gate indicative of specific binding and activation. Gating for sorting may also include gating based on the presence of both a cavity-containing and capped particle 18 and the presence of alive BiTE-secreting cell and T cell, using viability7stains or cellspecific markers.

[0105] Following sorting of capped particles 18 into tubes or index sorting into wells of a w ell plate, BiTE-secreting cell clones may be expanded, and / or nucleic acids may be extracted and sequenced to identify constructs with optimal functionality. The intensity' of the bound BiTE and cytokine signals and types of cytokines may also be used to prioritize clones based on their secretion levels and potential efficacy.

[0106] In some embodiments, following loading of BiTE-secreting cells and capping, capped particles 18 are incubated, and the binding of secreted BiTE molecules to target antigen-expressing cells is detected by fluorescent labeling of e.g., a His tag or other BiTE moiety. T cells introduced and activated by the BiTEs to secrete cytokines create an additional detection parameter, with cytokine-bound to capped particles 18 sorted by FACS2025-267-2based on a threshold or gate on fluorescence intensity. In a related embodiment, cytotoxic effects are assessed by detecting target cell killing of cells bound by BiTEs using a live / dead stain, and sorting based on a threshold of dead stain and presence of one or more viable BiTE-secreting cells is performed to isolate the most functional BiTE constructs.

[0107] In some embodiments, the FACS gates include additional forward scatter and side scatter gates to detect capped particles 18 containing cells, and / or gates in other fluorescence channels to detect capped particles 18 containing at least one target cell, one T cell, and one BiTE-secreting cell. Sorted events are regrown, or nucleic acids are extracted from BiTE-secreting cells to sequence and optimize constructs for improved functionality. Assays may be conducted for various BiTE designs, including those with novel binding domains, dualtargeting BiTEs, or BiTEs with enhanced half-life and stability.

[0108] Multiplexing secretion assays. Capped particles 18 have unique spatial localization of binding moieties on the cavity-containing particle 10 and capping particle 16 which enable enhanced multiplexing for detection of multiple secretions from a loaded cell simultaneously with fewer fluorescence channels. In one embodiment six different secretions from a cell are analyzed using three fluorescence channels. In this embodiment, three different secretion capture molecules specific to three separate secretions (e.g., antibodies, receptors) are bound to the cavity -containing particle 10 and three additional different secretion capture molecules specific to three additional secretions are bound to the capping particle 16. A cell binding motif (e.g., antibody, antigen, pMHC molecule, MHC-like molecule) is added to the cavity-containing particle 10 and the cell is specifically loaded based on an affinity interaction. Then a capping particle 16 is added to form a capped particle 18 containing the cell therein. The cell is incubated (e.g., for 30 min, 1 hr, 3 hrs, or 6 hrs) to accumulate secretions that bind to secretion capture molecules on the cavity-containing particle 10 and capping particle 16. The secretions are labeled using detection molecules that are fluorescently labeled, such as antibodies specific to the secretions. In this embodiment, six different labels with interpretable colors or spectral characteristics may be used for the six different detection molecules. However, since the capping particle 16 and cavity-containing particle 10 are spatially distinct, only three separate fluorophores may be used, where three detection molecules targeting secretions on the cavity-containing particle 10 are distinct and separable in an optical imaging or flow cytometry' system, and the same three fluorophores are used to label detection molecules targeting secretions on the capping particle 16. The spatial localization can be discerned when introducing the capped particles 18 to an imaging2025-267-2cytometer (e.g., Amnis ImageStream) or an image-activated cell sorter (e.g., BD FACSDiscover). Analysis or gating based on the co-localization of a signal with the brightfield image or fluorescent labeling of the cavity-containing particle 10 or capping particle 16 enables linking a particular color and intensity of the capped particle event to a specific secretion level. The spatial localization may also be observed in the peak shape information of the capped particle 18 as it passes through the standard focused excitation beam of the lasers used in standard flow cytometers and flow sorters. Importantly, the asymmetric shape of the capped particle 18 leads to alignment in the flow cytometry focuser to enable uniform readout of the capping particle 16 and cavity -containing particle 10 in a linear sequence. Although an embodiment with six secretions is described, other embodiments may include more secretions and detection fluorophores. E.g., using spectral flow cytometers, up to 40 different fluorophores can be resolved, which would correspond to 80 different secretions in the capped particle 18 (40 on the cavity-containing particle 10 and 40 on the capping particle). To avoid cross talk between detection molecules, such as detection antibodies across the different secretions on different surfaces, a related embodiment may also employ nELISA based approaches where capture and detection antibodies are co-localized on the cavity-containing particle 10 or capping particle 16 through a complementary' oligonucleotide bridge (see https: / / www.biorxiv.org / content / 10.1101 / 2023.04.17.535914v2, incorporated by reference). When a secretion is present it links these antibodies together, and a detection step is performed by introducing an oligonucleotide that hybridizes with the oligonucleotide bridge, labeling the detection antibody and opening and displacing the bridge oligonucleotides. If no secretion is present the detection antibody transports away from the interface and is not detected.

[0109] Single-cell secretome assay. Capped particle-based assays can also provide unique advantages for performing single-cell whole-secretome assays, when coupled with downstream mass spectrometry7. Single cells are loaded into cavity-containing particles 10. Cavity-containing particles 10 are then capped with capping particles 16. Capping particles 16 are modified with nonspecific binding elements for many or all secreted proteins. For example, lectins, such as concanavalin A or wheat germ agglutinin (WGA) may be used to bind sugar moieties on secreted proteins. Biotinylated proteins from turboID expressing cells may be bound to streptavidin bound to capping particles 16. Or sugar specific antibodies may be used to bind glycosylated proteins selectively7or non-selectively. Alternatively, a panel of2025-267-2multiple antibodies targeting different classes of proteins may be incorporated onto the capping particles 16. Cells are incubated in the capped particles 18 to accumulate secretions over a period of time (e.g., 3 hours, 6 hours, 1 day). Secretions are accumulated and bind to the capping particle 16 as seen in FIG. 7. The capping particles 16 may then be removed from the capped particles 18 using techniques described herein, such as vigorous pipetting, vortexing, addition of chemicals to break noncovalent interactions between capping particles 16 and cavity-containing particles 10, and the like. Free capping particles 16 containing the secretome or a subset of many secretions from a single cell may then be analyzed by introducing the capping particle 16 into a mass spectrometry system and obtaining a readout of peptide ion abundances. A computer accumulates and analyzes this dataset of peptide / ion abundances, performs algorithmic alignment to known proteins and generates a secretome for the single cell. Operation on the capping particle 16 follows similar protocols to operating on a small volume sample or a single-cell sample for mass spectrometry. Multiple single capping particles 16 can be analyzed in sequence to determine the secretomes of many single cells.

[0110] Multi-cell assays

[0111] Multi-cell fluorescent reporter assay. Capped particle-based assays can also be used for unique multi-cell assays, in particular ones that generate or quench fluorescent signals. One potential application of this is functional screening of antibodies using an ASC secreting antigen-specific antibodies and a reporter cell line expressing the antigen and engineered to produce a fluorescent signal upon functional interactions of an antibody with antigens on the cell surface (e.g., a T cell reporter that expresses a fluorescent protein like GFP upon activation of the nuclear factor of activated T cells, NF AT, pathway). In one embodiment, cavity -containing particles 10 are functionalized with antibodies specific to the ASC and reporter cell (e.g., anti-CD45, anti-CD138). In a preferred embodiment ASCs and reporter cells are stained or labeled with separate CellTracker dyes, calcein dyes, fluorescently-labeled antibodies against cell surface markers on the ASC and reporter cells or other fluorescent markers which have different colors or emission spectra which are distinguishable. The labeled ASCs and reporter cells are loaded into the cavity -containing particles 10, such that one ASC and one or more reporter cells are present. In some embodiments the cavity-containing and / or capping particles 16 include anti-IgG capture molecules to capture and localize secreted IgG. Antigen-specific IgG may also directly bind and accumulate on the reporter cell expressing antigen. In some embodiments, no anti-IgG capture molecules are incorporated on the capped particles 18. Cavilv-containing particles 102025-267-2are then capped with capping particles 16. Cells are incubated within the capped particle 18 for a period of time (e.g., 1 hour, 6 hours, 24 hours, 48 hours, 60 hours, or longer to accumulate sufficient fluorescence from the reporter cell line). During this incubation time ASCs are secreting antibodies which may or may not interact with antigens on the reporter cell line. For antibodies that interact and activate a signaling pathway in the reporter cell line, a fluorescent protein is produced and accumulated in the reporter cell line. Capped particles 18 are introduced into the flow7sorter (usually processes at rates of >100 events / sec) to enrich for capped particles 18 containing both an ASC and one or more activated, fluorescent reporter cells based on gates on corresponding fluorescent signals (ASC fluorescent label, reporter cell fluorescent label, reporter cell and genetically-encoded fluorescent protein signal). In some embodiments a fluorescently -labeled anti-IgG molecule is used to label secreted and bound IgG within the capped particle 18 and used as another gate to identify events with ASCs secreting IgG or to normalize other fluorescent reporter signals with respect to secreted IgG levels. Capped particles may be sorted into well plates and each well linked back to specific fluorescence signals for that event using index sorting features on flow sorting instruments. Individual nucleic acid amplification reactions may be performed in each w ell of the w ell plate containing a capped particle 18. These reactions may be barcoded with oligonucleotide sequences specific to the reaction volume, to enable linking back to the fluorescence signatures of the event. Capped particles 18 may also be bulk sorted and introduced into single-cell sequencing library preparation instruments or methods, such as from 10X Genomics, Parse, Scale or the like. Enriched ASCs mRNA, cDNA or DNA are then sequenced using barcoded single-cell sequencing to identify matched antibody heavy and light chains that induce a functional response in the reporter cell. These antibodies may have therapeutic potential to act as agonists inducing the activation of a signaling pathway. Use of a capped particle 18 vs. an open cavity-containing particle 10 (see https: / / www.biorxiv.org / content / 10.1101 / 2024.08.15.608174vl, which is incorporated by reference) for such an assay may enable longer-term culture without cell loss to enhance fluorescent reporter signals as both gene expression and translation has a delay time. In addition, larger quantities of antibody are expected to accumulate in the capped particle 18 without cross contamination of neighboring capped particles 18, leading to stronger stimulation of the reporter cells and a clearer signal. In some embodiments, multiple reporter cells including different fluorescent reporter proteins may be loaded into each capped particle 18 and a threshold on fluorescence intensity on both fluorescent protein channels is used2025-267-2during flow sorting to identify functional antibodies with higher confidence and lower false positive rates, especially if the reporter has some baseline level of signal in a fraction of cells. In some embodiments, as described below (and see FIG. 9), the capping particle 16 may also include a barcoded oligonucleotide capture moiety to capture released mRNA from the ASC for barcoded cDNA generation (e.g., the oligonucleotide capture moiety may include oligonucleotides with unique molecular identifier sequences, a poly T capture sequence (or heavy / light chain-specific capture sequences), and the same unique cell-specific barcode sequence for each capping particle). In this embodiment single-cell library preparation is conducted within the capped particles 18 through introducing a lysis buffer to induce the release of mRNA and capture on the capping particle, followed by reverse transcription for generation of barcoded cDNA. cDNA is then sequenced to recover the matched heavy and light chain of the antibody secreted by the ASC that led to activation of the reporter cell. In some embodiments the gene expression signature of the reporter cell is also analyzed following sequencing to obtain detailed insight on the effect of the antibody with the known sequence on the target-expressing cell's transcriptome or function.

[0112] Single-cell RNA-sequencing assay. In one embodiment a single-cell RNA-sequencing assay is performed using barcoded capped particles 18, following similar approaches as described in Drop-seq (https: / / pubmed.ncbi.nlm.nih.gov / 26000488 / ), or through techniques used in droplet microfluidic single-cell sequencing approaches (https: / / patents.google.com / patent / US20230087127Al / ), which are incorporated by reference. Advantages of using capped particles 18 include that no droplet microfluidic devices or instruments to operate them are needed to encapsulate single cells and barcoded beads into droplets in a sequential manner. FIG. 9 illustrates an illustrative workflow for performing single-cell sequencing with capped particles 18. Cells are loaded and compartmentalized simultaneously in bulk mixing processes as described herein. Single cells are first loaded into cavity -containing particles 10 through simple mixing, pipetting or seeding steps. For the capped-particle workflow, the sequential steps of loading cells, removing unbound cells, and adding barcoded capping particles 16 to form compartments leads to less contamination of capping particles 16 with free oligonucleotides that may bind to capping particles 16. Optionally, the cavity-containing particles 10 having binding moieties in the cavity 12 to selectively bind specific populations of cells, e.g., using antibodies specific to cell surface proteins, extracellular matrix (ECM) molecules that allow integrin binding, or peptide-major compatibility complex (pMHC) for antigen-specific T2025-267-2cells. In some embodiments cavity-containing particles 10 include one or more targetexpressing cells with binding moieties for the cells. In other embodiments, a first cell type and a second cell type are co-loaded in cavity-containing particles 10. Following loading at limiting dilutions to ensure mostly single cells are loaded in cavity-containing particles 10, cavity -containing particles 10 are capped with barcoded capping particles 16 as described herein. The plurality of barcoded capping particles 16 each having a unique oligonucleotide barcode and nucleic acid capture moiety (e.g., poly-T capture sequences, target gene-specific capture sequences). A plurality of uniquely barcoded capping particles 16 may be manufactured as described herein and known in the art e.g., using split-pool synthesis. In some embodiments the capping particles 16 also include unique molecular indices (UMIs) -randomized oligonucleotide sequences - as part of the nucleic acid capture moiety. Cells may be optionally cultured or incubated for a period of time in capped particles 18. During this time, secretions from the loaded single cells may be captured in respective cavity -containing particles 10 and / or capping particles 16 through capture moieties, such as antibodies located therein. Captured secretions may be labeled with fluorescent and / or oligonucleotide labeled detection molecules. Optionally, cell-containing capped particles 18 can be sorted based on the presence of cells and / or secretions to create downstream enriched populations of capped particles 18 containing cells.

[0113] Cells within capped particles 18 are then permeabilized or lysed by mixing the capped particles 18 with lysis reagents to release nucleic acids, such as mRNA. mRNA released from each single cell is captured on the respective capping particle 16 associated with each single cell. Hydrogel materials of the cavity -containing particles 10 and / or capping particle 16 are preferably permeable to lysis reagents and not released nucleic acids, or transport of released nucleic acids is limited by the capping process as described herein. In some embodiments, the capping particle 16 is removed from the cavity -containing particle 10 using agitation / mixing or the capping particle 16 is solubilized or degraded to release mRNA hybridized to oligonucleotide barcodes into solution.

[0114] Reverse transcriptase is next added to create cDNA of the released mRNA which is pooled, amplified, and sequenced. The cDNA can be amplified through polymerase chain reaction or other nucleic acid amplification processes to create cDNA libraries. cDNA is then sequenced, e.g., using next-generation sequencing. The capping particle barcode associated reads are then used to link cDNA / mRNA (and optionally secretions, through barcodes on the secretion detection antibody) to each single cell. In related embodiments, a diverse set of2025-267-2cavity -containing particles 10 also may include unique particle-type oligonucleotide barcodes for each specific particle and / or particle type. These particle-type barcodes can be released on a stimulus (light, chemical, enzyme) to hybridize to the oligonucleotide barcodes on the barcoded capping barcodes. Preferably this stimulus occurs during the lysis step of the cells contained within the capped particles 18. This enables a pool of different reactive moieties (e.g., pMHC molecules, antigens, ECMs, peptide drugs, nucleic acids, antibodies) on the diverse set of cavity-containing particles 10 to be separately barcoded. Linkage of gene expression with this particle-type barcode and cell-specific barcode enables characterizing the effect of the reactive moiety on single-cell mRNA expression, e.g., for understanding specific activation or modulation of gene expression at scale. In one embodiment the particle-type barcode also includes a poly-A region or unique complementary sequence region to oligonucleotide barcodes on the capping particle 16 to enable direct hybridization upon release. Single-cell sequencing workflows using capped particles 18 as described herein may be applied to various applications know n to those skilled in the art, and described herein, such as for matched heavy / light chain antibody sequencing, matched alpha / beta chain TCR sequencing, Perturb-seq CRISPR knockout library' assays, SEC-seq, characterization of cell-to-cell interactions upon co-loading two cells, and other related applications.

