WO2025155691A1

WO2025155691A1 - Plastic nanoparticle compositions and methods of use thereof

Info

Publication number: WO2025155691A1
Application number: PCT/US2025/011826
Authority: WO
Inventors: Emily K. BOWMAN; Christian SILTANEN; Emily D. DUNCAN; Sabrina EDWARDS; Miles Gander; Johan A. KERS
Original assignee: Birch Biosciences Inc
Current assignee: Birch Biosciences Inc
Priority date: 2024-01-17
Filing date: 2025-01-16
Publication date: 2025-07-24
Anticipated expiration: 2026-07-17

Abstract

Provided are labeled particles, such as an organic polymer having one or more luminescent labels, compositions thereof, and methods of using labeled particles for monitoring degradation reactions.

Description

PLASTIC NANOPARTICLE COMPOSITIONS

AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63/621,759, filed January 17, 2024, entitled “PLASTIC NANOPARTICLE COMPOSITIONS AND METHODS OF USE THEREOF,” which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Bio-enzymatic plastic recycling uses enzymes as “molecular scissors” to hydrolyze plastic into monomers or oligomers that can be recovered and used as a drop-in replacement for plastic manufacturing. Bio-enzymatic recycling could enable manufacture of 100% recycled plastic with the same performance properties as virgin plastic produced from petrochemicals, with significant reductions in energy consumption and greenhouse gas emissions.

[0003] Conventional approaches for monitoring plastic degradation and screening for plastic - degrading enzymes involve the use of surrogate assays, such as nitrophenol assays and synthetic fluorescent polyurethane analogue probe systems, which do not directly measure the degradation of the plastic substrate of interest.

SUMMARY

[0004] The present disclosure, in some aspects, provides compositions and methods for monitoring degradation reactions by directly measuring degradation of a plastic material, such as an organic polymer (e.g., a polyester-containing polymer). In some embodiments, the disclosure provides compositions comprising polymeric beads having labeled plastic particles uniformly encapsulated therein, and methods of using the same. [0005] In some aspects, the disclosure provides compositions comprising a labeled particle and a polymeric bead. In some embodiments, the labeled particle comprises an organic polymer comprising one or more luminescent labels. In some embodiments, the labeled particle is encapsulated in the polymeric bead. In some embodiments, the compositions comprise a surfactant. In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the surfactant comprises a copolymer (e.g., a poloxamer). [0006] In some embodiments, a labeled particle of the compositions and methods described herein comprises an organic polymer comprising one or more luminescent labels (e.g., one or more fluorescent dyes). In some embodiments, the organic polymer comprises a polyether- containing polymer, a polyester-containing polymer, a polypropylene-containing polymer, or a combination thereof. In some embodiments, the organic polymer comprises polyethylene glycol, polyethylene terephthalate, polyester-polyurethane, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene isosorbide terephthalate, polylactic acid, polyhydroxyalkanoate, polybutylene succinate, polybutylene succinate adipate, polybutylene adipate terephthalate, polyethylene furanoate, polycaprolactone, poly(ethylene adipate), polyethylene naphthalate, or a combination thereof. In some embodiments, the organic polymer comprises polyethylene terephthalate.

[0007] In some embodiments, the labeled particle has a diameter of less than about 5,000 nm (e.g., less than about 4,000 nm, less than about 3,000 nm, less than about 2,500 nm, less than about 2,000 nm, or less than about 1,500 nm). In some embodiments, the labeled particle has a diameter of less than about 1,000 nm (e.g., less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 100 nm). In some embodiments, the labeled particle has a diameter of between about 100 nm and about 1,000 nm, between about 200 nm and about 800 nm, between about 100 nm and about 500 nm, between about 150 nm and about 350 nm, or between about 200 nm and about 250 nm.

[0008] In some embodiments, a polymeric bead of the compositions and methods described herein comprises polyacrylamide, agarose, polyethylene glycol, alginate, and/or polystyrene. In some embodiments, the polymeric bead comprises polyacrylamide. In some embodiments, at least one oligonucleotide tag is attached to the polymeric bead. In some embodiments, the at least one oligonucleotide tag comprises one or more of a sequence encoding an enzyme, a restriction site (e.g., BSAII), a poly-T sequence, a molecular barcode sequence, a sample index sequence, a primer site sequence, and a promoter sequence.

[0009] In some embodiments, the polymeric bead has a diameter of less than about 250 pm (e.g., less than about 225 pm, less than about 200 pm, less than about 175 pm, less than about 150 pm, less than about 125 pm, less than about 100 pm, less than about 75 pm, less than about 70 pm, less than about 50 pm, less than about 25 pm, or less than about 10 pm). In some embodiments, the polymeric bead has a diameter of between about 10 pm and about 250 pm, between about 10 pm and about 200 pm, between about 50 pm and about 150 pm, between about 100 pm and about 200 |am, between about 10 pm and about 75 jam, or between about 25 |am and about 70 |am.

[0010] In some embodiments, a polymeric bead has a diameter of less than about 250 pm, and a labeled particle encapsulated therein has a diameter of less than about 1,000 nm. In some embodiments, the polymeric bead has a diameter of less than about 100 pm, and the labeled particle has a diameter of less than about 300 nm. In some embodiments, the polymeric bead has a diameter of between about 25 pm and about 70 pm, and the labeled particle has a diameter of between about 200 nm and about 250 nm.

[0011] In some aspects, the disclosure provides a composition comprising a plurality of droplets, where at least one droplet comprises a labeled particle and a polymeric bead as described herein. In some embodiments, at least one droplet comprises a hydrolase. In some embodiments, at least one droplet comprises a host cell comprising a heterologous polynucleotide encoding the hydrolase. In some embodiments, at least one droplet comprises a nucleic acid encoding the hydrolase.

[0012] In some aspects, the disclosure provides methods of preparing an encapsulated particle. In some embodiments, a method of preparing an encapsulated particle comprises: providing a first fluid comprising a first mixture that comprises a labeled particle (e.g., a labeled particle that comprises an organic polymer comprising one or more luminescent labels), a surfactant (e.g., a surfactant comprising poloxamer 407 (PLURONIC® F-127)), and a polyacrylamide precursor; and generating a droplet comprising the first mixture and a polymerization catalyst, where the polymerization catalyst initiates polymerization of the polyacrylamide precursor to produce a polyacrylamide bead having the labeled particle encapsulated therein. In some embodiments, the method of preparing the encapsulated particle further comprises recovering the polyacrylamide bead from the droplet to obtain a solution comprising the polyacrylamide bead and the surfactant. In some embodiments, the method of preparing the encapsulated particle further comprises removing the surfactant from the solution.

[0013] In some aspects, the disclosure provides methods of monitoring a degradation reaction. In some embodiments, a method of monitoring a degradation reaction comprises: contacting a hydrolase with a labeled particle, where the labeled particle comprises an organic polymer comprising one or more luminescent labels; and detecting a characteristic signal from the one or more luminescent labels, where the characteristic signal is indicative of degradation of the organic polymer by the hydrolase. [0014] In some embodiments, detecting the characteristic signal comprises detecting a decrease in luminescence from the one or more luminescent labels. In some embodiments, the decrease in luminescence is indicative of degradation of the organic polymer by the hydrolase. In some embodiments, the decrease in luminescence comprises a decrease in luminescence intensity over time. In some embodiments, the decrease in luminescence comprises an increase in luminescence intensity over time.

[0015] In some embodiments, contacting the hydrolase with the labeled particle comprises contacting the hydrolase with a polymeric bead comprising the labeled particle. Thus, in some embodiments, the labeled particle is encapsulated in a polymeric bead in accordance with the compositions described herein.

[0016] In some aspects, the disclosure provides methods of screening enzymes for degradation activity. Accordingly, in some aspects, the disclosure provides methods comprising: (a) generating a plurality of droplets, at least one droplet of the plurality comprising: an enzyme; and a labeled particle comprising an organic polymer that comprises one or more luminescent labels; (b) sorting the plurality of droplets based on a characteristic signal detected from the one or more luminescent labels, wherein the characteristic signal is indicative of degradation of the organic polymer by the enzyme; and (c) determining the identity of the enzyme in one or more droplets obtained from the sorting.