[0115] Molecular assays in capped particles

[0116] In vitro transcription and translation (IVTT) to link proteins and oligonucleotide barcodes in capped particles. In various embodiments, capped particle assays may leverage in vitro transcription and translation to link oligonucleotide sequence and protein sequence, which are both localized in capped particles 18. A plurality of different protein-coding oligonucleotide sequences can be incorporated into a plurality of cavitycontaining particles 10 and / or capping particles 16, where each particle has one or more types of protein-coding sequences. The cavity-containing particles 10 and / or capping particles 16 also include a binding agent to bind a produced protein. The produced protein may incorporate a specific tag, such as His tag, FLAG tag, strep-tag, etc. Upon in vitro transcription and translation, the produced protein is localized to the capped particle 18 that encodes its production with the unique protein-coding oligonucotide. Various assays may then be performed with the capped particles 18 as described herein to characterize the encoded proteins and their function. Following the assay and sorting / selection of a subset of capped particles 18 based on the assay results, the oligonucleotides on the capped particles 18 may be sequenced to identify the associated protein or proteins therein. Capped particles 182025-267-2enable molecular assay reagents, such as enzymes, nucleotides, and detection molecules, to be compartmentalized or transported within the cavity 12 of the capped particles 18.Following capping of cavity-containing particles 10 with capping particles 16, this compartmentalization prevents cross-contamination and enables parallel reactions, facilitating the multiplexing of samples and increasing production of many protein-functionalized capped-particles with linked protein-encoding oligonucleotides. The cavity -containing particles 10 can be functionalized with DNA or RNA oligonucleotides, through covalent linkages, hybridization or other high-specificity affinity-based interactions, including biotinstreptavidin chemistry. These barcodes allow for unique identification and tracking of individual particles, enabling high-resolution data collection across complex assays. In vitro transcription and translation assays, which are commonly used in synthetic biology, protein engineering, and drug development, cavity-containing particles 10 can be barcoded with oligonucleotides and / or fluorophores and functionalized with capture antibodies to isolate proteins being produced, such as secreted, intracellular, or membrane-bound proteins.Capping particles 16 may also be barcoded using oligonucleotides or fluorophores. ensuring compatibility with various experimental setups. Downstream processes can include DNA sequencing, uncapping the particles, releasing their contents, and performing functional assays with cells, such as viability7, proliferation, or metabolic activity tests.

[0117] In one embodiment using the capped particle system, a large-scale protein-protein, protein-DNA, or protein-RNA interaction assay is performed using in vitro transcription and translation. In this embodiment, a plurality of cavity -containing particles 10 are functionalized w ith a li brary of protein-encoding oligonucleotides encoding binding proteins, protein mutants, or Al-designed protein binders. A plurality of capping particles 16 are functionalized with a second library of protein-encoding oligonucleotides encoding a second set of target proteins, protein mutants, or Al-designed protein binders. One of the first or second library of protein-encoding oligonucleotides also includes oligonucleotides encoding for an affinity tag to enable capture within the capped particle system (e.g., on the cavitycontaining particle 10 or capping particle 16). The second of the first or second library of protein-encoding oligonucleotides, not functionalized with affinity tags, also includes encoding for a labeling moiety (e g., fluorescent protein, affinity tag for binding of a detection fluorophore). The cavity-containing particle 10 or capping particles 16 also include a protein capture moiety to capture the proteins from the first library in their respective capped particles 18 through the affinity tag (e.g., an anti-His, anti-FLAG) antibody that is2025-267-2incorporated. In vitro transcription and translation mix is added and cavity -containing particles 10 and capping particles 16 are mixed to form capped particles 18. Proteins from the first library are produced and bind selectively to the capped particles 18 with their associated protein-encoding oligonucleotides. Proteins from the second library are produced and may bind to the protein within the capped particle 18 from the first library. Optionally, a fluorescently-labeled detection antibody is introduced to bind to the proteins from the second library. Capped particles 18 may then be analyzed and sorted in a flow sorter to identify events associated with a binding pair of proteins. Capped particles 18 with a fluorescence intensify above or below a threshold or within a particular gate may be sorted in a tube or vessel or a single capped particle 18 may be sorted into individual wells of a well plate. The protein-encoding oligonucleotide sequences from both the cavity-containing and capping particle 16 may be analyzed to identify binding partners, which may include a nucleic acid amplification step, followed by a sequencing step. Analysis of events in an individual well enables linking of the two protein-encoding oligonucleotide sequences and associated binding proteins. In a related embodiment, one of the first or the second library includes a single type of biomolecule (protein, DNA, RNA, lipid, or sugar molecule) bound to the cavity -containing or capping particles 16, and the assay is used to characterize the second library of proteins, encoded by oligonucleotides, binding to the first biomolecule, following IVTT.

[0118] In a similar embodiment using the capped particle system, a large-scale protein-DNA or protein-RNA interaction assay is performed using in vitro transcription and translation. In this embodiment, a plurality of cavity-containing particles 10 are functionalized with a 1 ibrary of protein-encoding oligonucleotides encoding DNA- or RNA-binding proteins, protein mutants, Cas proteins, or Al-designed nucleic acid binders. A plurality of capping particles 16 are functionalized with a second library of DNA or RNA sequences that serve as target nucleic acids for interaction screening. Alternatively, cavitycontaining particles 10 are functionalized with the second library and capping particles 16 with the first library. The library of protein-encoding oligonucleotides also includes oligonucleotides encoding a labeling moiety (e.g.. fluorescent protein, affinity tag). In vitro transcription and translation mix is added, and cavity -containing particles 10 and capping particles 16 are mixed to form capped particles 18. Proteins from the first library are produced and may bind selectively within the capped particles 18 to associated oligonucleotides of specific sequences. The nucleic acid targets on the second library may bind to the protein within the capped particle, facilitating detection of protein-DNA or2025-267-2protein-RNA interactions. Optionally, a fluorescently-labeled detection probe (e.g., anti-tag antibody) is introduced to label the interaction complex. Capped particles 18 may then be analyzed and sorted in a flow sorter to identify events associated with a binding interaction between a protein and a DNA or RNA sequence. Capped particles 18 with a fluorescence intensify above or below a threshold or within a particular gate may be sorted into a tube or vessel, or single capped particles 18 may be sorted into individual wells of a well plate. The oligonucleotide sequences from both the cavity-containing and capping particles 16 may be analyzed to identify binding partners, which may include a nucleic acid amplification step followed by a sequencing step. Analysis of events in an individual well enables linking of the protein-encoding oligonucleotide sequences and the associated nucleic acid sequences, identifying specific protein-DNA or protein-RNA interactions.

[0119] Enzyme activity assay. In one embodiment, the capped particle system may be utilized to perform enzyme activity assays. Cavity-containing particles 10 are functionalized with enz mes or with substrates specific to the enzyme of interest. These enzymes or substrates may be immobilized onto the surfaces of the cavity-containing particle 10 through covalent bonding, affinity interactions, or physical adsorption. Enzymes are introduced into the system, either as purified preparations, lysates, or secreted components from cells (cells also may be contained within the capped particle), and become compartmentalized within the cavity-containing particles 10. If substrates are not immobilized on the cavity-containing particles 10, they may also be introduced in solution. The cavity-containing particles 10 are subsequently capped with capping particles 16 to form sealed capped particles 18, creating isolated microenvironments for enzyme-substrate interactions.

[0120] In some embodiments, enzyme activity is detected by monitoring the conversion of the substrate to a product within the capped particles 18. For example, substrates may be designed to generate a measurable signal upon enzymatic conversion, such as a colorimetric, fluorescent, or chemiluminescent signal. See FIG. 14. Fluorogenic substrates, for instance, may be cleaved by the enzyme to produce a fluorescent product that accumulates within the capped particle 18. The signal intensify is directly proportional to the enzymatic activity within each particle.

[0121] In certain embodiments, the capped particle system may allow multiplexed enzy me activity' assays by using cavity-containing particles 10 functionalized with different substrates or introduction of different fluorogenic, chromogenic, or related substrates that are optically distinguishable following enzymatic turnover. Each substrate may be uniquely barcoded with2025-267-2a fluorescent label or oligonucleotide, enabling simultaneous detection of multiple enzy me activities in a single assay. This capability is particularly useful for high-throughput screening of enzyme inhibitors, activators, or other modulators.

[0122] After incubation, capped particles 18 may be analyzed using flow cytometry', fluorescence microscopy, or other suitable detection methods to measure enzy matic activity7at the single-particle level. In addition, capped particles 18 with a signal above a threshold or within a particular gate may be sorted to recover barcode information or genetically -encoded information about the specific enzyme, for an enzyme produced and released by an encapsulated cell.

[0123] Binding assay. In one embodiment, the capped particle system may be utilized for performing binding assays to assess the interaction between biomolecules, such as proteinprotein, protein-ligand, antigen-antibody, or nucleic acid-protein interactions. Cavitycontaining particles 10 are functionalized with one binding partner (e.g., a protein, nucleic acid, or small molecule) immobilized on their surface through covalent coupling, affinity interactions, or physical adsorption. The second binding partner, such as a target molecule in a sample or solution, is introduced into the system and becomes localized within the cavitycontaining particles 10 if there is a binding event. Following the potential binding event, the cavity -containing particles 10 are interacted with capping particles 16 to form capped particles 18, creating isolated compartments with reduced or halted transport out of the capped particle 18 cavity 12.

[0124] In some embodiments, binding events may be detected by labeling one of the binding partners with a detectable tag, such as a fluorescent dye, chemiluminescent label, an enzyme, or radiolabel. For example, fluorescently labeled ligands or antibodies may bind to their target immobilized within the capped particle, generating a signal proportional to the extent of binding. In a preferred embodiment one of the binding partners is an enzyme, such as horseradish peroxidase, galactosidase, carboxylesterase, protease, or the like, which converts a precursor molecule into an optically visible detector molecule, such as a fluorogenic substrate, a polymer, or a precipitate (e.g., FIG. 15), or a chromogenic substrate.

[0125] In certain embodiments, the capped particle system may support multiplexed binding assays. Different populations of cavity-containing particles 10 can be functionalized with distinct binding partners and uniquely barcoded with fluorescent labels oroligonucleotides.2025-267-2

[0126] After incubation, capped particles 18 may be analyzed using flow cytometry, fluorescence microscopy, or other suitable detection methods to measure enzymatic activity at the single-particle level. In addition, capped particles 18 with a signal above a threshold or within a particular gate may be sorted to recover barcode information or genetically -encoded information about the specific enz me, for an enzyme produced and released by an encapsulated cell.

[0127] Nucleic acid amplification assay. Capped particle systems are also suitable to perform nucleic acid amplification assays, and in particular digital nucleic acid amplification assays. In these assays target oligonucleotide molecules from a sample are compartmentalized within capped particles 18 and a reaction is performed to amplify the target oligonucleotides and retain the products of amplification within the capped particles 18. The retained amplicons are stained using nucleic acid labels or dyes, such as complementary oligonucleotides containing a fluorophore or nucleic acid binding dyes such as Acridine Orange, EvaGreen, SYBR Green, SYTO dyes, TOTO dyes or the like. The capped particles 18 may then be analyzed using a flow cytometer to detect the intensity of the capped particle 18 as a measure of the amount of localized amplified nucleic acids. In some embodiments the capped particles 18 are sorted based on a threshold of fluorescence intensity and / or other gating on the capped particles 18 in scatter and intensity7. Capped particles 18 with an intensity above a threshold related to the presence of amplified DNA may be sorted and the amplified DNA subsequently sequenced. The sequence of the DNA may be used to identify an organism, disease, or cell type associated with the target oligonucleotide. In some embodiments a plurality of capped particles 18 are formed and a subset of the plurality7of capped particles 18 have a single target oligonucleotide localized within a capped particle 18 which is amplified to perform a digital nucleic acid amplification assay. A digital assay enables counting of target oligonucleotides. In some embodiments, cavity-containing particles 10 and / or capping particles 16 may include oligonucleotide capture probes that are complementary7to one or more target oligonucleotides. This enables the pull down and localization of target oligonucleotides from a large sample prior to amplification and detection, improving the sensitivity of the assay. A unique nucleic acid and / or fluorescent barcode may be associated with each oligonucleotide capture probe for a particular target oligonucleotide sequence. The nucleic acid amplification may be performed using reagents and amplification techniques known in the art, including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and rolling circle amplification (RCA), and2025-267-2their associated reagents. In some embodiments the amplicon is bound within the capped particle 18 through hybridization reactions with oligonucleotide capture probes. In other embodiments, the amplicons of a nucleic acid amplification reaction is sized to remain localized within the capped particle 18 because of the sealed nature of the capped particle, such as using RCA or LAMP, which generates much larger amplicons than the primers used. Oligonucleotide capture probes on the cavity-containing particle 10 and / or capping particle 16 may also include primers configured to hybridize and create complementary strands to the target oligonucleotide. Nucleic acid amplification may be performed using thermal cycling, increasing temperature, or introduction of enzymes and mixes suited for amplification as known in the art. Following amplification, the resulting amplicons may either remain bound to the capped particles 18 or be captured, particularly when the amplicon is large enough to be retained. Nucleic acids may be stained using intercalating dyes, such as SYBR Green or EvaGreen and analyzed by microscopy, flow cytometry, or imaging, where counting of amplified target oligonucleotides within capped particles 18 above a threshold intensity' compared to the total number of capped particles 18 may provide accurate digital counting and concentration determination of target oligonucleotides in a sample.

[0128] Immunoassay. In various embodiments, the capped particle system may be used to perform immunoassays, including direct, indirect, sandwich, and competitive ELISAs. The system enables compartmentalization of reagents and supports high-throughput and multiplexed detection with enhanced sensitivity and specificity. In one embodiment, the capped particle system is configured for a sandwich ELISA, wherein an affinity agent (e.g., antibody, aptamer, de novo designed binder protein, molecularly imprinted polymer) is immobilized onto the surface of the cavity -containing particle 10 and / or capping particle 16. The cavity-containing particle 10 and / or capping particle 16 is then exposed to a sample to bind an analyte. A detection molecule conjugated (e.g., antibody, aptamer, de novo designed binder protein, molecularly imprinted polymer) to a signal generating molecule (chromophore, fluorophore or enzy me) is then exposed to the bound analyte. The cavitycontaining particle 10 and capping particle 16 are then mixed to form capped particles 18. A signal proportional to the amount of analyte bound is generated through the addition of a signal generating reagent, such as a Anorogenic enzyme substrate, polymer precursor, or other enz me substrate. The generated signal is localized within the compartment or cavity' 12 of the capped particle 18 and is prevented from transporting out. These signals can be fluorescent (e.g.. resorufin, rhodamine, fiuorescein), chemiluminescent (e.g., luminol).2025-267-2colorimetric (e.g., precipitates such as the products of NBT-BCIP with alkaline phosphatase, large polymer chains), or detectable through non-imaging means, like long DNA amplicons. A plurality of capped particles 18 are then analyzed using flow cytometry, microscopy, or analysis techniques applied to analyze individual cells or particles. Capped particle events with a fluorescence or scatter signal above a threshold level are counted and indicate the presence of an analyte, the level of the fluorescence or scatter signal may indicate the concentration of the analyte present in the sample. In related embodiments to conduct a digital ELISA, a fraction (0.1% to 30% or 0.01% to 50%) of the cavity-containing particles 10 and / or capping particles 16 bind a single analyte molecule. In this embodiment the counting of capped particles 18 with signal above the threshold can be used to count the number of analyte molecules in the sample or to create a count of analyte molecules correlated to the concentration of analyte in the sample.