[0017] In some embodiments, at least one droplet comprises a nucleic acid encoding the enzyme. In some embodiments, at least one droplet comprises a host cell comprising the nucleic acid. In some embodiments, (c) comprises sequencing the nucleic acid to determine the identity of the enzyme. In some embodiments, (a) comprises: incubating the nucleic acid under conditions to express the enzyme in the at least one droplet; and adding the labeled particle to the at least one droplet.

[0018] In some embodiments, the method comprises, prior to (b): incubating the enzyme and the labeled particle under degradation conditions. In some embodiments, the degradation conditions comprise one or more conditions selected from the group consisting of a temperature of between about 30 °C and about 90 °C, a pH below about 7.0, and an increased plastic recalcitrance.

[0019] In some embodiments, (b) comprises sorting the plurality of droplets using fluorescence-activated droplet sorting (FADS). In some embodiments, (b) comprises sorting the plurality of droplets using fluorescence-activated cell sorting (FACS).

[0020] In some embodiments, the labeled particle is a labeled particle as described herein. For example, in some embodiments, the labeled particle is encapsulated in a polymeric bead. In some embodiments, at least one oligonucleotide tag is attached to the polymeric bead. In some embodiments, the at least one oligonucleotide tag comprises one or more of a sequence encoding the enzyme, a restriction site, a poly-T sequence, a molecular barcode sequence, a sample index sequence, a primer site sequence, and a promoter sequence. In some embodiments, the method comprises, prior to (b): attaching the at least one oligonucleotide tag to a nucleic acid encoding the enzyme in the at least one droplet.

[0021] The details of certain embodiments of the disclosure are set forth in the Detailed Description. Other features, objects, and advantages of the disclosure will be apparent from the Examples, Drawings, and Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying Drawings, which constitute a part of this specification, illustrate several embodiments of the disclosure and together with the accompanying description, serve to explain the principles of the disclosure.

[0023] FIGs. 1A-1B show example results for beads in microdroplets with enzyme- secreting cell supernatants. FIG. 1A shows images of polyethylene terephthalate (PET) nanoparticle- filled polyacrylamide beads encapsulated with three different enzymes of decreasing PETase activity from left to right. FIG. IB shows an example of data obtained from an enzyme ladder on a microfluidic sorting instrument (Styx, Atrandi Biosciences, Inc.). The example data represent four different cell culture supernatants containing enzymes with high to low activity (results shown left to right) on PET nanoparticle beads, which were incubated in droplets with cell supernatants for 18 hours at 50 °C, resulting in full clearance by the highest-activity enzyme.

[0024] FIGs. 2A-2B show example results from the preparation of PET nanoparticlecontaining polyacrylamide beads. FIG. 2A shows plots of degradation activity for enzymes using nanoparticle-containing beads prepared without a step of surfactant removal. FIG. 2B shows plots of degradation activity for enzymes using nanoparticle-containing beads treated with a surfactant removal step.

[0025] FIGs. 3A-3E schematically illustrate a microfluidic screening process. In the example process shown, an expression system is encapsulated in pico-liter sized microdroplets and incubated to achieve sufficient protein expression (FIG. 3A), and fluorescent dye-stained plastic nanoparticles are encapsulated in polyacrylamide beads optionally modified to contain primers (FIG. 3B). The incubated enzyme samples of FIG. 3A and beads of FIG. 3B are merged (FIG. 3C). As shown in FIG. 3D, the merged sample containing beads without (top) or with (bottom) primers is incubated to allow for degradation of the plastic inside of the beads. As shown in FIG. 3E, the incubated samples of FIG. 3D are analyzed via fluorescence activated droplet sorting (FADS) or fluorescence activated cell sorting (FACS), depending on the method of DNA retrieval being used, with an optional intervening step of PCR amplification of samples if a primer-laden bead was used (bottom). [0026] FIG. 4 depicts results showing DNA conjugation to nanoparticle-containing beads.

DETAILED DESCRIPTION

[0027] Among other aspects, the disclosure provides labeled particles, compositions comprising labeled particles and polymeric beads, and methods of using the same. In some embodiments, labeled particles and compositions thereof can be useful for evaluating bio- enzymatic degradation of plastic materials, which allows for the identification and optimization of enzymes, including improved enzyme variants, for degrading plastic materials.

Plastic Particle Compositions

[0028] Aspects of the disclosure relate to the development of polymeric beads which can be densely and uniformly packed with labeled particles. In some aspects, the disclosure provides compositions comprising a labeled particle and a polymeric bead, where the labeled particle is encapsulated in the polymeric bead. As described in Examples 1-3, such compositions can be used as substrates in degradation reactions to provide direct and improved readouts for evaluating degradation activity of enzymes, such as hydrolases.

[0029] In some embodiments, a labeled particle described herein comprises an organic polymer. As used herein, an organic polymer refers to a compound or mixture of compounds comprising monomers that are linked by covalent chemical bonds and comprise one or more carbon atoms (e.g., one or more carbon-containing groups). Within the context of the disclosure, an organic polymer includes natural or synthetic polymers, which can comprise a single type of monomer (e.g., homopolymers) or a mixture of different monomers (e.g., copolymers or heteropolymers).

[0030] In some embodiments, an organic polymer comprises polyester (e.g., polyethylene terephthalate), polyamide (e.g., nylon), polycarbonate, low-density polyethylene (e.g., polyethylene having a density of up to 930 kg/m³, such as 917-930 kg/m³), linear low-density polyethylene (e.g., linear polyethylene having a density of up to 930 kg/m³, such as 917-930 kg/m³), high-density polyethylene (e.g., polyethylene having a density of at least 930 kg/m³, such as 930-970 kg/m³), polypropylene, polystyrene, polyvinyl chloride, acrylic or polymethyl methacrylate, polyurethane, polycaprolactone, polylactic acid (PLA), polyglycolide (PGA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyanhydride, polyurea, poly(lactic-co-glycolic acid) (PLGA), polydioxanone (PDS), elastane (e.g., spandex, lycra), or a combination thereof.

[0031] In some embodiments, an organic polymer comprises a polyester-containing polymer, a polyether-containing polymer, a polypropylene-containing polymer, or a combination thereof. In some embodiments, an organic polymer comprises a polyester-containing polymer (e.g., a polymer comprising one or more esters or polyesters, which can be one or more esters or polyesters of the same type or a mixture of esters or polyesters of a different type). In some embodiments, an organic polymer comprises polyethylene glycol, polyethylene terephthalate, polyester-polyurethane, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene isosorbide terephthalate, polylactic acid, polyhydroxyalkanoate, polybutylene succinate, polybutylene succinate adipate, polybutylene adipate terephthalate, polyethylene furanoate, polycaprolactone, poly(ethylene adipate), polyethylene naphthalate, or a combination thereof. In some embodiments, an organic polymer comprises polyethylene terephthalate.

[0032] In some embodiments, an organic polymer of a labeled particle described herein comprises one or more luminescent labels (e.g., one or more fluorescent dyes). In some embodiments, a labeled particle comprises a plurality of luminescent labels. In some embodiments, a labeled particle comprises at least two (e.g., three or more, four or more, five or more, ten or more, 100 or more, 1,000 or more, 2-100, 5-25, 10-50, 20-75, 2-10, 10-25, 50- 100, 100-1,000, 100-500, 500-5,000, 1,000-10,000, 1,000-5,000) luminescent labels. In some embodiments, a labeled particle comprises up to ten (e.g., up to five, up to three, 1-10, 2-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) luminescent labels.

[0033] As used herein, a luminescent label refers to a fluorophore or a dye. In some embodiments, a luminescent label comprises an aromatic or hetero aromatic compound, such as a pyrene, anthracene, naphthalene, naphthylamine, acridine, stilbene, indole, benzindole, oxazole, carbazole, thiazole, benzothiazole, benzoxazole, phenanthridine, phenoxazine, porphyrin, quinoline, ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamine, xanthene, or other like compounds. Examples of dyes for use in accordance with a labeled particle of the disclosure include, without limitation, fluorescein or rhodamine dyes (e.g., 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'- dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX)), naphthylamine dyes, coumarin dyes (e.g., 3-phenyl-7-isocyanatocoumarin), acridine dyes (e.g., 9-isothiocyanatoacridine, acridine orange), cyanine dyes (e.g., Cy®3, Cy®3.5, Cy®3B), Cy®5, Cy®5.5, Cy®A), BODIPY® dyes, benzoxazoles, stilbenes, pyrenes, and the like, such as 4-dimethylamino-4'-nitrostilbene (DANS), 9-(diethylamino)-5H- benzo[a]phenoxazin-5-one or 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile Red).