[0129] In another embodiment, the capped particle system can be employed to perform a direct ELISA. In this embodiment, the system includes cavity -containing particles 10, capping particles 16, or a combination of both, functionalized with moieties to encourage non-specific or specific analyte binding to the cavity 12 or capped particle 18 surface. The functionalized particles may then be exposed to a sample (e.g., serum, whole blood, other biological fluid) under conditions that promote the binding of analytes in the sample to the particles. Following this, the particles may then be incubated with a detection molecule (e.g., antibody, aptamer, de novo designed binder protein, molecularly imprinted polymer) conjugated to a signal-generating molecule (e.g., chromophore, fluorophore, enzyme), wherein the detection molecule specifically binds to the captured analyte. In some embodiments, the cavity-containing particles 10 and capping particles 16 are combined to form capped particles 18, wherein the compartmentalization of reagents within the capped particle 18 mitigates cross-reactivity and enhances specificity. A detectable signal may be generated upon exposure to a signal-generating reagent, such as a fluorogenic substrate, enzy me substrate, or polymer precursor, wherein the signal is confined within the capped particle 18 and inhibited from transporting into the surrounding medium. The generated signal may be fluorescent (e.g., resorufin, rhodamine, fluorescein), chemiluminescent (e.g., luminol), colorimetric (e.g., precipitates such as the products of NBT-BCIP with alkaline phosphatase, large polymer chains), or detectable through non-imaging means, such as long DNA amplicons. A plurality of capped particles 18 may be analyzed using flow cytometry, microscopy, or other analytical techniques applied to analyze individual cells or particles.2025-267-2Capped particle events with a fluorescence or scatter signal above a threshold level are counted and indicate the presence of an analyte, and the level of the fluorescence or scatter signal may indicate the concentration of the analyte present in the sample. In related embodiments to conduct a digital ELISA, a fraction (0.1%, 30%, 0.01%, 50%) of the cavitycontaining particles 10 and / or capping particles 16 bind a single analyte molecule. In this embodiment, the counting of capped particles 18 with a signal above the threshold can be used to determine the number of analyte molecules in the sample or to create a count of analyte molecules correlated to the concentration of analyte in the sample.

[0130] In another embodiment, the capped particle system is employed to perform an indirect ELISA. In this embodiment, the system includes cavity-containing particles 10, capping particles 16. or a combination of both functionalized with one or more antigens configured to selectively bind a primary antibody as an analyte. The functionalized cavitycontaining particles 10 may then be exposed to a sample (e.g., serum, whole blood, other biofluids) under conditions that promote the specific binding of the primary antibody to the antigen presented on the cavity 12 of the cavity-containing particle 10 and / or capping particle 16. Following this, the particles may then be incubated with a secondary antibody conjugated to a signal-generating molecule (e.g., chromophore, fluorophore, enzyme), wherein the secondary anti-species antibody binds to the primary antibody. In some embodiments, the cavity-containing particles 10 and capping particles 16 are combined to form capped particles 18. wherein the compartmentalization of reagents within the capped particle 18 mitigates cross-reactivity and enhances specificity'. A detectable signal may be generated upon exposure to a signal-generating reagent, such as a fluorogenic substrate, enzyme substrate, or polymer precursor, wherein the signal is confined within the capped particle 18 and prevented from diffusing into the surrounding medium. The generated signal may be fluorescent (i.e. resorufin, rhodamine, fluorescein), chemiluminescent (e.g., luminol), colorimetric (e.g., precipitates such as the products of NBT-BCIP with alkaline phosphatase, large polymer chains), or detectable through non-imaging means, like long DNA amplicons. A plurality of capped particles 18 may be analyzed using flow cytometry, microscopy, or other analytical techniques applied to analyze individual cells or particles. Capped particle events with a fluorescence or scatter signal above a threshold level are counted and indicate the presence of an analyte, the level of the fluorescence or scatter signal may indicate the concentration of the analyte present in the sample. In related embodiments to conduct a digital ELISA, a fraction (0.1% to 30% or 0.01% to 50%) of the cavity -containing particles 10 and / or capping particles2025-267-216 bind a single analyte molecule. In this embodiment the counting of capped particles 18 with signal about the threshold can be used to count the number of analyte molecules in the sample or to create a count of analyte molecules correlated to the concentration of analyte in the sample.

[0131] In a further embodiment, the capped particle sy stem may support competitive ELISA for detecting small molecules, such as hormones or metabolites, where sandwiching with two antibodies is not feasible. Cavity -containing particles 10, capping particles 16 or a combination of the two may be functionalized with affinity agents (e.g., antibody, aptamer, de novo designed binder protein, molecularly imprinted polymer) for a specific small target. Following this, a sample (e.g., serum, whole blood, sweat, saliva, water for quality monitoring, food samples) is added, followed by a solution containing a look-alike antigen conjugated to signal-generating molecule (e.g., chromophore, fluorophore, enzyme) that competes with the sample antigen for antibody binding sites. A signal generating reagent is then introduced, such as a Anorogenic enzyme substrate, polymer precursor, or other enzyme substrate. The generated signal is localized within the compartment or cavity 12 of the capped particle 18 and is prevented from transporting out. These signals can be Auorescent (e.g., resorufm, rhodamine, Auorescein), chemiluminescent (e.g., luminol), colorimetric (e.g., precipitates such as the products of NBT-BCIP with alkaline phosphatase, large polymer chains), or detectable through non-imaging means, like long DNA amplicons. The signal generated is inversely proportional to the amount of analyte present in the sample. This approach is particularly useful for applications requiring high sensitivity in the detection of small molecules. A plurality of capped particles 18 are then analyzed using Aow cytometry, microscopy, or analysis techniques applied to analyze individual cells or particles. Capped particle events with a Auorescence or scatter signal above a threshold level are counted and indicate the absence of an analyte, the level of the Auorescence or scatter signal may indicate the concentration of the analyte vs analyte-signal-generation molecule complex present in the sample.

[0132] In additional embodiments, the capped particle system may enable multiplexing by functionalizing different particle populations with distinct barcodes. This facilitates the simultaneous detection of multiple targets from multiple sample inputs within a single assay.See FIG. 11. In this embodiment both the cavity -containing particle 10 and the capping particle 16 can be barcoded with a Auorophore, chromophore, or nucleic acid oligo. In a preferred embodiment, the barcode for the cavity -containing particle 10 will correspond to2025-267-2the antibody or antigen attached to the cavity 12 and the barcode for the capping particle 16 will correspond to a specific sample. In this embodiment, a cavity-containing particle barcode will indicate what analyte (e.g., polymer, protein, nucleic acid, small molecule) or affinity agent (e.g., antibody, aptamer, de novo designed binder protein, molecularly imprinted polymer) is bound to that specific cavity-containing particle 10. Both the analyte / affinity agent and barcode may be functionalized on the surface of the cavity -containing particle 10 at the same time. A mixture of multiple types of cavity-containing particles 10 with different analyte / affinity’ agents and barcodes may be mixed and split to perform multiplexed assays. One set of mixed cavity-containing particles 10 can be introduced to a sample (e.g., serum, whole blood, sweat, saliva, water for quality monitoring, food) and a capping particle 16 with a specific known barcode (e.g., fluorophore. chromophore, nucleic acid oligo) will be added as well that is specific to that sample. Then, capped particles 18 may be formed and extraneous fluid can be removed. In a preferred embodiment, capped particles 18 from multiple samples (including separately barcoded capping particles 16) can then be combined and a signal generation reagent may be introduced, such as a fluorogenic enzyme substrate, polymer precursor, or other enzyme substrate. In alternative embodiments, the signal generation reagent can be introduced before mixing capped particles 18 from multiple samples, then capped particles 18 from multiple samples may be mixed. The generated signal may be fluorescent (i.e. resorufin, rhodamine, fluorescein), chemiluminescent (e.g., luminol), colorimetric (e.g., precipitates such as the products of NBT-BCIP with alkaline phosphatase, large polymer chains), or detectable through non-imaging means, like long DNA amplicons, and will be confined within the capped particle 18 and inhibited from transporting into the surrounding medium. A plurality of capped particles 18 may be analyzed using flow cytometry, microscopy, or other analytical techniques applied to analyze individual cells or particles. Capped particle events with a fluorescence or scatter signal above a threshold level are counted and indicate the presence of an analyte, the level of the fluorescence or scatter signal may indicate the concentration of the analyte present in the sample. Fluorescence in one or more other channels indicates the presence of a particular capping particle barcode and a threshold or gate on the one or more other fluorescence channels is used to identify the particular sample and assign the data on the presence and level of analyte to a particular sample based on the capping particle barcode. In related embodiments to conduct a digital ELISA, a fraction (0.1% to 30% or 0.01% to 50%) of the cavity-containing particles 10 bind a single analyte molecule. In this embodiment the counting of capped particles 18 with a2025-267-2specific sample barcode signature and signal above the threshold can be used to count the number of analyte molecules in a specific barcoded sample or to create a count of analyte molecules correlated to the concentration of analyte in the specific barcoded sample.

[0133] Image-activated cell analysis and sorting of capped particles. In some embodiments capped particles 18 containing cells and / or products of reactions are analyzed or sorted using imaging cytometers or image-activated cell sorters. Images of capped particles 18 may provide unique information about reactions or cellular processes useful for an assay. For example, localization of a signal from a secretion or reaction to the capping particle 16, cavity-containing particle 10, or inner compartment can provide unique information for multiplexed assays or assays where multiple different secretions or reactions are spatially captured and localized to the capping particle, cavity-containing particle, and / or inner compartment. Having spatial localization enables further spatial multiplexing that may be challenging without the imaging data. Imaging data may also be used to ensure proper capping of a capped particle 18. Imaging data may also be used determine the signal accumulated on the surface of or within particular sub-populations of cells within the inner compartment of the capped particle, such as target expressing cells for antibody discovery, or reporter cells producing a fluorescent reporter in response to another cell, its secretions, or other reactions occurring within the inner compartment. Imaging can also be used to barcode reactions to a higher number of unique spatial and / or spectral barcodes. Spatial barcodes may include capping particles 16 and / or cavity-containing particles 10 of different sizes. Spectral barcodes may include one or more fluorophores with unique spectral characteristics. Spectral barcodes may be different on the capping particle 16 and cavity-containing particle, and spatially localized using imaging to enhance the total number of barcodes in a geometric fashion. In addition, spectral barcodes may be combined with barcodes related to capping particle size or other features (e.g., index of refraction), creating 1000s to millions of unique spatial and spectral barcodes.

[0134] Image analysis operations can then be performed on images of capped particles 18 to identify a particular reaction result, cell result, or barcode determination based on image analysis metrics. Image analysis operations to create image analysis metrics may include spatial correlations between different fluorescence and scatter / brightfield channels. Image analysis operations may include a threshold on the level of correlation between signals in a particular spatial region of the capped particle 18 image. Image analysis operations may also include identifying regions that are not spatially correlated. A different metric identifying2025-267-2signal in one channel that is not present in another channel may be used to identify the presence of staining on more than one cell or surface of the capped particle 18. For example, this may include the presence of antibody signal on a target cell but not an antibody-secreting cell. Image analysis operations may also include a masking operation, where a region of interest is masked based on signal in one channel and signal in a second channel is identified in the region of interest or outside of the region of interest. A threshold metric may be identified based on the signal within the region of interest or outside the region of interest. The masking operation can be used to identify one or more components of the capped particle 18 including the capping particle 16, the cavity -containing particle 10, one or more cell types within the inner compartment, and / or the inner compartment of the capped particle 18 based on a label or stain unique to each of the components. The label or stain may include a fluorescent dye. For example, a region of interest defining the capping particle location of the capped particle 18 image may be determined using a threshold on an intensity in a fluorescent channel corresponding to a fluorophore embedded in the capping particle 16. Then a binding amount of a test antibody to antigen on a capping particle 16 is determined by the intensity of an anti-IgG antibody conjugated with a second fluorophore that labels the test antibody and is co-located on the capping particle 16 within the region of interest. See e.g., FIG. 16. An intensify of the second fluorophore in the region of interest above a threshold may be used for determining if a test antibody with affinity to antigen is present and the capped particle event should be sorted. A sorting operation based on a threshold of one or more image analysis metrics may be performed to sort one or more capped particles 18 based on the combination of these spatial features. Sorting may be performed using commercial instruments such as the FACSDiscover™ S8, or the Nanocellect VERLO instrument, or other image-activated cell sorter instruments.

[0135] Analysis of capped particles with single-cell sequencing assays and instruments. In some embodiments capped particles 18 having cells therein are introduced into instruments or assay methods designed for single-cell analysis, such as single-cell sequencing library preparation, flow cytometers, large particle analyzers, or the like. In one embodiment, capped particles 18, a subset of which each includes a single cell, and optional oligonucleotide barcodes are sized so that they are compatible to be introduced into a singlecell sequencing instrument based on formation of separate compartments surrounding the capped particles 18, each containing a unique barcode sequence. Compatible instruments include the 10X Genomics Chromium instrument and GEM or GEM-X chips. Capped2025-267-2particle 18 dimensions are such that they are smaller than the minimum microchannel dimensions of the single-cell sequencing chips (e.g., 50 micrometers in the dimensions along one axis of the capped particle). In related embodiments capped particles 18 are prepared for single-cell sequencing based on a split and pool process for barcoding of the capped particle 18 or cells therein, such as provided commercially by Parse Biosciences or Scale Biosciences. In related embodiments each of a subset of capped particles 18 includes two different cell types (cell type 1 and cell type 2) interacting and capped particles 18 are introduced into a single-cell sequencing instrument or method to evaluate the combined transcriptome changes due to cell-cell communication and interactions within the capped particles 18. Separate capped particles 18 including cell type 1 and cell type 2 are also introduced into the instrument or method and gene expression of the combined system is deconvolved using the separate gene expression of cell type 1 and gene expression of cell type 2.

[0136] Detailed Examples.

[0137] Example: Screening hybridoma to isolate clones with antigen-specific antibodies. In this example, hybridoma that secrete antibodies that bind to an antigen are sorted. Hybridoma are first loaded into cavity-containing particles 10 through antibodies on the cavity -containing particles 10 that bind to CD45 on the hybridoma cell surface. A capping particle 16 containing the antigen is then used to form capped particles 18 containing hybridoma therein and bind secreted IgG from the encapsulated hybridoma. The IgG bound to antigen is stained with an anti-IgG antibody with afluorophore and events with secretion are sorted by FACS. A 40 pL sample (500,000 particles) of 65 pm outer diameter cavitycontaining particles 10 made from 17.5% w / v 5k molecular weight PEG and a 20% w / v gelatin coating within the cavity 12 is functionalized with 10 mM biotin-NHS for 12 hours. The cavity-containing particles 10 are then functionalized with 300 pg / mL streptavidin for 30 minutes, followed by a 50 pg / mL solution of biotinylated anti-CD45 antibody (anti-mouse) for 1 hour. In parallel, a 40 pL sample (2,000,000 particles) of 40 pm diameter capping particles 16 made from 7.5% w / v PEG and 1.5% w / v gelatin are similarly functionalized with 10 mM biotin-NHS for 12 hours, followed by 300 pg / mL of streptavidin for 30 minutes. The capping particles 16 are then functionalized with 25 pg / mL biotinylated antigen, in this case, biotinylated hen egg lysozyme (HEL), for 45 minutes at room temperature. All reactions are conducted at room temperature unless otherwise noted. 300,000 HyHEL5 hybridomas are loaded into the functionalized cavity -containing particles 10 via mixing and agitation in a2025-267-2well of a 12-well plate for 90 minutes. Every 30 minutes the cells and cavity-containing particles 10 are manually mixed via pipetting to promote cell loading into the cavitycontaining particles 10. Loose cells are removed from the mixture by passing the crude suspension through a 37 pm filter. Following filtration, cell-loaded cavity-containing particles 10 are mixed with the capping particles 16 in a 0.5 mL Eppendorf tube, which is filled to a final volume of 400 pL using PBS containing 0.05% pluronic F-127 and 0.5% bovine serum albumin (BSA). The tube is then centrifuged at 350 RCF for 1 minute and 30 seconds in order to form capped particles 18. The capped particles 18 are transferred to a 6 well plate and incubated with IMDM media containing 10% fetal bovine serum and 1% antibiotic-antimycotic (Thermo Fisher 15240062) for 30 minutes for secreted antibodies to accumulate within the capped particle 18 and bind to the capping particle 16. The particles are next incubated with an initial 100 pL solution of 40 pg / mL anti-mouse IgG DyLight 650 (Abeam 98715) antibody and 5 pM calcein AM (cell stain) for 30 minutes in the dark. The cell-loaded capped particles 18 can then be analyzed via fluorescence microscopy and / or flow cytometry.