[0034] In some embodiments, a luminescent label comprises a quencher-fluorophore pair which produces an increase in fluorescence upon hydrolysis in accordance with methods described herein. In some embodiments, a quencher-fluorophore pair comprises a quenching molecule, such as Dabcyl (4-(dimethylaminoazo)benzene-4-carboxylic acid) or a dark quencher (e.g., a Black Hole Quencher® dye), paired with a fluorescent dye described herein or known in the art.

[0035] In some embodiments, a luminescent label comprises an amide- or ester-conjugated dye which produces an increase in fluorescence upon hydrolysis in accordance with methods described herein. For example, in some embodiments, a labeled particle comprises 4- methylumbelliferyl laurate (4-MUL). Methods for preparing and using labeled plastic polymers comprising 4-MUL are known in the art (see, for example, Ruthi, et al. Front. Microbiol. 2023, May 10:14:1178474. doi: 10.3389/fmicb.2023.1178474. eCollection 2023, the relevant contents of which are incorporated herein by reference).

[0036] In some embodiments, a labeled particle of the disclosure has a diameter of less than about 1,000 nm. In some embodiments, the diameter of a labeled particle can refer to a diameter of one particle among a population of particles, or the diameter can refer to an average diameter of more than one particle among a population of particles. In some embodiments, a labeled particle of the disclosure has a diameter of less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 100 nm. In some embodiments, the labeled particle has a diameter of between about 50 nm and about 1,000 nm, 100 nm and about 1,000 nm, between about 200 nm and about 800 nm, between about 100 nm and about 500 nm, between about 100 nm and about 300 nm, between about 150 nm and about 350 nm, between about 150 nm and about 250 nm, or between about 200 nm and about 250 nm. In some embodiments, a labeled particle of the disclosure has a diameter of less than about 5,000 nm. In some embodiments, a labeled particle of the disclosure has a diameter of less than about 4,000 nm, less than about 3,000 nm, less than about 2,500 nm, less than about 2,000 nm, or less than about 1,500 nm (e.g., 1,000-5,000 nm, 1,000-2,500 nm, 2,000-4,000 nm, or 2,500-5,000 nm).

[0037] In some embodiments, a labeled particle of the disclosure is prepared from a plastic material comprising one or more polyesters. In some embodiments, the plastic material is in a crystalline or semi-crystalline form. In some embodiments, the plastic material is in an amorphous form. In some embodiments, the plastic material is plastic waste in a suitable form for preparing a labeled nanoparticle described herein. For example, in some embodiments, the plastic material is plastic waste that has been processed to a more suitable form for preparing a labeled particle described herein (e.g., pellets, flakes, powder, discs, sheets). In some embodiments, the plastic material comprises microplastic particles that are dye-labeled and milled or suspended in a slurry for encapsulation. Methods for preparing labeled particles of the disclosure are known in the art. See, for example, Rodriguez- Hemandez, et al. Environ. Sci.: Nano, 2019, 6, 2031 (describing methods of preparing labeled polyethylene terephthalate particles); Cassano, et al. ACS Appl. Nano Mater. 2021, 4, 1551-1557 (describing methods of preparing labeled polypropylene particles); Alferiev, et al. ACS Appl Polym Mater. 2022, 4(2): 1196-1206 (describing methods of preparing labeled polylactic acid particles); and Stanton, et al. Environ. Sci. Technol. Eett. 2019, 6(10): 606- 611 (describing methods of staining microplastic particles), the relevant contents of each of which are incorporated herein by reference.

[0038] In some embodiments, a labeled particle of a composition described herein is encapsulated in a polymeric bead. In some embodiments, a polymeric bead comprises polyacrylamide, agarose, polyethylene glycol, alginate, and/or polystyrene. In some embodiments, the polymeric bead comprises polyacrylamide. In some embodiments, the composition comprises a labeled polyethylene terephthalate particle encapsulated in a polyacrylamide bead.

[0039] In some embodiments, a polymeric bead of the disclosure has a diameter of less than about 250 pm. In some embodiments, the diameter of a polymeric bead can refer to a diameter of one polymeric bead among a population of polymeric beads, or the diameter can refer to an average diameter of more than one polymeric bead among a population of polymeric beads. In some embodiments, a polymeric bead of the disclosure has a diameter of less than about 225 pm, less than about 200 pm, less than about 175 pm, less than about 150 pm, less than about 125 pm, less than about 100 pm, less than about 75 pm, less than about 70 pm, less than about 50 pm, less than about 25 pm, or less than about 10 pm. In some embodiments, the polymeric bead has a diameter of between about 1 pm and about 250 pm, between about 10 pm and about 250 pm, between about 10 pm and about 200 |am, between about 50 pm and about 150 pm, between about 150 pm and about 250 pm, between about 100 pm and about 200 |am, between about 10 pm and about 100 pm, between about 10 pm and about 75 pm, between about 25 pm and about 70 pm, or between about 50 pm and about 70 |am.

[0040] In some embodiments, a polymeric bead has a diameter of less than about 250 pm, and a labeled particle encapsulated therein has a diameter of less than about 1,000 nm. In some embodiments, the polymeric bead has a diameter of less than about 200 pm, and a labeled particle encapsulated therein has a diameter of less than about 500 nm. In some embodiments, the polymeric bead has a diameter of less than about 100 pm, and a labeled particle encapsulated therein has a diameter of less than about 300 nm. In some embodiments, the polymeric bead has a diameter of between about 10 pm and about 100 pm, and a labeled particle encapsulated therein has a diameter of between about 200 nm and about 300 nm. In some embodiments, the polymeric bead has a diameter of between about 25 pm and about 70 pm, and a labeled particle encapsulated therein has a diameter of between about 200 nm and about 250 nm.

[0041] In some embodiments, a polymeric bead comprises one or more labeled particles described herein. In some embodiments, a polymeric bead comprises a single labeled particle. In some embodiments, a polymeric bead comprises two or more labeled particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, labeled particles). In some embodiments, a polymeric bead comprises 1,000 labeled particles or fewer (e.g., 100-1,000, 250-1,000, or 500-1,000 labeled particles). In some embodiments, a polymeric bead comprises 500 labeled particles or fewer. For example, in some embodiments, the polymeric bead comprises at least 1 and up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, or up to 25 labeled particles (e.g., 1-500, 100-500, 250-500, 300-400, or 100-200 labeled particles). In some embodiments, a polymeric bead has a diameter of between about 50 pm and about 100 pm (e.g., about 70 pm) and comprises between about 300 and 400 (e.g., about 350) labeled particles.

[0042] In some embodiments, a labeled particle of a polymeric bead is characterized by a zeta potential of between about -40 mV and about -60 mV (e.g., -40 to -50 mV, -50 to -60 mV, -45 to -55 mV, or about -50 mV). Methods of characterizing particles and determining zeta potential are known in the art. For example, labeled particles of the disclosure can be analyzed by dynamic light scattering using the appropriate instrumentation (e.g., Zetasizer) to determine stability based on trends appropriate for a particular organic polymer. Particle stability and fitness for encapsulation can be evaluated using desired parameters, such as poly dispersion index (Pdl) and standard deviation of size that should be between 0.1-0.3 for Pdl and within 10% of the size for standard deviation. Zeta potential can provide a measure for stability over time for labeled particles, such as PET nanoparticles, which are typically considered suitable for encapsulation when characterized by a zeta potential in the range of - 40 mV to -60 mV.

[0043] In some embodiments, a polymeric bead of the disclosure comprises one or more modifications. As described in Example 3, compositions described herein can be used in enzyme screening methods that can be performed by relating the identity of a particular enzyme (genotype) to its degradation activity for a labeled particle (phenotype). To facilitate PCR-based identification during such methods, the polymeric bead can be modified to comprise an oligonucleotide tag, such as a barcoded primer, for isolation and/or PCR amplification of a gene encoding the enzyme.