[0138] Sorting of hybridoma based on antigen-specific IgG secretion. Cell-loaded capped particles 18 that have been previously stained with a fluorescent anti-IgG antibody and cellular stain can be analyzed and sorted using flow cytometry and FACS. Single control samples of capping particles 16 stained with anti-mouse IgG DyLight 650 antibody, calcein AM-stained HyHEL5 cells, and unstained cavity -containing particles 10 are used to perform compensation and establish flow cytometry gates. Plotting forward scatter width (FSC-W) signal against side scatter height (SSC-H) signal is used to identify free capping particles 16, free cavity-containing particles 10, and capped particles 18. See FIG. 6. Capped particles 18 can be distinguished from capping particles 16 and cavity-containing particles 10 by their elevated FSC-W signal relative to the single particle control samples. After gating for capped particles 18, this population is plotted using FSC-W and FITC area (FITC-A) signal. This yields two subpopulations; one with an elevated FITC-A signal (cell-loaded) and one with a relatively lower FITC-A signal (capped particle 18 without a cell). The top population with high FITC-A signal is gated and a histogram of its DyLight 650 area (DL-A) is plotted. The top 1%, 5%, 10%, etc. DL-A population is gated and used to sort the top producing antigenspecific clones from the secretion assay.

[0139] Example: Hybridoma growth assays. In this example, single hybridoma clones are loaded and grown within capped particles 18 and the colonies with the highest growth are2025-267-2sorted by FACS. First, 40 pL (500,000 particles) of 65 pm outer diameter cavity-containing particles 10 made from 17.5% w / v 5k molecular weight PEG and a 20% w / v gelatin coating within the cavity 12 are functionalized with 10 mM biotin-NHS, followed by 300 pg / mL streptavidin. Cavity-containing particles 10 are next functionalized with 50 pg / mL of biotinylated anti-mouse CD45 antibody. The functionalized cavity -containing particles 10 are loaded with 300,000 hybridoma cells via constant agitation in a 12 well plate for 90 minutes. Every 30 minutes the well is mixed vigorously via pipetting to promote cell loading into the cavity-containing particles 10. Unbound cells are removed by straining the crude suspension through a 37 pm filter. Following filtration, a 40 pL (2,000,000 particles) sample of 35 pm diameter capping particles 16 is capped onto the cavity -containing particles 10 by mixing them together in a 0.5 mL Eppendorf tube brought to 400 pL total volume in PBS. The particles are centrifuged together in a swing bucket centrifuge at 350 RCF for 1 minute and 30 seconds. The cell-loaded capped particles 18 are incubated in a standard cell culture incubator at 37°C for 3-5 days in a 6 well plate supplemented with IMDM media containing 10% FBS and 1% anti-anti. Cells are next stained with 5 pM calcein AM for 30 minutes in the dark. The cell-loaded capped particles 18 are then brought to a flow cytometer and sorted based on FITC-A signal intensity. The top 10% of FITC-A-positive particles are sorted to enrich the fastest growing clones in the population.

[0140] Example: Single-Cell RNA-Sequencing for TCR Discovery Using Capped Particles

[0141] Materials and Reagents:1. Cavity-containing particles: Biotinylated cavity -containing particles 10 (Partillion Bioscience, 50 micron EZM Cavity-containing particles 10) coated with streptavidin (Thermo Fisher, Cat# 21135)2. Peptide-MHC monomers: Biotinylated peptide-MHC monomers (MBL International) 3. Capping particles: 25 micron streptavidin-functionalized PEG microparticles conjugated with unique oligonucleotide barcodes (Manufactured using Split-Pool Synthesis) 4. Anti-interferon gamma capture antibody: biotin-conjugated anti-IFN-y (BioLegend, Cat# 506504)5. Detection antibody: A cocktail of BrilliantViolent and oligo-conjugated anti-IFN-y detection antibody (BioLegend, TotalSeq™-B1003 anti-human IFN-y Antibody) 6. Lysis buffer: Single-cell Lysis Buffer (Takara Bio, Cat# 635013)7. Reverse transcription and PCR reagents: SMART-Seq v4 Ultra Low Input RNA Kit (Takara, Cat# 634891)8. FACS sorting buffer: PBS + 2% FBS (Thermo Fisher. Cat# 10437010) +1% Pluronic 9. Single-cell sorting system: Sony SH800S2025-267-210. Next-generation sequencing platform: Illumina NovaSeq 6000 (Illumina)

[0142] Step 1: Preparation of Cavity-Containing Particles and Capping Particles1. Resuspend biotinylated cavity-containing particles 10 in PBS and wash three times by centrifugation at 500 x g for 5 min.2. Incubate cavity-containing particles 10 with streptavidin (50 pg / mL) for 30 min at room temperature.3. Wash excess streptavidin with PBS three times.4. Incubate streptavidin-coated cavity-containing particles 10 with biotinylated peptide- MHC monomers (5 pg / mL) for 1 hour at 4°C.5. Wash and resuspend cavity-containing particles 10 in PBS + 1% Pluronic.6. Add anti-IFN-y capture antibody to streptavidin-functionalized capping particles 16 (5 pg / mL) and incubate for 1 hour at 37°C.7. Wash and resuspend cavity-containing particles 10 in PBS + 1% Pluronic.

[0143] Step 2: Loading of T Cells into Cavity-containing particles1. Isolate peripheral blood mononuclear cells (PBMCs) from blood using density gradient centrifugation.2. Load PBMCs at a concentration of 5 x 10A5 cells / mL in PBS + 2% FBS.3. Mix PBMCs with peptide-MHC functionalized cavity-containing particles 10 at a 1.6: 1 ratio to load antigen-specific T cells.4. Incubate for 30 min at 37°C to allow T cell binding through TCR interaction with cognate peptide MHC.5. Wash with PBS and filter through a 30 micron filter to remove unbound cells.

[0144] Step 3: Capping of Cavity-containing particles with Functionalized Capping Particles1. Mix cavity-containing particles 10 containing bound T cells with streptavidin- functionalized capping particles 16 at a ratio of 1:5.2. Agitate and centrifuge to create stably capped particles 183. Wash and filter through a 30 micron to remove unbound capping particles 16.4. Wash and resuspend in DMEM.

[0145] Step 4: IFN-y Secretion Detection and Sorting1. Stimulate T cells through peptide-MHC interaction on the cavity-containing particles 10 for 3 hours at 37°C.2. Introduce fluorescent and oligonucleotide-conjugated detection antibody against IFN-y (1 pg / mL) and incubate for 30 min at 4°C.3. Wash and resuspend in sorting buffer.2025-267-24. Analyze on Sony SH800S using fluorescence gating:1. Forward and side scatter width to identify capped cavity-containing particles 10 containing a T cell.2. Sort on events with fluorescence signal in the IFN-y channel above a threshold of two standard deviations above no secretion capture control capping particles 16.5. Collect sorted capped particles 18 as a pool and transfer into lysis buffer.

[0146] Step 5: Cell Lysis and RNA Capture1. Mix sorted capped particles 18 with Takara Single Cell Lysis Buffer according to manufacture recommendations.2. Incubate at room temperature for 10 min.3. Capture released mRNA on oligonucleotide barcoded capping particles 16.4. Wash and resuspend in 100 pL reverse transcription reaction buffer per 100,000 capped particles 18.

[0147] Step 6: Reverse Transcription and cDNA Amplification1. Perform reverse transcription using the SMART-Seq v4 Ultra Low Input RNA Kit:1. Incubate at 42°C for 90 min.2. Inactivate at 70°C for 10 min.2. Amplify cDNA using SMART-Seq v4 Kit:1. 25 cycles of PCR (98°C for 10 sec, 63°C for 30 sec, 72°C for 2 min).3. Purify amplified cDNA using AMPure XP Beads (Beckman Coulter. Cat# A63881).

[0148] Step 7: Library Preparation and Sequencing1. Prepare sequencing libraries using the Nextera XT DNA Library Prep Kit (Illumina, Cat# FC-131-1096).2. Normalize libraries and load onto the Illumina NovaSeq 6000.3. Sequence at 100 bp paired-end reads with a target depth of 50,000 reads per cell.

[0149] Step 8: Data Processing and TCR Discovery1. Use Cell Ranger (lOx Genomics) or other software for alignment and gene expression analysis.2. Identify TCR alpha and beta chain sequences from paired-end reads.3. Link TCR alpha and beta chain sequences with interferon gamma secretion levels by associating reads with shared capping particle barcode reads.4. Validate high-confidence TCRs through functional assays.

[0150] Example: Multiplexed Cardio-biomarker ELISA for Prognosis of Major Adverse Cardiac Events in an Elderly Population2025-267-2

[0151] Materials and Reagents:1. Cavity-containing particles: Biotinylated cavity -containing particles 10 (Partillion Bioscience. 50-micron EZM Nanovials) coated with streptavidin (Thermo Fisher, Cat# 434301)2. Capping particles: 25-micron biotin-functionalized PEG microparticles3. Antibody pairs: Antibodies specific to cardiac biomarkers, including:1. Troponin I (Tnl) (Fly Test, Cat# 19C7 & 560)2. N-terminal B-type natriuretic peptide (NTproBNP) (HyTest, Cat# 15C4cc & 13G12cc)3. C-reactive protein (CRP) (HyTest, Cat# 4C28 & 4C28cc)4. PBSP: PBS + 0.1% Pluronic F127 (Sigma-Aldrich, Cat# P2443)5. Sample matrix: Human serum or plasma samples collected from multiple patients 6. Blocking buffer: PBS + 1% BSA (Thermo Fisher, Cat# B14)7. Signal-generating reagent: QuantaRed Enhanced Chemifluorescent HRP Substrate Kit (Thermo Fisher, Cat# 15159)8. Sorting buffer: PBS + 2% FBS + 1% Pluronic (Thermo Fisher, Cat# A5670701) 9. Multiplex flow cytometer: BD FACSymphony™ A5 SE or similar

[0152] Step 1: Preparation of Cavity-Containing Particles and Capping Particles 1. Resuspend biotinylated cavity-containing particles 10 in PBSP.2. Incubate cavity-containing particles 10 with streptavidin (10 pg / mL) for 30 min at room temperature.3. Wash excess streptavidin with PBSP three times.4. Split the cavity-containing particles 10 into three different tubes. Follow instructions below for cavity-containing particle incubation:1. Tube 1: Incubate streptavidin-coated cavity -containing particles 10 with biotinylated Anti-cTnl (10 pg / mL) and SA-AF350 (5 pg / mL) for 1 hour at 4°C.2. Tube 2: Incubate streptavidin-coated cavity -containing particles 10 with biotinylated Anti-NTproBNP (10 pg / mL) and SA-AF488 (5 pg / mL) for 1 hour at 4°C.3. Tube 3: Incubate streptavidin-coated cavity -containing particles 10 with biotinylated Anti-CRP (10 pg / mL) and SA-AF647 (5 pg / mL) for 1 hour at 4°C.5. Wash (three times) and resuspend cavity-containing particles 10 in PBSP. Set aside until sample loading step.6. Split biotin-functionalized capping particles 16 into a minimum of 2 and up to 5 different tubes, depending upon how many patient samples are expected.7. To each capping particle tube, add a single streptavidin-AlexaFluor conjugate (5 pg / mL each) for 30 minutes at 37°C. Make sure to record yvhich batch of capping particles 162025-267-2is associated with which color. Colors can include AF350, AF488, AF532, AF568, and AF647.8. Wash (three times) and resuspend capping particles 16 in PBSP.

[0153] Step 2: Sample Loading, Biomarker Capture, and Detection Antibody Capture1. Collect serum or plasma samples from multiple patients.2. Dilute samples 1 :5 in PBS + 1% BSA to reduce matrix effects.3. Add cavity -containing particles 10 from each tube to each patient sample at a 1 : 10 ratio.4. Incubate at room temperature for 1 hour with gentle agitation.5. Wash cavity -containing particles 10 three times with PBSP to remove unbound proteins.6. Incubate cavity -containing particles 10 with a mixture of all three detection antibodies (1.5 pg / mL) for 1 hour at room temperature with gentle agitation.7. Wash cavity-containing particles 10 three times with PBSP to remove unbound antibodies.

[0154] Step 3: Capping of Cavity-Containing Particles with Functionalized Capping Particles1. Add one color of cap to each patient sample, making sure to record which patient sample is associated with which cap at a 1:5 ratio.2. Agitate gently and centrifuge at 2000 x g for 2 min to facilitate stable capping.3. Wash and filter through a 30-micron filter to remove unbound capping particles 16. 4. Resuspend in PBSP.

[0155] Step 4: Signal Detection and Flow Cytometry Analysis1. Incubate capped cavity -containing particles 10 with the QuantaRed Enhanced Chemifluorescent HRP Substrate Kit according to the manufacturer’s instructions.2. Allow the chemifluorescent reaction to proceed for 30 min at room temperature.3. Wash and resuspend in sorting buffer.4. Analyze capped particles 18 using a multiplex flow cytometer:1. Identify capped cavity -containing particles 10 by forward and side scatter parameters.2. Detect biomarker-specific signal using fluorescence channel corresponding resorufin, the product of the HRP Substrate Kit.3. Determine which biomarker signal is being read using color signal from the cavity -containing particle (AF350 for cTnl, AF488 forNTproBNP, and AF647 for CRP).4. Determine which patient each sample belongs to by identifying the fluorescence signal on the capping particles 16 attached to the cavitycontaining particle 10.2025-267-25. Set gating thresholds based on control samples lacking analytes.6. Sort and collect capped particles 18 with fluorescence signals above a threshold corresponding to biomarker positivity.

[0156] Step 5: Multiplexed Data Analysis1. Export flow cytometry data and assign signal intensities to individual biomarkers.2. Correlate fluorescence intensity with biomarker concentrations using standard curves.3. Analyze patient-specific biomarker profiles to assess cardiovascular risk.