[0044] Accordingly, in some embodiments, a polymeric bead of the disclosure comprises at least one oligonucleotide tag attached to the polymeric bead. In some embodiments, the at least one oligonucleotide tag comprises a sequence encoding an enzyme (e.g., a hydrolase). In some embodiments, the at least one oligonucleotide tag comprises a restriction site (e.g., a sequence recognized by a restriction enzyme, which can bind and cleave the oligonucleotide tag at the restriction site). In some embodiments, the at least one oligonucleotide tag comprises a poly-T sequence (e.g., a sequence of contiguous thymine bases capable of hybridizing to a poly-A tail of an RNA molecule). In some embodiments, the at least one oligonucleotide tag comprises a target- specific sequence (e.g., a sequence capable of hybridizing to a target sequence, such as a target gene). In some embodiments, the polymeric bead comprises a plurality of oligonucleotide tags, where each oligonucleotide tag of the plurality comprises a different target- specific sequence (e.g., sequences capable of hybridizing to different target sequences, such as different regions of one or more target genes). In some embodiments, the at least one oligonucleotide tag comprises a molecular barcode sequence (e.g., a random or otherwise unique sequence that allows for deduplication of sequence reads to account for bias during amplification). In some embodiments, the at least one oligonucleotide tag comprises a sample index sequence (e.g., a sequence that uniquely identifies a sample or source, such as a single cell or sample of cells, which facilitates multiplex analysis of sequencing results). In some embodiments, the at least one oligonucleotide tag comprises a primer site sequence (e.g., a known sequence for which primers can be designed to hybridize and extend from in an amplification and/or sequencing reaction). In some embodiments, the at least one oligonucleotide tag comprises a promoter sequence (e.g., a T7 promoter sequence that allows for in vitro transcription using T7 RNA polymerase).

[0045] Methods for preparing polymeric beads of the disclosure are provided herein and known in the art. In some embodiments, a polymeric bead is prepared by dissolving a labeled particle described herein in a monomeric precursor, and the resulting mixture is incubated under polymerization conditions to promote formation of bead-encapsulated particles. For example, in some embodiments, a polyacrylamide bead is prepared by dissolving a labeled particle in acrylamide monomer(s), and the resulting mixture is incubated under conditions to promote formation of polyacrylamide bead-encapsulated particles. See also, for example, Wang, et al. Adv. Sci. 2020, 7, 1903463 (describing methods of preparing polyacrylamide beads); and Zilionis, et al. Nat. Protoc. 2017, 12, 44-73 (describing methods of preparing hydrogel beads containing barcoded primer oligonucleotides), the relevant contents of each of which are incorporated herein by reference.

[0046] In some embodiments, a composition of the disclosure comprises a surfactant. For example, in some embodiments, a surfactant can be used in the preparation of bead- encapsulated particles to maintain particle stability during encapsulation. Accordingly, in some embodiments, a composition comprising bead-encapsulated particles further comprises a surfactant. In some embodiments, when a surfactant is used during preparation of bead- encapsulated particles, the surfactant can be subsequently removed from the composition. For example, as described herein, compositions of the disclosure can be used for monitoring enzymatic degradation reactions. In some instances, a surfactant can inhibit or otherwise interfere with enzymatic activity, and it therefore may be desirable to remove the surfactant before the composition is used to monitor enzymatic degradation reactions.

[0047] In some embodiments, the surfactant is a non-ionic surfactant. In some embodiments, the surfactant comprises a copolymer, such as a triblock copolymer (e.g., a poloxamer). Poloxamers (also known by the trade name PLURONIC®) are low molecular weight triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). The absolute and relative sizes of the PEO and PPO blocks can be varied to provide surfactant compounds having desired properties suitable for different implementations according to the disclosure. In some embodiments, the surfactant comprises poloxamer 407 (PLURONIC® F-127).

[0048] In some aspects, the disclosure provides a composition comprising a plurality of droplets, where at least one droplet comprises a labeled particle and a polymeric bead as described herein. In some embodiments, a droplet refers to an aqueous droplet, such as a droplet comprising a water-in-oil emulsion.

[0049] In some embodiments, the at least one droplet comprises a hydrolase. As used herein, a hydrolase (corresponding to EC 3 in the EC number classification of enzymes) refers to an enzyme that can use water to break a chemical bond. In some embodiments, the hydrolase is a PET hydrolase. As used herein, a “polyethylene terephthalate hydrolase” or “PET hydrolase” refers to a hydrolase enzyme that can degrade polyethylene terephthalate, abbreviated “PET.”

[0050] In some embodiments, the at least one droplet comprises a host cell comprising a heterologous polynucleotide encoding a hydrolase. In some embodiments, a host cell is any cell capable of replicating, transcribing, and/or translating a heterologous polynucleotide for producing a recombinant hydrolase. In some embodiments, the host cell is a prokaryotic cell (e.g., Escherichia coli, Bacillus subtilis, Salmonella typhimurium, a species of Streptomyces, or any prokaryotic cell known in the art to be suitable for expressing and/or isolating a recombinant protein, such as a hydrolase of the disclosure). In some embodiments, the host cell is a eukaryotic cell (e.g., yeast cell, mammalian cell, plant cell, avian cell, amphibian cell, plant cell, fish cell, or insect cell, or any eukaryotic cell known in the art to be suitable for expressing and/or isolating a recombinant protein, such as a hydrolase).

[0051] In some embodiments, a host cell has been or is transformed or transfected with, or otherwise contained or contains, a heterologous polynucleotide, vector, or expression cassette that encodes a hydrolase. In some embodiments, the heterologous polynucleotide is introduced into a host cell using any method known in the art, including transformation, transfection, and transduction. It should be appreciated that any heterologous polynucleotide associated with the disclosure can be expressed transiently in a host cell or can be integrated into the genome of a host cell.

[0052] In some embodiments, the at least one droplet comprises a nucleic acid encoding a hydrolase. The nucleic acid can be naturally-occurring or non-naturally occurring.

[0053] In some embodiments, the at least one droplet comprises a vector comprising a nucleic acid encoding a hydrolase. The nucleic acid can be naturally-occurring or non- naturally occurring. Examples of vectors suitable for use in accordance with the disclosure include, without limitation, plasmids, phagemids, phasmids, cosmids, viruses, artificial chromosomes, shuttle vectors, expression vectors, and the like. Any vector suitable for expression of polynucleotides described herein may be compatible with aspects of the disclosure. [0054] In some embodiments, the at least one droplet comprises an expression cassette comprising a nucleic acid encoding a hydrolase. The nucleic acid can be naturally-occurring or non-naturally occurring. In some embodiments, the expression cassette comprises a vector of the disclosure in operable linkage with a nucleic acid of the disclosure. In some embodiments, the expression cassette comprises one or more expression control elements known in the art, including, without limitation, enhancers, promoters, factor-specific binding sites, terminators, and/or ribosome binding sites.

Methods of Encapsulating Plastic Particles

[0055] In some aspects, the disclosure provides methods of preparing an encapsulated particle, such as a labeled plastic particle encapsulated in a polymeric bead, which can be used to directly measure enzymatic degradation according to a method of the disclosure. As described in Example 2, such methods of encapsulating advantageously provide beads having pore sizes that are sufficiently small enough to retain labeled particles while being sufficiently large enough to allow enzymes to enter the beads to degrade the labeled particles. [0056] In some embodiments, a method of preparing an encapsulated particle comprises: providing a first fluid comprising a first mixture that comprises a labeled particle, a surfactant, and a polyacrylamide precursor; and generating a droplet comprising the first mixture and a polymerization catalyst, where the polymerization catalyst initiates polymerization of the polyacrylamide precursor to produce a polyacrylamide bead having the labeled particle encapsulated therein. In some embodiments, the labeled particle is as described herein (e.g., a labeled particle that comprises an organic polymer comprising one or more luminescent labels). In some embodiments, the surfactant is as described herein (e.g., a surfactant comprising poloxamer 407 (PLURONIC® F-127)).