[0157] Example: Antimicrobial Susceptibility Testing (AST) using capped particles

[0158] Materials and Reagents1. Cavity-containing particles: Biotinylated cavity -containing particles 10 (Partillion Bioscience, 50-micron EZM Nanovials, Cat# EZM-50-Biotin)2. Biotinylated spherical capping particles: Fluorescently barcoded with therapeutic agents (synthesis described below)3. Microbial sample: Patient-derived bacterial isolate (e.g., E. coli, P. aeruginosa, S. aureus) obtained from blood, urine, or sputum4. Microbial growth medium: Mueller-Hinton broth (Thermo Fisher, Cat# CM0405B) 5. Viability and metabolic activity dyes:1. Live / dead stain: Propidium Iodide (PI, Thermo Fisher, Cat# P1304MP, Ex 535 nm / Em 617 nm)2. Metabolic dye: Resazurin (Sigma- Aldrich, Cat# R7017, Ex 570 nm / Em 590 nm)3. Viability dye: SYTO 9 (Invitrogen, Cat# S34854, Ex 485 nm / Em 498 nm) 6. Fluorescence-conjugated streptavidin for barcoding:1. Streptavidin-APC (BioLegend, Cat# 405207, Ex 650 nm / Em 660 nm) 2. Streptavidin-Brilliant Violet 421 (BioLegend, Cat# 405225, Ex 405 nm / Em 421 nm)3. Streptavidin-PE (BioLegend, Cat# 405203, Ex 488 nm / Em 578 nm) 7. Antibiotics panel for AST testing:1. Beta-lactams: Ampicillin, Cefepime, Meropenem2. Aminoglycosides: Gentamicin. Tobramycin3. Fluoroquinolones: Ciprofloxacin, Levofloxacin4. Glycopeptides: Vancomycin5. Polymyxins: Colistin6. Tetracyclines: Doxycycline, Tigecycline7. Sulfonamides: Trimethoprim-sulfamethoxazole8. Culture vessels: 96-well U-bottom plates (Coming, Cat# 3795)9. Washing buffers: PBS with 1% BSA and 0.05% Tween-20 (Sigma-Aldrich. Cat#P4417)2025-267-2

[0159] Step 1. Preparation of Barcoded Capping Particles

[0160] Loading Therapeutic Agents1. Prepare a 10 mg / mL solution of each antibiotic in PBS. Filter sterilize (0.22 pm, Millipore, Cat# SLGP033RS).2. Label capping particles 16 with streptavidin-conjugated fluorophores (APC, BV421, or PE) at 0.01, 0.1, 1, or 10 pg / mL to encode barcode information.3. Incubate at RT for 15 min, followed by washing in PBS twice. Incubate 100 pL of capping particles 16 (IxlO7particles / mL) with antibiotic solutions at concentrations spanning three orders of magnitude (0.1, 1, 10, 100 pg / mL) for 30 min at RT. Store at 4°C until use.

[0161] Step 2. Preparation of Cavity-Containing Particles

[0162] Microbial Isolation1. Collect a clinical isolate from biological fluids (e.g., blood, urine, sputum).2. Centrifuge sample at 400g for 1 min, aspirate supernatant, and resuspend in 500 pL Mueller-Hinton broth.3. Adjust bacterial concentration to 5 x 106CFU / mL using OD600 measurement.4. Mix 100 pL microbial suspension with 100 pL of barcoded capping particles 16 (IxlO7particles / mL) and 20 pL of cavity-containing particles (IxlO6particles / mL).5. Vortex for 5 sec and centrifuge at 400g for 2 min.6. Reverse strain sample using a 40 pm cell strainer (Corning, Cat# 431750) to remove excess capping particles 16 and unbound microbial cells.

[0163] Step 3. Incubation and Drug Release

[0164] Culture Conditions1. Transfer 100 pL of cavity-containing particle / microbe mix into 96-well plates containing 100 pL Mueller-Hinton broth per well.2. Incubate at 37°C for 3 hours in a humidified incubator.

[0165] Controlled Drug Release1. Trigger antibiotic release by maintaining culture at 37°C for the duration of incubation.2. Ensure capped particles 18 remain bound for localized drug delivery.

[0166] Step 4. Assay Readout and Analysis

[0167] Microscopy and Flow Cytometry1. Use capping particles 16 and free cavity-containing particles 10 to determine gating controls.2025-267-22. Analyze fluorescent barcoding of capped particles 18 using a Sony SH800S (or equivalent) with the following filter sets:1. APC (Ex 650 nm / Em 660 nm)2. BV421 (Ex 405 nm / Em 421 nm)3. PE (Ex 488 nm / Em 578 nm)3. Sort capped particles 18 using FSC-Width and SSC-Height gating.4. Identify populations carrying different drugs and concentrations via fluorescence barcode deconvolution.

[0168] Dose-Response and MIC Determination1. Assess bacterial viability via fluorescence intensity of viability dyes:1. Propidium iodide (dead cells, Ex 535 nm I Em 617 nm)2. Resazurin (metabolic activity, Ex 570 nm / Em 590 nm)2. Compute minimum inhibitory concentration (MIC) from dose-response curves using Prism (GraphPad).3. Identify resistant clones using scatter and fluorescence gating (high drug concentration, high growth signal).

[0169] Sorting and Further Analysis1. Isolate rare resistant clones using FACS gating for high fluorescence in viability dyes.2. Collect sorted resistant populations for whole-genome sequencing (Illumina, Nextera XT Kit, Cat# FC-131-1096)3. Perform phenotypic validation in follow-up growth assays.

[0170] Experimental

[0171] Results

[0172] Generating suspendable compartments through complementary docking of shaped particles

[0173] To create a microscale assay platform capable of analyzing and isolating single cells and multi-cell interactions in a high-throughput manner, a two-particle interlocking system was developed composed of larger diameter (bowl-shaped) cavity -containing particles 10 and smaller diameter spherical capping particles 16. See FIG. 17A. Cavity-containing particles 10 are hydrogel microparticles with cavities 12 that are sized to hold cells. Each hydrogel cavity -containing particle 10 can be functionalized with biomolecules to capture cells and / or their secretions. Through mixing and centrifugation processes it was found that smaller capping particles 16 are able to stably dock within the cavities 12 of the cavity -containing particles 10, capping the cavity-containing particles 10 and creating a multitude of nanoliter-scale volume compartments surrounded by the hydrogel materials of the cavity-2025-267-2containing particle 10 and capping particle 16. These capped particles 18 impart unique functions to the compartment depending on how each component (the cavity -containing particle 10 and capping particle 16) is chemically modified to attach proteins, antigens, antibodies and other molecules (FIG. 17A). A standard workflow enables encapsulation of cells within the capped cavity -containing particles 10, which can be obser ed through standard microscopy (FIG. 17B) or flow cytometry, as described later. Thousands to millions of capped particles 18 can be formed in parallel in solution creating sealed compartments for clonal cell culture, on-particle assays for growth or secretion, and cell-cell interaction studies (FIG. 17C).

[0174] Both cavity-containing particles 10 and capping particles 16 of varying sizes are fabricated using a microfluidic droplet generator 20 of water-in-oil droplets made from a polyethylene glycol (PEG)-gelatin aqueous two-phase system (FIG. 17D). Phase separation of PEG and gelatin is concentration dependent, following a binodal curve. At high concentrations above the binodal curve, PEG and gelatin phase separate to create bowlshaped cavity’ -containing particles 10 after ultraviolet (UV) crosslinking, whereas at low concentrations below the binodal curve phase separation does not occur, resulting in spherical capping particles 16. The microfluidic device dimensions were adjusted to manufacture cavity -containing particles 10 and capping particles 16 of varying sizes. Cavity -containing particles 10 of tw o sizes were manufactured for experiments described herein, ~40 pm and ~70 pm. Because of the stability of the droplet generator 20, the outer diameter of the cavitycontaining particles 10 remained consistent in size with coefficients of variation of 2.48% and 2.02% for the two types of cavity-containing particles 10 respectively (FIGS. 23A-23B). Varying capping particle sizes were also manufactured from 30 to 40 pm with CVs of 2.81% and 2.44% (FIGS. 23C-23D). Although capping of cavity-containing particles 10 was achieved across a range of capping particle diameters spanning 25 pm to 90 pm, capping particles 16 of 30 pm diameter were used to cap 40 pm cavity' -containing particles 10 and capping particles 16 of 40 pm diameter to cap 70 pm cavity-containing particles 10. Cavity containing particles 10 and capping particles 16 were functionalized with biotin using sulfo-N-hydroxysuccinimide-biotin (NHS-biotin) conjugation to free lysine residues on the entrapped gelatin, to enable downstream cell and molecular capture (FIG. 24).

[0175] Process parameters and chemistries that enhanced the docking process betw een the cavity -containing particle 10 and capping particle 16 and its stability are described. To assemble capped particles 18, cavity -containing particles 10 are mixed with capping particles2025-267-216 and induced to interact through centrifugation steps in a wash buffer (WB) composed of phosphate buffer saline and Pluronic F-127 or media. Although the capping process could not be visualized under centrifugation, the mixed system of cavity-containing particles 10 and capping particles 16 was observed to be settling under gravity to inform processes expected to occur during centrifugation, but at a slower rate. A few insights from these observations were: (i) cavity -containing particles 10 settle and orient with their cavities 12 facing upwards due to their asymmetric center of mass, (ii) capping particles 16, which are less cross-linked and less dense, then settle into the cavities 12 of cavity -containing particles 10, and (iii) because both cavity-containing particles 10 and capping particles 16 can rotate under force moments, capping particles 16 that fall off-center into a cavity 12, appear to re-align the cavity-containing particle to form a capping event. Based on these unexpected observations it is surmised that capping rates are high even with the stochastic nature of the random encounters between capping particles 16 and cavity-containing particles 10, because the system has a broad range of starting conditions and trajectories that favor coupling.

[0176] In some preferred configurations to maximize the formation of capped particles 18, the size or diameter of the capping particle 12 is less than the outer diameter of the cavitycontaining particle 10. In preferred configurations, the size or diameter of the capping particle 12 is greater than the narrowest diameter of the cavity 12 that opens to the surface of the cavity-containing particle 10. In addition, in a preferred configuration to form capped particles 12, the cavity diameter at the opening to the external environment of the cavity -containing particle 10 is less than a diameter of the cavity 12 internal to the cavity-containing particle 10. That is, a more enclosed cavity 12 of the cavity -containing particle is preferred in the formation of capped particles 18.

[0177] Next, experimental conditions were modulated to investigate capping efficiency, beginning with the ratio of capping particles 16 to cavity -containing particles 10 (FIG. 18 A). When mixing unfunctionalized capping particles 16 of 40 pm diameter with cavitycontaining particles 10 of 70 pm outer diameter at a ratio of 5: 1 capping rates of 9.3% ± 5.4% were observed as measured by flow cytometry (FIG. 18A). Capping rates appeared to saturate around 10% for even higher ratios, such as 10:1. The ability to interact with open cavity-containing particles 10 was enhanced by mixing and repeat centrifugation steps, with increasing rates of capping from 8.7% ± 4.1% to 18.0% ± 2.5% with subsequent centrifugations (FIG. 18B). Centrifugation speed or time had minimal effects on formation of capped particles 16 (FIG. 25 A). Similarly, the vessel size in which capping was performed2025-267-2did not significantly impact capped particle 18 formation (FIG. 25B). Although physical docking between the flexible hydrogels enables stable capping, molecular affinity also was found to further stabilize interacting particles 10, 16. Streptavidin-biotin interactions on the complementary particles 10, 16 promoted capped particle 18 formation.

[0178] Interestingly, it was found that streptavidin functionalization of capping particles 16 interacting with biotinylated cavity -containing particles 10 led to highest capping rates ( 17.7% ± 4.0%), demonstrating moderate improvement over streptavidin on cavitycontaining particles 10 interacting with biotinylated capping particles (11.9% ± 5.1%), streptavidin on both particle types (10.7% ± 3.1%) and statistically significant improvement over no functionalization (4.7% ± 0.6%) (FIG. 18C). For these experiments, a single centrifugation step was performed with a capping particle to cavity-containing particle ratio of 5:1. Additionally, increasing the centrifugation steps from one centrifugation step (8.7% ± 4.1%), two centrifugation steps (16.5% ± 0.9%), and three centrifugation steps (18.0% ± 2.5%) was also demonstrated to improve capping efficiency in a statistically significant manner (FIG. 18B). While these various factors were evaluated on capping performance for larger 70 pm cavity-containing particles 10, well suited for mammalian cell assays, it was found that using smaller particles (40 pm cavity-containing particles 10 and 30 pm capping particles 16) further improved capping efficiency . The trend of the impact of functionalization on the capping efficiency was preserved for the 40 pm cavity-containing particles 10 and 30 pm capping particles 16. where streptavidin functionalization of capping particles 16 interacting with biotinylated cavity-containing particles 10 had the highest capping efficiency (68.3% ± 34.8%), an improvement over streptavidin on cavity-containing particles 10 interacting with biotinylated capping particles (25.1% ± 10.7%) and statistically significant improvement over streptavidin on both particle types (7.5% ± 2.4%) and no functionalization (2.4% ± 1.9%) (FIG. 18D). Increasing the numerical ratio of capping particles to cavity-containing particles were also demonstrated to improve the capping efficiency, although the effect was not statistically significant (FIG. 18E). Additionally, increasing the centrifugation steps from one centrifugation step to two and three centrifugation steps was also demonstrated (FIG. 18F). Transitioning the assay from wash buffer used for optimization studies to yeast culture media (YM) also showed improved capping efficiency (40.7% ± 6.6%) (FIG. 18G). Combining the conditions identified above that resulted in improved capping efficiency (i.e., 40 pm cavity-containing particles 10 and 30 pm capping particles 16, streptavidin functionalization on capping particles, three2025-267-2centrifugation steps, and a 10: 1 numerical ratio of capping particles to cavity -containing particles) resulted in high capping efficiencies ranging from 73.3% to 92.4% across different batches of capping particles and cavity -containing particles (FIG. 18M). Taken together, these methods enable consistent capping efficiency and rapid generation of hundreds of thousands to millions of compartments.

[0179] The docking of a capping particle 16 onto the cavity 12 of the cavity-containing particle 10 opening 14 forms a sealed microscale compartment that limits convective mixing and restricts the diffusion of larger molecules, while still permitting exchange of smaller nutrients and reagents. To characterize molecular transport across the capped particles 18, fluorescent dextrans of varying molecular weights were introduced prior to capping and subsequently removed any unencapsulated dextran by washing. Dextrans smaller than 50 kDa readily diffused through the hydrogel matrix and / or the interface between the capping particle 16 and the cavity-containing particle 10 (FIG. 18H), indicating permeability to small solutes. In contrast, dextrans larger than 500 kDa were largely retained within the cavity712, remaining confined for hours to days in the majority7of capped particles 18. These results highlight the ability of capped particles 18 to act as semi-permeable microcompartments, selectively isolating larger biomolecules while allowing controlled molecular exchange.

[0180] Capped particle compatibility with standard flow sorter technology

[0181] Capped particles 18 were compatible with analysis by flow cytometry and flow sorting without breakup of the composite asymmetric particle, similar to cavity-containing particles 10 alone. The ability to analyze and sort capped particles 18 using standard FACS (Sony SH800S) was investigated. First, both capping particles 16 and cavity7-containing particles 10 were functionalized with streptavidin Alexa Fluor 568 and streptavidin Alexa Fluor 647, respectively, after biotinylation (FIG. 181). Following docking, capped particles 18 exhibited composite fluorescence profiles containing signal from both component particles 10, 16, confirming successful assembly and maintenance of the docked form through fluid dynamic shear present during flow analysis (FIG. 18 J). This labeling allowed the generation of distinct fluorescent profiles for all three particle types (capping particles 16, cavitycontaining particles 10, and capped particles 18) which could be gated to identify the unique forward scatter (FSC) and side scatter (SSC) profiles of each (FIG. 18K). In particular, all of the three particle types had distinct FSC-width values (FIG. 18K). Interestingly , the asymmetric capped particles 18 did not yield a large distribution of scatter width profiles as would be expected for random orientations in the flow stream. Notably, the distinct FSC2025-267-2width value measured was larger than either the cavity -containing particles 10 or capping particles 16 alone. These results suggest that capped particles 18 may align under the hydrodynamic sheath flow of the Sony SH800S microfluidic sheath flow chip, such that the long axis of the oblong capped particle 18 is directed along the flow direction.