[0057] In some embodiments, the method of preparing the encapsulated particle further comprises recovering the polyacrylamide bead from the droplet to obtain a solution comprising the polyacrylamide bead and the surfactant. In some embodiments, the droplet comprises a water-in-oil droplet of an emulsion, and recovering the polyacrylamide bead comprises contacting the emulsion with an emulsion breaking buffer and/or a deemulsification buffer. In some embodiments, the emulsion breaking buffer comprises perfluorooctanol (e.g., perfluorooctanol in 3M™ NOVEC™ 7500 fluid). In some embodiments, the de-emulsification buffer comprises a detergent (e.g., 1% Triton in a PBS buffer). [0058] In some embodiments, the method of preparing the encapsulated particle further comprises removing the surfactant from the solution. As described in Example 3, the surfactant can be used in the preparation of bead-encapsulated particles to maintain particle stability during encapsulation, but it was discovered that the presence of the surfactant in a degradation reaction can inhibit or otherwise interfere with enzymatic activity. In some embodiments, removing the surfactant from the solution comprises incubating the solution in a buffer that does not comprise the surfactant. In some embodiments, removing the surfactant from the solution comprises rinsing the solution with a buffer that does not comprise the surfactant. In some embodiments, the buffer that does not comprise the surfactant comprises PBS.

[0059] In some embodiments, the method of preparing the encapsulated particle further comprises, prior to generating the droplet: providing a second fluid comprising the polymerization catalyst, where the first and second fluids are immiscible. In some embodiments, the first fluid is an aqueous fluid, and the second fluid comprises an oil. In some embodiments, the first fluid comprises an oil, and the second fluid is an aqueous fluid. In some embodiments, generating the droplet comprises combining the first fluid and the second fluid to generate an emulsion.

[0060] In some embodiments, the polyacrylamide precursor comprises acrylamide and bisacrylamide. In some embodiments, the polyacrylamide precursor comprises acrylamide to bis-acrylamide in a ratio of between about 5:1 and about 25:1 (e.g., between about 5:1 and about 20:1, between about 10:1 and about 25:1, between about 10:1 and about 20:1, between about 15:1 and about 25:1, between about 15:1 and about 20:1). In some embodiments, the polyacrylamide precursor comprises acrylamide to bis-acrylamide in a ratio of about 19:1. In some embodiments, the polyacrylamide precursor comprises acrylamide and bis-acrylamide at a concentration of between about 5% and 25% (e.g., 5-20%, 5-15%, 5-10%, 10-25%, 10- 20%, 10-15%, 15-25%, 15-20%) in the first fluid. In some embodiments, the polyacrylamide precursor comprises acrylamide and bis-acrylamide at a concentration of about 20% in the first fluid.

[0061] In some embodiments, the polymerization catalyst comprises tetramethylethylenediamine (TEMED) and/or ammonium persulfate (APS). In some embodiments, the polymerization catalyst comprises TEMED, and the first fluid comprises APS. In some embodiments, the polymerization catalyst comprises APS, and the first fluid comprises TEMED. In some embodiments, the first mixture comprises at least one oligonucleotide tag as described herein. In some embodiments, the polyacrylamide bead is attached to the at least one oligonucleotide tag.

Methods of Monitoring Degradation

[0062] In some aspects, the disclosure provides methods of monitoring a degradation reaction. Advantageously, methods of the disclosure directly measure enzymatic degradation of a plastic substrate by a hydrolase. Previous approaches for monitoring enzymatic degradation have been limited to detection of specific degradation products and/or indirect measurements through surrogate assays. As demonstrated in Examples 1-3 of the disclosure, the methods described herein measure degradation directly, which improves the ability to identify enzymes or enzyme variants that have improved degradation activity and are capable of accessing and uniformly breaking down a complex polymer matrix. Moreover, the methods described herein are not limited by detection of specific degradation products and can therefore be adapted to any synthetic polymer of interest.

[0063] In some embodiments, a method of monitoring a degradation reaction comprises contacting a hydrolase with a labeled particle that comprises an organic polymer comprising one or more luminescent labels; and detecting a characteristic signal from the one or more luminescent labels, where the characteristic signal is indicative of degradation of the organic polymer by the hydrolase.

[0064] In some embodiments, the method comprises contacting a hydrolase with a composition described herein. For example, in some embodiments, the method comprises contacting the hydrolase with a labeled particle as described herein. In some embodiments, the method comprises contacting the hydrolase with a polymeric bead comprising a labeled particle (e.g., a labeled particle encapsulated in a polymeric bead). However, it should be appreciated that the methods of the disclosure can be performed using a labeled particle that is not encapsulated in a polymeric bead.

[0065] In some embodiments, methods of the disclosure monitor degradation by detecting a characteristic signal from one or more luminescent labels of a labeled particle. As used herein, a characteristic signal refers to a detectable signal that is indicative of degradation of the labeled particle. In some embodiments, the characteristic signal comprises a change in luminescence. For example, in some embodiments, a labeled particle of the disclosure can be detected by exposing the labeled particle to an excitation light source and detecting emission from one or more luminescent labels of the labeled particle. In some embodiments, degradation of the labeled particle produces a change in the emission signal, such as a decrease in emission intensity resulting from the loss of luminescent label(s) concomitant with degradation of the particle. In this way, the change in luminescence is indicative of degradation of the labeled particle (e.g., degradation of the organic polymer of the labeled particle). In some embodiments, degradation of the labeled particle produces an increase in emission intensity, for example, where the luminescent labels comprise a quencher- fluorophore pair in which fluorescence quenching is reversed upon degradation.

[0066] In some embodiments, a change in luminescence comprises a change in luminescence over time. In some embodiments, the luminescent signal can be monitored in real-time or at two or more different time points to determine a change in luminescence over time. In some embodiments, a change in luminescence can be determined by evaluating a luminescent signal at one time point relative to a reference value (e.g., a value based on the literature or a control experiment).

[0067] In some embodiments, a change in luminescence comprises a decrease in luminescence intensity over time. In some embodiments, a change in luminescence comprises an increase in luminescence intensity over time. As used herein, luminescence intensity relates to the number of photons per unit time that are emitted by one or more luminescent labels of a labeled particle. In some embodiments, luminescence intensity is detected as emission from a plurality of luminescent labels of a labeled particle.

[0068] As described in Example 3, compositions and methods of the disclosure can be utilized for screening enzymes (e.g., hydrolases) for degradation activity. Thus, in some aspects, the disclosure provides methods of screening, which may be performed using a microfluidic -based format. In some embodiments, a method of the disclosure comprises: (a) generating a plurality of droplets, at least one droplet of the plurality comprising: an enzyme; and a labeled particle comprising an organic polymer that comprises one or more luminescent labels; (b) sorting the plurality of droplets based on a characteristic signal detected from the one or more luminescent labels, wherein the characteristic signal is indicative of degradation of the organic polymer by the enzyme; and (c) determining the identity of the enzyme in one or more droplets obtained from the sorting.

[0069] In some embodiments, the at least one droplet comprises a nucleic acid encoding the enzyme. In some embodiments, the at least one droplet comprises a host cell comprising the nucleic acid (e.g., a host cell comprising a heterologous polynucleotide encoding the enzyme). In some embodiments, determining the identity of the enzyme comprises sequencing at least part of the nucleic acid encoding the enzyme to determine the identity of the enzyme. Sequencing data obtained according to the methods described herein may be used to predict amino acid sequences for additional enzymes having increased degradation activity or enhanced attributes. For example, in some embodiments, the obtained sequencing data is used to generate an activity measurement proxy based on population enrichment from NGS data. In some embodiments, the obtained sequencing data is used as training data for machine learning models. Predictive modeling using sequencing data is known in the art, see, e.g., Peterman N, Levine E. Sort-seq under the hood: implications of design choices on large-scale characterization of sequence-function relations. BMC Genomics. 2016 Mar 9;17:206.

[0070] In some embodiments, generating the plurality of droplets comprises: incubating the nucleic acid under conditions to express the enzyme in the at least one droplet; and adding the labeled particle to the at least one droplet.

[0071] In some embodiments, the method comprises, prior to sorting the plurality of droplets: incubating the enzyme and the labeled particle under degradation conditions. In some embodiments, the degradation conditions comprise one or more conditions selected from the group consisting of a temperature of between about 30 °C and about 90 °C, a pH below about 7.0, and an increased plastic recalcitrance. As used herein, plastic recalcitrance refers to a decrease in enzymatic degradation caused by one or more properties, such as polymer crystallinity, cross-linking, additives, or other polymer properties that limit enzyme accessibility.

[0072] In some embodiments, sorting the plurality of droplets comprises sorting the plurality of droplets using fluorescence-activated droplet sorting (FADS). In some embodiments, sorting the plurality of droplets comprises sorting the plurality of droplets using fluorescence- activated cell sorting (FACS).