[0182] It was further established that capped particles 18 could be sorted without disrupting the docking between the capped particles 16 and the cavity-containing particles 10. Because of the larger particle sizes compared to single cells and cavity-containing particles 10 alone the drop delay increment, which modulates the time between detection and the electrostatic deflection of a sorted droplet, was modulated to achieve maximum sorting performance. When using a Sony SH800S sorter, sort efficiency and purity metrics increase to a maximum using the Single Cell sort mode as the drop delay is adjusted, respectively (FIGS. 26A-26B). A maximum sort yield of -50% and sort purity of -80% of 40 pm capped particles 18 were achieved at the optimal drop delay, which approaches expected sort yields for other large particles. Reduced sorting yield was observed for larger capped particles 18 with a shifted optimal drop delay. Sorted capped particles 18, gated on FSC-width. remained intact through hydrodynamic focusing, droplet-in-air sorting and recovery into a well plate demonstrating the robustness and stability of capping (FIG. 18L). When gating based on FSC-width other particle populations could also be sorted and successfully identified (FIG.18L). Together, these results demonstrate that capped particles 18 can be directly integrated into FACS-based pipelines, allowing for enrichment and recovery for downstream assays and analysis.

[0183] Capped cavity-containing particles enable single-cell growth and selective enrichment

[0184] Capped particles 18 compartmentalize growing colonies of cells supporting longitudinal tracking and enrichment based on growth rate. A growth assay was first performed using HyHEL5 hybridomas encapsulated within capped cavity -containing particles 10. Following nanovial functionalization with biotinylated anti-mouse CD45, cells were counted, loaded into cavity-containing particles 10 based on binding to surface expressed CD45, subsequently isolated through capping and incubated for growth (FIG. 19A). To measure the encapsulated cells, Calcein AM (622 Da) was used, which is permeable to capped cavity-containing particles 10 and can stain cells after being transported through the hydrogel and / or microparticle interface. Flow cytometry analysis revealed a distinct subpopulation of cell-loaded, capped cavity -containing particles 10, identifiable by2025-267-2high calcein AM fluorescence and high FSC-width (FIG. 19A). When gating this population, sorting, and imaging it, the majority of events included capped cavity-containing particles 10 containing cells. Capped cavity-containing particles 10 with high calcein AM signal were sorted and cultured for 96 hours to monitor clonal expansion. Within this time, HyHEL5 cells proliferated wi thin the compartment, forming visible colonies that were retained (FIG. 19B). By the 96-hour time point, the mechanical force of the proliferating cells led to the displacement of the capping particle 16 from some cavity-containing particles 10, allowing the colony to erupt from the compartment (FIG. 19B) and colonize the well. This enables dow nstream recovery and facile recovery of cells compared to chemical de-emulsification steps required for microdroplets, as cells simply grow out of the compartment and expand into a standard culture vessel. These results indicate that capped cavity-containing particles 10 accommodate several rounds of mammalian cell division while maintaining physical containment.

[0185] Colonies of hybridoma were selected based on relative growth rate using the capped cavit -containing particles 10 to enrich highly proliferative single cells. HyHEL5 cells were again loaded into cavity-containing particles 10 and capped followed by 72 hours of culture to limit eruption from the capped particles 18. After staining with calcein AM, the average number of cells per compartment were compared following gating and sorting based on different thresholds of fluorescence intensity. Cavity-containing particles 10 in the top 20% of the calcein AM signal distribution contained, on average, four cells per compartment, while those in the bottom 10% averaged approximately one cell per compartment (FIG. 19C).

[0186] Using similar approaches, populations of yeast and bacteria cells with varying growth rates were able isolated and sorted in 40 pm cavity-containing particles 10 with 30 pm capping particles 16. S. cerevisiae were loaded into cavit -containing particles 10 via sealing with capping particles 16, cultured for 16 hours, sorted and selected based on growth using SSC-height signal, which reflected the density of the growing culture within the capped particle 18 (FIG. 19D). Notably, compartmentalizing S'. cerevisiae within capped particles 18 enables formation of larger colonies than in cavity -containing particles 10 alone by physically entrapping the cells (FIG. 19D, middle panel). In another example, E. co / i were loaded directly into the cavity 12 of cavity -containing particles 10 without any capture antibody, capped, and incubated over 5 hours, at which point the bacteria displaced the cap sufficiently to break containment (FIG. 19E). E. coli expressing GFP loaded into capped particles 18 for varying incubation periods could be identified and FACS-sorted based on overall GFP2025-267-2fluorescence of a colony and was confirmed by microscopy (FIGS. 27A-27B). These results demonstrate the ability to identify and isolate single-cell-derived colonies of mammalian cells, yeast, and bacteria based on growth from a mixed population in a high-throughput manner without the need for microfluidic systems at the time of the assay.

[0187] Conducting single-cell secretion assays in capped cavity-containing particles reduces crosstalk

[0188] The enclosed cavity 12 of capped particles 18 confined secreted proteins within each local compartment improving the signal-to-noise ratio (SNR) in single-cell antibody secretion assays. COMSOL simulations of hybridomas loaded into cavity-containing particles 10 generated higher local antibody concentrations within capped particles 18 compared to uncapped cavity-containing particles 10 over the same secretion period (FIGS. 28A-28B). The concentration of secretions increased by 3.1 -fold within the cavity 12 of capped particles 18 compared to the uncapped cavity-containing particle 10 when integrating over the cavity spaces of both particles. HyHEL5 hybridomas, which secrete mouse IgG antibodies against hen egg lysozyme (HEL), were loaded into both capped particles 18 and uncapped cavitycontaining particles 10 functionalized with anti -mouse CD45 antibodies. In the capped configuration, biotinylated HEL antigen was conjugated to the spherical capping particle 16, while in the uncapped control it was bound to the surface of the cavity -containing particle 10 along with anti-CD45 (FIG. 20 A). Cells were loaded into both systems for 90 minutes to enable adhesion and capped particles 18 were formed. A fraction of the cavity-containing particles 10 remained uncapped, serving as controls. IgG-specific signal on cavity-containing particles 10 and on the capping particles 16 docked onto cavity -containing particles 10 both were on average elevated for flow cytometry' analyzed events containing HyHEL5 cells compared to background cavity -containing particles 10 or capped particles 18 without cells (FIG. 20B). Fluorescence microscopy of the uncapped and capped configurations reveals the secretion capture profile of both particle types (FIG. 20C). The decreased signal in the capped particle 18 formulation is believed to stem from the reduced gelatin concentration of capping particles 16 (2% (w / v)) compared to cavity-containing particles 10 (20% (w / v)), limiting the number of biotin-streptavidin sites for antibody capture and detection. However, the capped particle 18 configuration exhibited an increased fold change enabling better discrimination between HyHEL5-loaded and empty capped particles 18 (SNR = 5.59) compared to uncapped cavity-containing particles 10 (SNR = 1.44), emphasizing its improved utility for specific secretion detection (FIG. 20D). The interlocking architecture likely limits convective2025-267-2mixing and transport of secreted antibodies, leading to increased local concentration and reduced signal accumulation in neighboring compartments. The system still remains permeable to fluorescent labeling antibodies during mixing / staining steps. Since capping particles 16 containing binding antigen are introduced following cell loading, crosstalk introduced during loading steps that occur for standard cavity-containing particles 10 may also be reduced. Following sorting capped particles 18 or uncapped cavity-containing particles 10 based on high fluorescence intensity signal for secretion specific signal, fluorescence microscopy of capped particles 18 reveals localized signal on caps associated with secreting cells, while crosstalk is observed directly on cavity-containing particles 10 without bound cells (FIG. 20C). Fluorescent signal localized near single cells exclusively within the capped particles 18 indicated efficient and spatially resolved capture of secreted antibodies. It was also found that when gelatin-containing biotinylated-HEL-conjugated capping particles 16 and cavity-containing particles 10 were incubated with varying dilutions of HyHEL5 supernatant, capping particles 16 exhibited a slightly larger dynamic range across dilutions from 60 to 4250 intensity values, compared with 200 to 3100 intensity values (FIG.29) and this extended range of intensity on capping particles 16 enabled resolution of different secretion amounts accumulating in a time-dependent manner over an order of magnitude in intensity7when incubating up to 6 hours (FIG. 30). Together, these results highlight the ability to limit crosstalk and introduce time dependent capture of secretions using capped particles 18 enhancing the ability to select functional cells within a complex background.

[0189] Measuring and sorting cells based on cell-cell interactions within capped cavity-containing particles

[0190] It was hypothesized that one could compartmentalize and maintain interacting cells within capped particles 18 to accumulate secreted molecules and amplify their interactions. Specifically, OKT3 hybridomas (which constitutively secrete anti-CD3 antibodies) were coloaded with NFAT-GFP Jurkat reporter cells, which express GFP upon T cell activation via CD3 binding and clustering (FIG. 21A). When both cells are confined within a single capped particle 18, the local accumulation of anti-CD3 secreted by the hybridoma was expected to activate the Jurkat cell, triggering GFP expression. Following incubation for 24 hours a marked shift in the GFP fluorescence was observed in capped particles 18 containing OKT3 cells compared to capped particles 18 with only Jurkat reporter cells by flow cy tometry (FIG.2 IB). Flow cytometry analysis confirmed that reporter cell activation was robust and2025-267-2distinguishable in capped particles 18 (12.6-fold average signal increase between OKT3+ / OKT3- containing capped particles 18, n = 3 biological replicates using different cultures of both 0KT3 and Jurkat reporter cells) (FIG. 21C). Fluorescence microscopy of sorted capped particles 18 with high GFP levels confirmed successful co-encapsulation of Jurkat and hybridoma pairs and generation of GFP fluorescence in the activated reporter cells when co-encapsulated (FIG. 2 ID).

[0191] To assess the utility of this platform to identify functional antibody clones in a background of non-functional antibodies a population spiking experiment was performed using an 80% background of HyHEL5 hybridomas (non-stimulatory) with a 20% population of OKT3 cells (FIG. 22A). After co-loading the mixed hybridomas with Jurkat NF AT-GFP cells, the cells were incubated for 24 hours to induce CD3-mediated activation and GFP expression. Following gating for Jurkat-loaded (CellTracker Deep Red positive) capped particles 18, an over 30-fold increase in the average GFP activation signal for capped particles 18 co-loaded with OKT3 cells (CellTracker Blue positive) was found compared to HyHELS cells (CellTracker Orange) (FIG. 22B), resulting in clear thresholds for selection between the two populations. Reflecting a blinded selection experiment, co-loaded capped and uncapped cavity -containing particles 10 were gated for the top NF AT-GFP area signal, the percentage of particles that were loaded with on-target OKT3 cells were identified. Purify up to 100% for OKT3 hybridoma could be achieved for the most stringent gates, and across every NF AT-GFP area gating stringency capped particles 18 yielded improved purity compared to uncapped controls (FIG. 22C). Fluorescence microscopy images of representative co-loaded capped particles 18 with OKT3 or HyHEL5 hybridoma reflected the selective induction of GFP expression in the reporter cells co-loaded with OKT3 (FIG. 22D).

[0192] Discussion

[0193] The capped particle system introduced here redefines the functionality of lab-on-a-particle systems by enabling stable compartmentalization, multi-cell assays, and growthbased selections — all within an accessible, scalable format. By combining cavity-containing particles 10 and capping particles 16 through complementary docking, sealed, suspendable vessels are created that bridge a critical gap in microscale biological experimentation.

[0194] A major advance of the capped particle system is its ability’ to support cell growth assays within a confined microenvironment. While prior “nanovial” platforms enabled secretion assays, their open architecture was less suited for capturing clonal expansion or measuring growth phenotypes. Here, the addition of a hydrogel capping particle 162025-267-2transforms the open cavity-containing particle 10 into a true microvessel — retaining single cells or small colonies over time, restricting exchange of larger molecules, and preserving secretions. This is believed to be the first demonstration of bacterial, yeast, and mammalian cell grow th within capped particles 18, opening powerful new applications in directed evolution, synthetic biology, and biomanufacturing optimization where growth rates are critical readouts.

[0195] Beyond growth, the capped particles 18 enable a new class of multi-cell assays. By co-encapsulating different cell types and sealing them together, secreted molecules can accumulate locally, amplifying functional interactions that would otherwise be diluted in bulk. This was demonstrated by co-loading antibody-secreting hybridomas with NFAT-GFP reporter T cells, achieving robust, selective activation dependent on local antibody production. This is believed to be the first time a reporter-based multi-cell assay has been implemented using cavity-containing particles 10, aided by the secure microenvironment established through capping with capping particles 16. Further multi-cell studies could be developed with this technology, such as T cell killing assays, antigen-presenting cell activation assays, and a myriad of other cell-cell communication studies.

[0196] The capped architecture also introduces an important design flexibility': the cavitycontaining particle 10 and capping particle 16 can be independently functionalized with different capture agents, biomolecules, or barcodes. It w as demonstrated that this design reduces noise in secretion assays of IgG antibodies and could apply to larger secreted molecules. Capping particles 16 and cavity-containing particles 10 can also be functionalized with moieties that impart orthogonal reactivity (i.e. , click chemistry' and biotin-streptavidin conjugation), enabling temporally-controlled and multiplexed secretion detection. Modularity extends the range of possible assays from single-cell secretion analysis to antibody, T cell receptor (TCR), and chimeric antigen receptor (CAR) discovery workflows, cell-cell interaction screening, and even colony or single-cell sequencing when capping particles 16 are oligo-barcoded. In principle, capped particles 18 could enable combined workflows where functional screening is followed by sequencing, for example by secretion-encoded single-cell sequencing (SEC-seq), to link function to genotype at the level of a growing colony, a cell-cell interaction pair, or a single cell.

[0197] Perhaps most remarkably, the simplicity of the capped particle platform stands out from other microfluidic compartmentalization technologies. Much of microfluidics and single-cell analysis relies on specialized instrumentation and expertise, hampering2025-267-2accessibility, operational flexibility (e.g., compatible cell types, reagent and gas exchange, cell recovery for downstream analysis), and throughput of assays. On the other hand, capped particles 18 are assembled and analyzed with standard laboratory tools — simple mixing, centrifugation, and flow cytometry. This operational simplicity, coupled with the robustness of the capped compartments even through sorting, positions the platform for broad adoption across biology, biotechnology, and medicine. Taken together, the capped particle system provides a new approach for performing scalable, accessible assays of growth, secretion, and interaction at the single-cell and colony level. By miniaturizing and democratizing the concept of a sealed experimental vessel, this platform extends the power of high-throughput biology — bringing the classic virtues of the test tube and the petri dish into the microscale era, and supercharging collection of functional cell data to power the biological Al models of the future.

[0198] Materials and Methods

[0199] Cavity-containing particle fabrication

[0200] The production of cavity -containing particles 10 commercially known as nano vials has been described in detail in previous publications. See e.g., G. Aubry, H. J. Lee, H. Lu, Advances in Microfluidics: Technical Innovations and Applications in Diagnostics and Therapeutics. Anal. Chem. 95, 444-467 (2023) and R. Ghosh, A. Amheim, M. Van Zee, L. Shang, C. Soemardy, R.-C. Tang, M. Mellody, S. Baghdasarian, E. Sanchez Ochoa, S. Ye, S. Chen, C. Williamson, A. Karunaratne. D. Di Carlo, Lab on a Particle Technologies. Anal. Chem. 96, 7817-7839 (2024), both of w hich are incorporated herein by reference. Cavitycontaining particles 10 were fabricated using a poly dimethylsiloxane (PDMS) microfluidic device (droplet generator 20) designed with a flow -focusing geometry' for droplet generation. The aqueous polyethylene glycol (PEG) phase consisted of 27.5% (w / v) 4-arm 5 kDa PEG-acrylate (Advanced BioChemicals, 4AP0902-lg) and 4% (w / v) lithium phenyl -2,4,6-trimethylbenzoylphosphinate (LAP) (Sigma, 900889), dissolved in phosphate-buffered saline (PBS) (Thermo Fisher, 14190250). This w as co-injected with a separate aqueous gelatin phase containing 20% (w / v) cold-water fish gelatin (Sigma. G7041100G) in sterile-filtered deionized water. An oil phase composed of 1% (w / w) 008-FluoroSurfactant (RAN Biotechnologies, 008-FluoroSurfactant-lG) in Novec™ 7500 fluorinated oil (3M, 7100134816) served as the continuous phase. Each solution w as loaded into individual syringes and introduced into the device via syringe pumps at flow- rates of 1 pL min'1(PEG), 1 pL min1(gelatin), and 15 pL min1(oil). Upon reaching a stable flow regime,2025-267-2monodisperse droplets were formed with spontaneous phase separation between the PEG-rich and gelatin-rich domains. Crosslinking of the PEG phase was initiated by UV exposure through a 10x microscope objective. Following droplet formation and photo-crosslinking, the emulsion was collected into a microcentrifuge tube. Residual surfactant was removed by triple washing with sterile-filtered Novec™ 7500. Droplets were then demulsified by adding a 20% (v / v) solution of Pico-Break (Sphere Fluidics, C082) in Novec™ 7500. Oil was aspirated, and remaining traces were eliminated through three hexane washes. Excess hexane was subsequently removed via aspiration, and particles were further purified by washing in sterile-filtered 70% (v / v) ethanol three times. To eliminate aggregates, the cavity -containing particles 10 were passed through a 70 pm cell strainer (Stem Cell Technologies, 27216), then incubated overnight at 4 °C in ethanol for sterilization. Cavity-containing particles 10 were then incubated overnight at 4 °C in a solution of 10 mM biotin-NHS (ApexBio, A8001). Prior to use, cavity -containing particles 10 were washed in a Pluronic-based buffer composed of 0.05% (w / v) Pluronic F-127 (Sigma, P2443), 1% (v / v) antibiotic-antimycotic, and 0.5% (w / v) bovine serum albumin (BSA) (Sigma, A7906). Cavity -containing particles 10 were stored at 4 °C in this buffer until use.

[0201] Capping particle fabrication

[0202] Capping particle 16 production was performed similarly to cavity -containing particle fabrication. Capping particles 16 were fabricated using a flow-focusing PDMS device with a 28 pm junction height to create 30 and 40 pm particles. The aqueous PEG phase used 12% (w / v) 4-arm 5 kDa PEG-acrylate and 4% LAP dissolved in PBS. The second aqueous phase used a 2% (w / v) solution of gelatin dissolved in deionized water. An oil phase composed of 0.5% (w / v) of Pico-Surf surfactant (Sphere Fluidics, C024) in Novec™ 7500 fluorinated oil was used as the continuous phase. The PEG and gelatin phases were mixed in a 1 : 1 volumetric ratio and loaded into two syringes with the oil phase in a third syringe. The solutions were perfused through the flow-focuser at flow rates of 0.5 pL min'1(PEG + gelatin), 0.5 pL min'1(PEG + gelatin), and 18 pL min'1(oil) for 30 pm particles and at 1 pL min'1(PEG + gelatin), 1 pL min'1(PEG + gelatin), and 15 pL min'1(oil) for 40 pm particles. Upon reaching a stable flow regime, monodisperse droplets were formed with a uniform distribution of PEG and gelatin throughout each droplet. Crosslinking was performed through UV exposure through a 10x microscope objective, which polymerized the PEG and embedded gelatin strands within the matrix. Following particle collection, residual surfactant was removed by triple washing with sterile-filtered Novec™ 7500. Droplets were then2025-267-2demulsified by adding a 20% (v / v) solution of perfluoro- 1 -octanol (PFO) (Sigma, 370533) in Novec™ 7500. Oil was aspirated, and remaining traces were eliminated through three hexane washes. Capping particles 16 were then sterilized, filtered, biotinylated, and transferred to a Pluronic-based buffer similarly to cavity -containing particles 10.

[0203] Capped particle formation

[0204] 1.8 x io570 pm cavity-containing particles 10 were incubated with 50 pL of 300 pg mL’1streptavidin (Thermo Fisher, 434302) in a 1.5 mL microcentrifuge tube. In a separate 1.5 mL microcentrifuge tube, 9.0 x |()- 40 pm capping particles 16 were incubated with 75 pL of 300 pg mL’1streptavidin. Both solutions are incubated for 30 minutes at room temperature, then washed three times with Pluronic washing buffer. Both particle types are then mixed together in a 0.5 mL Eppendorf tube and resuspended to a final volume of 300 pL in Pluronic washing buffer. The tube containing capping particles 16 and cavity-containing particles 10 is then centrifuged at 600 RCF for 90 seconds, creating a mixed population of capped particles 18, free cavity-containing particles 10, and free capping particles 16. For testing the optimal condition for forming capped particles 18, a variety of different conditions were tested: the numeric ratio of capping particles to cavity-containing particles 10 (2: 1, 5:1. and 10:1), frequency of centrifugation steps (1~3), different combinations of capping particles and cavity-containing particles 10 with or without 300 pg mL’1streptavidin functionalization, varying the relative centrifugal force (RCF) of the centrifugation step (150 RCF~ 1400 RCF), centrifugation duration (30 sec ~ 120 sec), and the volume of vessel to perform capping formation (0.5 mL, 1 mL, and 5 mL). All flow cytometric analysis was performed using a Sony Biotechnology7SH800S Cell Sorter. Alexa Fluor 588 streptavidin fluorophore was excited using a 561 nm laser filtered through a 617 / 30 filter. Alexa Fluor 647 streptavidin fluorophore was excited using a 638 nm laser filtered through a 665 / 30 filter. Sorting was performed on single-cell sort mode with a drop delay set to 15 (from a machine-calibrated setting of 18). Capping percentages were defined as capped events / (nanovial events + capped nanovial events), where all events were identified using the distinct forward scatter profile of the different particle populations. Capped particle sort efficiency and purity metrics were calculated by sorting 100 capped particle events based on FSC width and SSC height profiles and counting the ratio of sorted capped particles 18 to desired number of capped cavity-containing particles 10 (sort efficiency) and the ratio of sorted capped particles 18 to all sorted particles (sort purity).

[0205] Culture of hybridoma cells and reporter T cells2025-267-2

[0206] OKT3 hybridomas were purchased from ATCC. HyHEL5 hybridomas were provided by Richard Wilson from the University of Houston Department of Biology and Biochemistry. Jurkat NFAT-GFP cells were provided by Zhiyuan Mao and Owen Witte from the University of California Los Angeles Department of Microbiology, Immunology, and Molecular Genetics. Hybridoma cells were cultured in IMDM cell culture media (Thermo Fisher 12440053) supplemented with 10% fetal bovine serum (Thermo Fisher A5669701) and 1% antibiotic-antimycotic (Thermo Fisher 15240062). Jurkat cells were cultured in RPMI 1640 cell culture media (Thermo Fisher 11875093). Media was sterile filtered using 0.22 pm, 500 mL Stericups (Thermo Fisher S2GPU05RE). All cells were quickly thawed from liquid nitrogen at 1 x 105cells per mL and passaged three times per week. Cells were maintained in a sterile incubator at 37 °C and 5% CO2 and passaged up to P20 before replacement with a fresh vial. All cells were assessed for >95% viability before proceeding with experiments.

[0207] Hybridoma growth assay

[0208] 1.8 x io570 pm cavity-containing particles 10 were incubated with 50 pL of 300 pg mL’1streptavidin in a 1.5 mL microcentrifuge tube for 30 minutes at room temperature, then washed three times with Pluronic washing buffer. Cavity-containing particles 10 were then incubated with 50 pL of 50 pg mL’1biotinylated anti-mouse CD45 antibody (Thermo Fisher, 13045182). In parallel, 9.0 x 10540 pm capping particles 16 were incubated with 75 pL of 300 pg mL’1streptavidin for 30 minutes at room temperature. Both particles 10, 16 were washed three times with Pluronic washing buffer. 3.0 x 1 ()- HyHEL5 cells were mixed in with the antibody-conjugated cavity -containing particles 10 in the well of a 24 well plate and placed on a rocker in an incubator at 37°C incubator for 90 minutes. Every 30 minutes the cells and cavity-containing particles 10 were mixed by pipetting to promote cell loading. Following loading, unbound cells were removed using a 37 pm cell strainer (STEMCELL Technologies, 27215) and cavity -containing particles 10 with and without cells were reverse strained into a 5 mL Eppendorf tube. This mixture is briefly centrifuged at 100 ref for 2 minutes and the supernatant is removed. The cell-loaded cavity -containing particles 10 are then transferred to a 0.5 mL Eppendorf tube along with the capping particles 16. Cells are then sealed in capped particles 18 by centrifuging the mixture at 300 ref for 90 seconds. The now capped cell-loaded particles 18 are transferred to the well of a 6 well plate that has been pre-filled with 5 mL of IMDM media. After three days of culture, the cell-loaded particles 18 are stained with 1 pg mL’1of calcein AM (Thermo Fisher, C3099) for 30 minutes. After2025-267-2washing two times with Pluronic washing buffer, capped particles 18 are transferred to FACS tubes for flow cytometry. Calcein AM was excited using a 488 nm laser fdtered through a 525 / 50 filter. After gating for capped particles 18 using FSC-width, capped particles 18 loaded with Calcein AM-stained cells were gated using Calcein AM-area signal and the bottom 10% and top 20% segments of this population were sorted.

[0209] E. coli culture, capped particle loading, and analysis

[0210] Escherichia coli strains were cultured in Difco™ LB (Luria-Bertani) broth. Miller (BD Biosciences) at 37°C with shaking speed of 250 rpm. Solid media culture plates were prepared with Difco™ LB Agar, Miller (BD Biosciences) in 10 cm petri dishes and incubated at 37°C. 40 pm cavity -containing particles 10 and 30 pm capping particles 16 were stored at concentrations of 2,500 particles / pL and 3,400 particles / pL, respectively, in 0.05% (w / v) Pluronic F-127 (Sigma- Aldrich) in PBS at 4°C. All experiments were conducted in 0.5 mL microcentrifuge tubes, and centrifugation steps were performed using a swing-bucket centrifuge. The general bacteria loading and capping protocol is as follo s: an aliquot of cavity-containing particles 10 was centrifuged at 630 RCF for 2 minutes to pellet the particles 10, and excess supernatant was carefully removed. Cavity-containing particles 10 and bacteria were mixed in a 1:3 nanovial-to-bacteria ratio and gently mixed using a 1000 pL pipette. The nanovial-bacteria suspension was centrifuged at 630 RCF for 2 minutes to promote bacterial loading into the cavities 12. To enhance loading efficiency, the pipette mixing and centrifugation steps were repeated twice more, for a total of three cycles.Following bacterial loading, excess supernatant w as removed, and capping particles 16 were added at a 1:2 nanovial-to-cap ratio. The mixture was gently mixed by pipetting and centrifuged at 630 RCF for 2 minutes to facilitate capping. This capping process was repeated twice more, for a total of three cycles, to maximize capping efficiency.

[0211] Culture and capping of S. cerevisiae

[0212] A strain of S. cerevisiae 651 with tw o plasmids HRPK.S (-U) and NRPKS + ACPTE (-L) was used. Cells stored at -80°C in glycerol stock solution were thawed in 5 mL of yeast extract peptone dextrose (YPD) cell media and cultured at 25°C with shaking speed of 360 rpm (Amerex Instruments). After an overnight incubation, the cell suspension was diluted 1 / 100 prior to all capped nanovial experiments. For encapsulating single A cerevisiae in capped particles 18, 200 pL of the diluted yeast cell suspension, 6.0 pL of 40 pm pelleted cavity-containing particles 10 (-180,000 cavity-containing particles 10) and 12.7 pL of 302025-267-2pm pelleted capping particles 16 (-900,000 capping particles 16) were added to a 0.5 mL microcentrifuge tube. Following a brief vortexing step (Vortex Genie 2), the suspension was centrifuged (Thermo Scientific, Legend Micro 21 Centrifuge) at 600 RCF for 60 seconds. The pellet was gently suspended in washing buffer, strained and washed with washing buffer using a 20 pm strainer (Sysmex, CellTrics 04-004-2325) to remove unbound cells, and then reverse strained with 2 mL of YPD media into a well of 6 well plate. Cells encapsulated in capped particles 18 were incubated for 12 hours at 25°C without shaking prior to analysis and sorting with flow cytometry. After the incubation, the capped particles 18 were collected and suspended in 400 pL of washing buffer and transferred to a flow tube for flow cytometry'. Flow cytometry' analysis and sorting was performed using a Sony Biotechnology SH800S Cell Sorter. The particle suspensions were gated for (1) capped nanovial events based on forward scatter (FSC) profile and (2) colony growth based on side scatter (SSC) profile, where elevated SSC profile corresponded to higher yeast biomass content within capped cavity -containing particles 10. Capped particles 18 were sorted on single-cell sort mode with a drop delay set to 17 (from a machine-calibrated setting of 19).

[0213] Hen egg lysozyme biotinylation

[0214] Recombinant hen egg lysozyme (HEL) (Aviva System Biology, OORA00201) was biotinylated using an EZ-Link™ Sulfo-NHS-LC-biotinylation kit (Thermo Fisher, 21435) following the manufacturer’s instructions. After purification, concentration was measured using a spectrophotometer and the protein was stored at -20°C.

[0215] Hybridoma secretion and binding assay

[0216] 2.5 x 1 C 70 pm cavity-containing particles 10 were incubated with 75 pL of 300 pg mL’1streptavidin in a 1.5 mL microcentrifuge tube for 30 minutes at room temperature, then washed three times with Pluronic w ashing buffer. Cavity-containing particles 10 were then incubated with 75 pL of 50 pg mL’1biotinylated anti-mouse CD45 antibody. In parallel, 1.25 x 10640 pm capping particles were incubated with 125 pL of 300 pg mL’1streptavidin for 30 minutes at room temperature. Following washing with Pluronic washing buffer, capping particles 16 were incubated with 125 pL of 25 pg mL’1biotin-HEL. All particles were washed with Pluronic washing buffer. 5.0 x io5HyHEL5 cells were loaded onto the cavity-containing particles 10 as described previously^ and unbound cells were strained out. After transfer to a 0.5 mL Eppendorf tube and mixing with the biotin-HEL capping particles 16, the cell-loaded capped particles 18 w ere sealed via centrifugation. The capped, cell-loaded particles 18 were transferred to the well of a 6 well plate that was pre-fdled with 5 mL2025-267-2of IMDM media and incubated for 30 minutes for secretion accumulation to occur. The particles 18were then stained with 125 pL of 50 pg mL1anti-mouse IgG DyLight 650 antibody (Abeam ab98715) and 1 pg mL'1of calcein AM for 30 minutes in the dark. After a final wash with Pluronic washing buffer, cell-loaded capped particles 18 were transferred to FACS tubes for flow cytometry7analysis. The DyLight 650 fluorophore was excited using a 638 nm laser filtered through a 665 / 30 filter. After gating for cell loaded, capped particles 18 as described previously, particles were plotted for DyLight 650 area signal.

[0217] Reporter T cell activation assay (bulk)

[0218] 2.5 x io370 pm cavity-containing particles 10 were incubated with streptavidin as described previously. Cavity-containing particles 10 were then incubated with 75 pL of 25 pg mL1biotinylated anti-mouse CD45 antibody and 25 pg mL1biotinylated anti-human CD45 antibody (BioLegend 368534) for 1 hour at room temperature and washed three times with Pluronic washing buffer. In parallel, 1.25 x 10640 pm capping particles 16 were incubated with streptavidin as described previously. 2.0 x io5OKT3 cells were labeled with 200 pM CellTracker Blue CMHC (Thermo Fisher C2111). 2.0 x io5Jurkat NFAT-GFP cells were labeled with 1 pM CellTracker Deep Red (Thermo Fisher C34565). After washing, cells were mixed together and loaded into cavity -containing particles 10 as described previously. After straining, capping particles 16 were added to cavity -containing particles 10 via centrifugation in a 0.5 mL Eppendorf tube. Cell-loaded, capped particles 18 were transferred to the well of a 6 well plate pre-loaded with 5 mL of a 50:50 volumetric mixture of complete IMDM and complete RPMI media and incubated for 24 hours to enable OKT3 antibody secretion and Jurkat reporter activation. Particles were transferred into Pluronic washing buffer and placed in FACS tubes for flow cytometry analysis. The CellTracker Blue CMHC fluorophore was excited using a 405 nm laser filtered through a 450 / 50 filter. The NFAT-GFP reporter fluorophore was excited using a 488 nm laser filtered through a 525 / 50 filter. The CellTracker Deep Red fluorophore was excited using a 638 nm laser filtered through a 665 / 30 filter. After gating for capped particles 18, events were gated for CellTracker Deep Red area and CellTracker Blue CMHC area signal. These events were then plotted for NFAT-GFP area signal to compare activation profiles of capped particles 18 with and without hybridomas.