[0073] In some embodiments, the labeled particle is a labeled particle as described herein. For example, in some embodiments, the labeled particle is encapsulated in a polymeric bead. In some embodiments, at least one oligonucleotide tag is attached to the polymeric bead. In some embodiments, the at least one oligonucleotide tag comprises one or more of a poly-T sequence, a molecular barcode sequence, a sample index sequence, a primer site sequence, and a promoter sequence. In some embodiments, the method comprises, prior to (b): attaching the at least one oligonucleotide tag to a nucleic acid encoding the enzyme in the at least one droplet.

[0074] The present disclosure is further illustrated by the following Examples, which should not be construed as limiting. EXAMPLES

Example 1. Monitoring Enzymatic Degradation of Labeled Plastic Nanoparticles

[0075] Homogeneous polyethylene terephthalate (PET) nanoparticles were prepared by dissolving PET substrates in an organic solvent and precipitating the solution in water to generate a dispersion of 200-250 nm plastic particles. During the final stage of particle generation, a hydrophobic fluorescent dye was doped into the polymer solution to generate homogeneously stained fluorescent PET nanoparticles.

[0076] The fluorescent PET nanoparticles were concentrated via centrifugation and suspended in a polyacrylamide gel precursor (10% 19:1 polyacrylamide) with surfactant (PLURONIC® F-127). PET nanoparticle-filled polyacrylamide beads were generated via a flow-focusing droplet generation device and isolated from the emulsion post-polymerization. The surfactant (PLURONIC® F-127), which was used to maintain nanoparticle stability during encapsulation, was subsequently removed from the beads using cold phosphate buffered saline (PBS) and an overnight incubation at 4 °C.

[0077] The PET nanoparticle-filled polyacrylamide beads were merged with microdroplets containing either whole cells secreting PETase enzymes, or cell supernatants of these cells expressing enzymes that have different levels of activity. The PET nanoparticle beads were incubated in droplets with cells or cell supernatants for 18 hours at 50 °C and subsequently analyzed by imaging, microfluidic sorting, and fluorescence determination (FIGs. 1A-1B). [0078] FIG. 1A shows images of PET nanoparticle-filled polyacrylamide beads encapsulated with three different enzymes of decreasing PETase activity from left to right. FIG. IB shows an example of data obtained from an enzyme ladder on a microfluidic sorting instrument (Styx, Atrandi Biosciences, Inc.). The example data represent four different cell culture supernatants containing enzymes with high to low activity (results shown left to right) on PET nanoparticle beads, which were incubated in droplets with cell supernatants for 18 hours at 50 °C, resulting in full clearance by the highest-activity enzyme.

[0079] The results of this example demonstrated that the fluorescent plastic nanoparticles provide a means for evaluating enzymatic activity by directly measuring degradation as a loss of fluorescence commensurate with plastic depolymerization. This improves the ability to identify only those enzymes or enzyme variants that have improved degradation activity, rather than simply any enzyme or enzyme variant that may have improved kinetics for breaking certain bonds without necessarily being capable of accessing and uniformly breaking down a complex polymer matrix. Additionally, the encapsulation of fluorescent nanoparticles in polyacrylamide gel beads is expected to keep plastic evenly suspended and prevent aggregation, allowing for improved fluorescence readout.

Example 2. Encapsulation of Fluorescent Nanoparticles in Polyacrylamide Gel Beads [0080] Extensive studies were performed to determine the conditions necessary to obtain beads having pores of sufficient size to allow enzymes to enter the beads to degrade the plastic nanoparticles while retaining the nanoparticles within the beads. This work was performed by evaluating different polyacrylamide ratios and different concentrations thereof using 19:1 polyacrylamide (BIO-RAD®) and 40% bis-acrylamide (BIO-RAD®) combined in the following ratios: 19:1 and 9.5:1. In addition, acrylamide percentage was adjusted to determine the appropriate density of the gel bead. Both 20% and 10% concentrations were tested in combination with the aforementioned ratios. The use of 10% of 19:1 polyacrylamide in the resuspension of fluorescent PET nanoparticles was found to provide nanoparticle-containing beads having an ideal pore size and density for maintaining enzyme activity relationships in regards to plastic degradation.

[0081] The packaging of nanoparticles in beads required a high concentration of surfactant (PLURONIC® F-127 (MILLIPORE SIGMA® P2443)). Experimental data showed that this surfactant inhibited enzyme activity, even when the nanoparticles were encapsulated suspended in the surfactant in the polyacrylamide beads. For example, FIG. 2A shows plots of degradation activity for enzymes using nanoparticle-containing beads prepared without a step of surfactant removal at the indicated ratios of acrylamide to bis-acrylamide. Here, an enzyme known to have relatively lower activity (“Enzyme A”) showed the most degradation (reduction in signal) as compared to more highly active enzymes (“Fast” PETase and “LCC”).

[0082] Surfactant removal was performed by incubating the beads in a large volume of PBS containing no surfactant at 4 °C for up to 48 hours and rinsing repeatedly with cold PBS to remove excess surfactant, which resulted in beads that maintained relative enzymatic activity relationships. FIG. 2B shows plots of degradation activity for the enzymes of FIG. 2A using nanoparticle-containing beads treated with the surfactant removal step. This treatment maintained the expected relative enzyme activity based on experimental data and results in the literature.

[0083] Example Protocol for Preparing PET-Nile Red Nanoparticle Beads

[0084] PET nanoparticles labeled with Nile Red (4 mL) were concentrated via centrifugation at 3000xg for 45-60 minutes. The resulting supernatant was removed, leaving -20-50 pL in the tube, and pellet was resuspended by the addition of 20 pL 1000X PLURONIC® F-127 (MILLIPORE SIGMA® P2443) surfactant. Once PET nanoparticles were fully resuspended, gel precursor was added to the tube (the 10% ammonium persulfate (APS) was added last to avoid destabilizing the nanoparticles): 250 pL 40% 20:1 bis-acrylamide (BIO-RAD®); an appropriate volume of acrydite-modified forward primer (if using DNA isolation system); QS to 900 pL with filtered/sterile H2O; and 100 pL 10% APS.

[0085] PET nanoparticle-filled polyacrylamide beads were generated via a flow-focusing droplet generation device and isolated from the emulsion post-polymerization. For this process, 1.5 mL of 1% TEMED in 2.5% RAN surfactant in Novec 7500 was prepared, and 15 pL of TEMED to 2.5% RAN surfactant in Novec 7500 was added. After loading onto a 60 pm droplet generation chip, droplets were generated with a diameter between 59-65 pm and collected in a 2 mL collection tube (beads were expected to swell slightly to approximately 70 pm in diameter). The aqueous phase was used to make approximately 1 mL of beademulsion that was allowed to incubate for about 30 minutes once collection was complete. Emulsion Breaking Solution was prepared by preparing 20% perfluorooctanol (PFO) in 3M™ NOVEC™ 7500 fluid and adding 2 mL PFO to 8 mL 3M™ NOVEC™ 7500 fluid. De-emulsification buffer was prepared as 1% Triton in IX PBS: 2.5 mL.

[0086] Emulsion breaking and bead recovery was performed according to the following: (1) Carrier oil phase was removed leaving just the packed emulsion in the tubes; (2) 750 pL-1 mL of 1% Triton in PBS was added on top of emulsion pack; (3) 500-750 pL of 20% perfluoroctonal (PFO) in Novec 7500 was added to emulsion/PBS mixture; (4) Vortexed vigorously for 30 seconds to 1 minute; (5) Spun down at 5,000xg for 1 minute; (5) Bottom % of PFO oil phase was removed; (6) Vortexed; (7) Spun down at 5,000xg for 1 minute; (8) Bottom % of PFO oil phase was removed; (9) Vortexed; (10) Spun down at 5,000xg for 1 minute; (11) Beads resuspended in aqueous phase by using a wide-bore 200-300 pL pipette tip and pipetting gently up and down; and (12) Resuspended beads collected in a new tube. [0087] The surfactant (PLURONIC® F-127), which was used to maintain nanoparticle stability during encapsulation, was subsequently removed from the beads. The beads were washed at least three times and allowed to incubate at 4 °C for at least 1-2 hours preferably overnight (before utilizing in enzyme degradation reaction) according to the following: (1) Spun down beads at 5,000xg for 1 minute; (2) Removed supernatant; (3) Resuspended pellet in 1 mL of cold IX PBS (not containing the surfactant); (4) Spun down; (5) Removed supernatant; (6) Resuspended in 1 mL of cold IX PBS; (7) Spun down; and (8) Removed supernatant. The resulting beads were filtered using a 70 pm cell filter/cold IX PBS (no surfactant) to wash them through, and filtered beads were recovered and RSS in at least 1 mL IX PBS before incubating at 4 °C for at least 2 hours, preferably overnight.