[0219] Reporter T cell activation assay (spiked)

[0220] 5.0 x 10570 pm cavity -containing particles 10 were incubated with 150 pL of 300 pg mL1streptavidin as described previously. Cavity -containing particles 10 were then2025-267-2incubated with 150 pL of 25 ig m ’1biotinylated anti-mouse CD45 antibody and 25 pig m ’1biotinylated anti-human CD45 antibody for 1 hour at room temperature and washed three times with Pluronic washing buffer. 2.5 x 10640 pm capping particles 16 were incubated with 250 pL of 300 pg mL’1streptavidin as described previously. 6.0 x 104OKT3 cells were stained with CellTracker Blue CMHC as described previously. 2.4 x 105HyHEL5 cells were stained with 20 pM CellTracker Orange CMRA. 5.0 x io5Jurkat NFAT-GFP cells were stained with CellTracker Deep Red as described previously. After washing excess cell stain, the different cells were mixed together and loaded into cavity-containing particles 10, strained, and capped as previously described. Following 24 hours of incubation for antibody secretion and Jurkat activation, particles w ere transferred to Pluronic washing buffer and placed in FACS tubes for flow cytometry. The CellTracker Orange CMRA fluorophore was excited with a 561 nm laser filtered through a 617 / 30 filter. After gating for capped particles 18, events w ere gated for the presence of Jurkat NFAT-GFP cells based on CellTracker Deep Red area signal. These events were then gated for CellTracker Orange CMRA area and CellTracker Blue CMHC signal. Events that were single positive for one of these markers were then plotted for NFAT-GFP area signal to compare the activation profiles of Jurkat-loaded capped particles 18 with either type of hybridoma co-loaded.

[0221] COMSOL simulation of IgG accumulation in capped cavity-containing particles

[0222] Secretion was modeled in a 2D axisymmetric COMSOL (Version 6.3) simulation using the Transport of Diluted Species module, assuming w ater as the solvent and purely diffusive transport. IgG was secreted at a constant rate of 1.71 x 10’12mol m’2s’1, equivalent to 1 pg hr'1per cell, based on literature estimates for mouse hybridoma cells. The diffusion coefficient for IgG was set to 4xl0-11m'2s’1, consistent with reported values for IgG in water or PBS. The simulation was evaluated over 2 hours to generate IgG concentration profiles within each particle. The capping particle 16 and the cavity -containing particle 10 were simulated to not be permeable to the IgG species. In the capped nanovial simulation, a gap of 100 nm was used to simulate limited transport out of the compartment through the nanovial lip region.

[0223] While embodiments of the present invention have been shown and described, various modifications may be made without departing from the scope of the present invention. The invention, therefore, should not be limited except to the following claims and their equivalents.

Claims

1. 2025-267-2What is claimed is:

1. A multi-particle system for capture and detection of cellular and molecular analytes comprising:a plurality' of cavity-containing particles including respective cavities therein that open to an external environment of the plurality of cavity -containing particles via respective openings; anda plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity-containing particles to at least partially seal or occlude the respective cavities from the external environment.

2. The multi-particle system of claim 1, wherein the plurality of cavitycontaining particles have a spherical envelope with an outer diameter of less than 100 pm and wherein the plurality of capping particles are spherical and have a diameter of less than 60 pm.

3. The multi-particle system of claim 2, wherein the plurality’ of cavity containing particles have an outer diameter of about 60 pm and a cavity diameter of about 32 pm and wherein the plurality of capping particles have a diameter of about 37 pm.

4. The multi-particle system of claim 1 , wherein the plurality’ of capping particles are spherical, ellipsoidal, or polyhedral.

5. The multi-particle system of claim 1, wherein the plurality of cavitycontaining particles comprise a hydrogel and wherein the plurality of capping particles comprise a hydrogel, a polymer, glass, a metal, or metal alloy.

6. The multi-particle system of claim 1, wherein the plurality of capping particles comprises a permeable hydrogel having a molecular weight cutoff within the range of 20 kD to 750kD.2025-267-27. The multi-particle system of claim 1, wherein the plurality of cavitycontaining particles comprises a permeable hydrogel having a molecular weight cutoff within the range of 20 kD to 750kD.

8. The multi-particle system of claim 1, wherein the plurality of cavitycontaining particles and the plurality of capping particles comprise binding or reactive moieties that facilitate the associating and capping of the plurality of cavity-containing particles with the plurality of capping particles.

9. The multi-particle system of claim 8, wherein the binding or reactive moieties comprise complementary binding moieties that interact and lead to non-covalent or covalent binding between the binding or reactive moieties.

10. The multi-particle system of claim 9, wherein the binding or reactive moieties are degradable in response to a chemical or other stimulus.

11. The multi-particle system of claim 1 , wherein the plurality of cavity -containing particles have respective cavities functionalized with one or more reactive or binding moieties.

12. The multi-particle system of claim 1 , wherein the plurality of capping particles are functionalized with one or more reactive or binding moieties.

13. The multi-particle system of claim I . wherein the plurality of cavitycontaining particles and / or the plurality of capping particles are barcoded.

14. The multi-particle system 13, wherein the barcodes are disposed on a surface of the respective cavities of the plurality of cavity-containing particles or a surface of the capping particles.

15. The multi-particle system 13, wherein the barcodes are disposed throughout the volume of the plurality of cavity-containing particles.2025-267-216. The multi-particle system of claim 1, wherein the plurality of cavitycontaining particles and / or the plurality of capping particles comprise magnetic material contained therein.

17. The multi-particle system of claim 1, wherein the plurality of cavitycontaining particles comprise between about 20% and 30% w / v 5 kDa 4-arm PEG acrylate.

18. The multi-particle system of claim 17, wherein the plurality of capping particles comprise between about 5% and 25% w / v 5 kDa 4-arm PEG acrylate.

19. The multi-particle system of claim 1, wherein the plurality of capping particles are degradable.

20. The multi-particle system of claim 1, wherein the plurality of cavitycontaining particles and the plurality of capping particles are disposed in an aqueous mixture and wherein the number of the plurality of capping particles is greater than the number of the plurality of cavity-containing particles.

21. The multi-particle system of claim 20, wherein the number of the plurality of capping particles is between 2 and 10 times larger than the number of the plurality of cavitycontaining particles.

22. The multi-particle system of claim 1, wherein the plurality of capping particles are configured to interact or associate with the openings of the plurality of cavity-containing particles to fully seal or occlude the respective cavities from the external environment.

23. The multi-particle system of claim 1, wherein the plurality of cavitycontaining particles comprise a cell binding moiety disposed on a surface of the plurality of cavity-containing particles located within respective cavities.

24. The multi-particle system of claim 1 or 23, wherein the plurality of cavity containing particles and / or the plurality of capping particles comprise a secretion capture2025-267-2moiety disposed on a surface of the plurality of cavity -containing particles located within respective cavities or on a surface of the plurality of capping particles.

25. The multi-particle system of claim 1, wherein the plurality of cavitycontaining particles comprise an antibody or aptamer disposed on a surface of the plurality of cavity-containing particles located within respective cavities.

26. A method of forming a multi -particle system for capture and detection of cellular and molecular analytes using capped particles comprising:associating (1) a plurality' of cavity -containing particles including respective cavities therein that open to an external environment of the plurality of cavity -containing particles via respective openings and (2) a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity-containing particles to at least partially seal or occlude the respective cavities from the external environment, wherein the associating results in a mixture; andmixing or agitating the mixture to form a plurality of capped particles.

27. The method of claim 26, wherein the mixture further comprises a sample.

28. The method of claim 27, wherein the sample comprises cells.

29. The method of claim 28, wherein the plurality of cavity-containing particles contain a single cell or a pair of cells therein.

30. The method of claim 27, wherein the sample comprises a biological sample.

31. The method of claim 26, wherein the mixing or agitating comprises one or more of: centrifugation, pipetting, vortexing, shaking.

32. The method of claims 29 or 30, further comprising exchanging molecules or reagents between the external environment and the respective cavities in the capped particles.2025-267-233. The method of claims 29 or 30, further comprising washing, centrifuging, or vortexing capped particles.

34. The method of any of claims 26-31, further comprising de-capping the plurality7of capped particles.

35. The method of claim 34, wherein the de-capping is performed by one or more of mixing, sonication, agitation, a chemical agent, enzyme, or degrading agent.

36. The method of claim 26, wherein the plurality of cavity-containing particles are functionalized with biotin and the plurality7of capping particles are functionalized with streptavidin.

37. The method of claim 26, wherein the plurality of cavity-containing particles are functionalized with streptavidin and the plurality7of capping particles are functionalized with biotin.

38. A method of performing cellular analysis comprising:generating a mixture of (1) cells, (2) a plurality of cavity-containing particles including respective cavities therein that open to an external environment of the plurality of cavity-containing particles via respective openings, and (3) a plurality of capping particles that are configured to interact or associate with the openings of the plurality7of cavity containing particles to seal or occlude the respective cavities from the external environment;mixing or agitating the mixture to form a plurality of capped particles with cells contained in the plurality7of capped particles;incubating the plurality7of capped particles containing the cells;labelling the cells or a secretion from the cells with a dye or fluorescent reporter; and flowing the capped particles containing the labelled cells or labelled secretion from the cells through a cell sorter to sort and enrich a sub-population of the capped particles containing the labelled cells or labelled secretion from the cells based on one or more signals from the cell sorter.2025-267-239. The method of claim 38, wherein the cells comprise mammalian cells, yeast cells, or bacteria cells.

40. The method of claim 38, wherein the plurality of capping particles are functionalized or loaded with one or more therapeutic agents and wherein the plurality of capping particles are barcoded with the identity of the one or more therapeutic agents.

41. The method of claim 38, wherein the plurality of cavity-containing particles include respective cavities that are functionalized with antibodies or other affinity agents and wherein the plurality of capping particles are functionalized with target antigens or peptides.

42. The method of claim 38, wherein the plurality of cavity-containing particles include respective cavities that are functionalized with antibodies or peptide-MHC monomers presenting specific antigenic peptides for T cells and wherein the plurality of capping particles are functionalized with antibodies or peptide-MHC monomers presenting antigenic peptides.

43. The method of claim 38, wherein the plurality of cavity-containing particles include respective cavities that are functionalized with antibodies or peptide-MHC monomers presenting specific antigenic peptides for T cells and wherein the plurality of capping particles are functionalized with antibodies for cytokines or other secreted effector molecules.

44. The method of claim 38, wherein the plurality of cavity-containing particles include respective cavities that are functionalized with one or more capture agents specific to secreted bispecific T cell engager molecules (BiTEs) or a cell that binds to BiTEs.

45. The method of claim 38, wherein the plurality of cavity -containing particles include respective cavities that are functionalized with a plurality of different secretion capture moieties and w herein the plurality of capping particles are functionalized with another plurality of different secretion capture moieties.2025-267-246. The method of claim 38, wherein the cell sorter is an image activated cell sorter and sorting is based, at least in part, on spatial localization of the signals from capped particles containing the labelled cells or labelled secretion from the cells.

47. The method of claim 46, wherein the spatial location of the capped particles includes the capped particle region, the cavity-containing particle region, or the cavity region.

48. A method of performing a cell secretome assay comprising:generating a mixture of (1) cells, (2) a plurality7of cavity-containing particles including respective cavities therein that open to an external environment of the plurality of cavity-containing particles via respective openings, and (3) a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity containing particles to seal or occlude the respective cavities from the external environment, wherein the plurality of capping particles are functionalized with non-specific binding elements for all or substantially all secreted proteins from the cells;mixing or agitating the mixture to form a plurality of capped particles with one or more cells contained in the plurality of capped particles:incubating the plurality of capped particles containing the one or more cells;de-capping the plurality of capped particles to generate a plurality of capping particles having accumulated secretions thereon; andanalyzing the plurality of capping particles having accumulated secretions thereon with a mass spectrometer.

49. A method of performing an enzyme assay comprising:generating a mixture of (1) enzymes, (2) a plurality of cavity-containing particles including respective cavities therein that open to an external environment of the plurality' of cavity-containing particles via respective openings, and (3) a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavitycontaining particles to seal or occlude the respective cavities from the external environment, wherein the plurality of cavity-containing particles and / or the plurality of capping particles contain enzyme substrates;mixing or agitating the mixture to form a plurality of capped particles with the enzymes located in the plurality of capped particles;2025-267-2incubating the plurality of capped particles, wherein the enzymes interact with the enzyme substrate to generate a colorimetric, fluorescent, or chemiluminescent signal;determining the signal level for each of the plurality of capped particles; and sorting the plurality of capped particles based at least in part on the determined signal level.

50. A method of performing single-cell RNA-sequencing assay comprising: generating a mixture of (1) cells of a first cell type, (2) a plurality of cavity -containing particles including respective cavities therein that open to an external environment of the plurality of cavity-containing particles via respective openings, and (3) a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity-containing particles to seal or occlude the respective cavities from the external environment, wherein the plurality' of capping particles have a unique oligonucleotide barcode and nucleic acid capture moiety;mixing or agitating the mixture to form a plurality of capped particles with single cells of the first cell type contained in the plurality of capped particles;lysing the single cells of the first cell type contained in the plurality of capped particles to release mRNA;capturing mRNA in the plurality of capped particles;forming cDNA from the captured mRNA and amplifying the same; and sequencing the amplified cDNA.

51. The method of claim 50, further comprising associating the sequence of the cDNA and / or mRNA with a specific cell based on the unique oligonucleotide barcode.

52. The method of claims 50 or 51, wherein the plurality of cavity -containing particles and / or the plurality of capping particles comprise one or more secretion capture moieties disposed thereon for capturing secretions from the single cells.

53. The method of any of claims 50-51, further comprising introducing a plurality of cells of a second cell type prior to mixing or agitating the mixture to form a plurality of capped particles with single cells of the first cell ty pe and one or more cells of the second cell type contained therein.2025-267-254. A kit comprising:a plurality of cavity-containing particles including respective cavities therein that open to an external environment of the plurality of cavity -containing particles via respective openings;a plurality of capping particles that are configured to interact or associate with the openings of the plurality of cavity-containing particles to at least partially seal or occlude the respective cavities from the external environment; andwherein the plurality of cavity-containing particles and the plurality of capping particles are located in either a common container or vessel or separate containers or vessels.

55. The kit of claim 54, wherein the plurality of cavity -containing particles and / or the plurality of capped particles are provided in separate containers or vessels as an aqueous suspension with a defined number of particles per volume such that w hen the entire suspensions from the two containers or vessels are mixed the plurality of cavity -containing particles and the plurality of capping particles are mixed at a pre-defined ratio.

56. The kit of claim 54, w herein the plurality of cavity -containing particles and / or the plurality of capping particles are provided in a dried or lyophilized state in separate containers or vessels.

57. The kit of claim 54, w herein the plurality of cavity -containing particles and the plurality of capping particles are contained in a common vessel or container as a dried or lyophilized mixed powder containing a pre-defined ratio of cavity-containing particles to capping particles.

59. The kit of claim 54, w herein the plurality of cavity -containing particles are contained in a first vessel or container and the plurality of capping particles are barcoded and contained in a plurality of separate vessels or containers with each separate vessel or container comprising a different barcode associated with the plurality’ of capping particles contained therein.

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