Example 3. A Microfluidic-Based Platform for Ultra-High-Throughput Screening of Plastic-Degrading Enzymes and Enzyme Variant

[0088] A microfluidic-based process was developed for screening plastic -degrading enzymes and enzyme variants. This process is schematically illustrated in FIGs. 3A-3E and described more fully in this example.

[0089] Encapsulation and Incubation of DNA expression system of Enzyme Library [0090] A system capable of expressing an enzyme of interest is encapsulated in pico-liter sized aqueous-in-oil droplets with an inducer for protein expression by a microbe. The subsequent emulsion is incubated from 16-48 hours to allow for cell proliferation and enzyme expression (FIG. 3A).

[0091] Generation of Homogeneous Plastic Nanoparticles Stained with Fluorescent Dyes [0092] Homogeneous plastic nanoparticles are generated by dissolving commercial plastic substrates in an organic solvent (DMSO) and precipitating the solution in water to generate a dispersion of 200-250 nm plastic particles (2). These particles increase the surface area of the substrate and allow for more rapid plastic depolymerization but have minimal toxicity and still mimic real-world plastic substrates. During the final stage of particle generation, a hydrophobic fluorescent dye is doped into the polymer solution, creating homogeneously stained fluorescent plastic particles (3, 4). As described in Example 1, these fluorescent particles are directly monitored for loss of fluorescence commensurate with plastic depolymerization, providing improved readout relative to conventional screening technologies (7, 8, 9).

[0093] Generation of Dispersed Plastic Nanoparticle Beads

[0094] The fluorescent particles are concentrated via centrifugation and suspended in a polyacrylamide gel precursor. Optionally, an acrydite-modified primer with homology to the gene of interest is added (5) to allow for PCR-based identification of gene variants. Beads are generated via a flow-focusing droplet generation device (FIG. 3B) and isolated from the emulsion post-polymerization (6). A surfactant is used to maintain nanoparticle stability during encapsulation. The surfactant inhibits enzyme activity and is removed from beads using cold phosphate buffered saline (PBS) and an overnight incubation at 4 °C.

[0095] Bead Merging with Enzyme Library Emulsion and Incubation for Enzyme Reaction [0096] Beads are merged with post-growth enzyme library emulsion (5) utilizing a microfluidic device (FIG. 3C), which is modified to allow for merging of incubated cells rather than single cells, and incubated under challenging conditions, such as temperature (e.g., 30-90 °C), decreased pH, and increased plastic recalcitrance (FIG. 3D). [0097] Optional DNA Hybridization to Beads

[0098] If beads are generated with acrydite-modified forward primers, an amplification or ligation step is performed before sorting to attach the DNA to the beads. This consists of addition of a reverse primer and PCR master mix, or GoldenGate master mix (NEB®), during the bead merging step above. After incubation to allow for plastic degradation, DNA-Bead hybridization is performed by either performing PCR via a thermal cycler or by performing a GoldenGate reaction or ligation reaction.

[0099] FIG. 4 shows results demonstrating DNA conjugation to nanoparticle-containing beads. Panel A shows results from a control experiment performed utilizing beads with conjugated primers to amplify purified DNA template, where beads were washed three times after first PCR step with NF H2O and then used as template in final PCR reaction. Panel B shows results from an experiment in which the first PCR reaction was performed in an emulsion containing primer-hybridized beads and purified DNA. Panel C shows results from an experiment in which individual cells were encapsulated and allowed to grow under conditions as described herein for library screening. DNA was conjugated via an in-emulsion PCR step, followed by breaking the emulsion, washing the beads, and performing a final PCR reaction on the sample. Negative controls for Panels A and C are the same and comprised of beads that never had the DNA conjugated to them. In Panel B, the negative controls consist of supernatant from the first bead wash as template in a PCR, and unconjugated beads. The 1 kbp product from the final iteration of the experiment (beads in droplets with cells, Panel C) was sequenced via Sanger sequencing and identified the Enzyme A gene.

[0100] Sorting on FADS or FACS

[0101] After enzyme activity incubation and optional DNA hybridization to beads, the library is sorted utilizing fluorescent signals (FIG. 3E). This can be performed in a fluorescence activated droplet sorter (FADS) which is available commercially or custom-built. When utilizing a FADS, DNA is hybridized to the beads as the cells/DNA for each enzymatic reaction will be contained within the same droplet. To achieve even higher throughputs, if DNA has been hybridized to the beads, the emulsion is broken post-hybridization reaction and washed (as described above). The beads are then sorted on a wide-nozzle FACS while still maintaining the enzyme activity-genotype relationship.

[0102] Sorting is performed via thresholding or binning, resulting in sorted populations that have measured fluorescent signals associated with them. The higher the number of bins these are split into, the more information is obtained about sequence-enzyme activity correlation.

[0103] DNA Isolation/ Amplification and Sequencing

[0104] For sorting on the FADS, DNA is isolated by breaking the emulsion with a low volume of molecular biology grade water. This is extracted from the continuous oil phase to produce the template DNA for amplification. Beads sorted on the FACS are used directly as template DNA for amplification. Amplification utilizes primers barcoded for NGS library preparation.

[0105] Post amplification or purification of DNA from the sorted material NGS libraries are created to sequence the variants appearing across the populations binned for activity. By applying the count data from the sequencer across the different sorted populations, an activity score for plastic degradation is calculated for each variant observed in the process. This scoring can be a weighted average of the counts in each bin similar to sort-seq applications for other FACS based assays (1). DNA sequences compatible with this process can be either long read (e.g., PacBio® sequencing technologies, Oxford Nanopore™ sequencing technologies) or short read (e.g., Illumina™ sequencing technologies).

[0106] Analysis and Machine Learning (ML) Pipeline

[0107] The obtained sequencing data is optionally used to train machine learning or artificial intelligence models, such as LLMs, autoencoders and/or diffusion models, to predict other plastic degrading enzymes with increased activity or enhanced attributes.

[0108] List of Cited References:

1. Peterman, Neil, and Erel Levine. "Sort-seq under the hood: implications of design choices on large-scale characterization of sequence-function relations." BMC genomics 17 (2016): 1-17.

2. Rodriguez-Hemandez, Ana, et al. “A novel and simple method for polyethylene terephthalate (PET) nanoparticle production.” Environ. Sci.: Nano, 2019,6, 2031- 2036.

3. Cassano, et al. “Inorganic Species-Doped Polypropylene Nanoparticles for Multifunctional Detection.” ACS Appl. Nano Mater. 2021, 4, 2, 1551-1557.

4. Alferiev, et al. “Robust Chemical Strategy for Stably Labeling Polyester-Based Nanoparticles with BODIPY Fluorophores.” ACS Appl. Polym. Mater. 2022, 4, 2, 1196-1206. 5. Zilionis, R., Nainys, J., Veres, A. et al. Single-cell barcoding and sequencing using droplet microfluidics. Nat Protoc 12, 44-73 (2017).

6. Wang, Y., Cao, T., Ko, J., Shen, Y., Zong, W., Sheng, K., Cao, W., Sun, S., Cai, L., Zhou, Y.-L., Zhang, X.-X., Zong, C., Weissleder, R., Weitz, D., Dissolvable Polyacrylamide Beads for High-Throughput Droplet DNA Barcoding. Adv. Sci. 2020, 7, 1903463.

7. Yidi Liu, Zhanzhi Liu, Zhiyong Guo, Tingting Yan, Changxu Jin, Jing Wu, Enhancement of the degradation capacity of IsPETase for PET plastic degradation by protein engineering, Science of The Total Environment, 2022, 834.

8. Xu, A., Liu, J., Cao, S., Xu, B., Guo, C. & Yu, Z. et al. (2023) Application of a novel fluorogenic polyurethane analogue probe in polyester-degrading microorganisms screening by microfluidic droplet. Microbial Biotechnology, 16, 474-480.

9. Puetz et al. “Validated High-Throughput Screening System for Directed Evolution of Nylon-Depolymerizing Enzymes” ACS Sustainable Chemistry & Engineering. DOI: 10.1021/acssuschemeng.3c01575.

EQUIVALENTS AND SCOPE

[0109] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0110] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. [0111] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0112] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0113] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0114] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0115] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the application describes “a composition comprising A and B,” the application also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

[0116] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0117] This application may refer to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art. 1 [0118] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

[0119] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Claims

CLAIMS What is claimed is:

1. A composition comprising: a labeled particle comprising an organic polymer that comprises one or more luminescent labels; and a polymeric bead, wherein the labeled particle is encapsulated in the polymeric bead.

2. The composition of claim 1, wherein the polymeric bead comprises polyacrylamide, agarose, polyethylene glycol, alginate, and/or polystyrene.

3. The composition of claim 1 or 2, wherein the polymeric bead comprises polyacrylamide.

4. The composition of any one of claims 1-3, wherein the polymeric bead has a diameter of less than about 250 pm.

5. The composition of any one of claims 1-4, wherein the labeled particle has a diameter of less than about 1,000 nm.

6. The composition of any one of claims 1-5, wherein the organic polymer comprises a polyether-containing polymer, a polyester-containing polymer, a polypropylene-containing polymer, or a combination thereof.

7. The composition of any one of claims 1-6, wherein the organic polymer comprises polyethylene glycol, polyethylene terephthalate, polyester-polyurethane, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene isosorbide terephthalate, polylactic acid, polyhydroxyalkanoate, polybutylene succinate, polybutylene succinate adipate, polybutylene adipate terephthalate, polyethylene furanoate, polycaprolactone, poly(ethylene adipate), polyethylene naphthalate, or a combination thereof.

8. The composition of any one of claims 1-7, wherein the organic polymer comprises polyethylene terephthalate.

9. The composition of any one of claims 1-8, wherein the one or more luminescent labels comprise one or more fluorescent dyes.

10. The composition of any one of claims 1-9, further comprising a surfactant.

11. The composition of claim 10, wherein the surfactant comprises poloxamer 407 (PLURONIC® F-127).

12. The composition of any one of claims 1-11, wherein at least one oligonucleotide tag is attached to the polymeric bead.

13. The composition of claim 12, wherein the at least one oligonucleotide tag comprises one or more of a sequence encoding an enzyme, a restriction site, a poly-T sequence, a molecular barcode sequence, a sample index sequence, a primer site sequence, and a promoter sequence.

14. A composition comprising a plurality of droplets, wherein at least one droplet comprises the composition of any one of claims 1-13.

15. The composition of claim 14, wherein the at least one droplet comprises a hydrolase.

16. The composition of claim 15, wherein the at least one droplet comprises a host cell comprising a heterologous polynucleotide encoding the hydrolase.

17. The composition of claim 15 or 16, wherein the at least one droplet comprises a nucleic acid encoding the hydrolase.

18. A method of preparing an encapsulated particle, the method comprising: providing a first fluid comprising a first mixture that comprises a labeled particle, a surfactant, and a polyacrylamide precursor; generating a droplet comprising the first mixture and a polymerization catalyst, wherein the polymerization catalyst initiates polymerization of the polyacrylamide precursor to produce a polyacrylamide bead having the labeled particle encapsulated therein; recovering the polyacrylamide bead from the droplet to obtain a solution comprising the polyacrylamide bead and the surfactant; and removing the surfactant from the solution.

19. The method of claim 18, further comprising, prior to generating the droplet: providing a second fluid comprising the polymerization catalyst, wherein the first and second fluids are immiscible.

20. The method of claim 19, wherein the first fluid is an aqueous fluid, and the second fluid comprises an oil.

21. The method of claim 19, wherein the first fluid comprises an oil, and the second fluid is an aqueous fluid.

22. The method of any one of claims 19-21, wherein generating the droplet comprises: combining the first fluid and the second fluid to generate an emulsion.

23. The method of claim 22, wherein recovering the polyacrylamide bead comprises: contacting the emulsion with an emulsion breaking buffer and/or a de-emulsification buffer.

24. The method of any one of claims 18-23, wherein removing the surfactant from the solution comprises: incubating the solution in a buffer that does not comprise the surfactant.

25. The method of any one of claims 18-24, wherein the polyacrylamide precursor comprises acrylamide and bis-acrylamide.

26. The method of claim 25, wherein the polyacrylamide precursor comprises acrylamide to bis-acrylamide in a ratio of between about 5:1 and about 25:1.

27. The method of claim 25 or 26, wherein the polyacrylamide precursor comprises acrylamide and bis-acrylamide at a concentration of between about 5% and 25% in the first fluid.

28. The method of any one of claims 18-27, wherein the polymerization catalyst comprises tetramethylethylenediamine (TEMED) and/or ammonium persulfate (APS).

29. The method of any one of claims 18-28, wherein the polymerization catalyst comprises TEMED, and the first fluid comprises APS; or wherein the polymerization catalyst comprises APS, and the first fluid comprises TEMED.

30. The method of any one of claims 18-29, wherein the first mixture comprises at least one oligonucleotide tag.

31. The method of claim 30, wherein the polyacrylamide bead is attached to the at least one oligonucleotide tag.

32. The method of any one of claims 18-31, wherein the surfactant comprises poloxamer 407 (PLURONIC® F-127).

33. The method of any one of claims 18-32, wherein the labeled particle comprises an organic polymer comprising one or more luminescent labels.

34. A method of monitoring a degradation reaction, the method comprising: contacting a hydrolase with a labeled particle, wherein the labeled particle comprises an organic polymer comprising one or more luminescent labels; and detecting a characteristic signal from the one or more luminescent labels, wherein the characteristic signal is indicative of degradation of the organic polymer by the hydrolase.

35. The method of claim 34, wherein the labeled particle has a diameter of less than about 1,000 nm.

36. The method of claim 34 or 35, wherein the organic polymer comprises a polyether- containing polymer, a polyester-containing polymer, a polypropylene-containing polymer, or a combination thereof.

37. The method of any one of claims 34-36, wherein the organic polymer comprises polyethylene glycol, polyethylene terephthalate, polyester-polyurethane, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene isosorbide terephthalate, polylactic acid, polyhydroxyalkanoate, polybutylene succinate, polybutylene succinate adipate, polybutylene adipate terephthalate, polyethylene furanoate, polycaprolactone, poly(ethylene adipate), polyethylene naphthalate, or a combination thereof.

38. The method of any one of claims 34-37, wherein the organic polymer comprises polyethylene terephthalate.

39. The method of any one of claims 34-38, wherein the one or more luminescent labels comprise one or more fluorescent dyes.

40. The method of any one of claims 34-39, wherein detecting the characteristic signal comprises: detecting a decrease in luminescence from the one or more luminescent labels, wherein the decrease in luminescence is indicative of degradation of the organic polymer by the hydrolase.

41. The method of claim 40, wherein the decrease in luminescence comprises a decrease in luminescence intensity over time.

42. The method of any one of claims 34-41, wherein contacting the hydrolase with the labeled particle comprises: contacting the hydrolase with a polymeric bead comprising the labeled particle.

43. The method of claim 42, wherein the polymeric bead comprises polyacrylamide, polyacrylate, agarose, polyethylene glycol, alginate, and/or polystyrene.

44. The method of claim 43, wherein the polymeric bead comprises polyacrylamide.

45. The method of any one of claims 42-44, wherein the polymeric bead has a diameter of less than about 250 pm.

46. A method comprising: a) generating a plurality of droplets, at least one droplet of the plurality comprising: an enzyme; and a labeled particle comprising an organic polymer that comprises one or more luminescent labels; b) sorting the plurality of droplets based on a characteristic signal detected from the one or more luminescent labels, wherein the characteristic signal is indicative of degradation of the organic polymer by the enzyme; and c) optionally determining the identity of the enzyme in one or more droplets obtained from the sorting.

47. The method of claim 46, wherein the at least one droplet comprises a nucleic acid encoding the enzyme.

48. The method of claim 47, wherein the at least one droplet comprises a host cell comprising the nucleic acid.

49. The method of claim 47 or 48, wherein (c) comprises sequencing the nucleic acid to determine the identity of the enzyme.

50. The method of any one of claims 47-49, wherein (a) comprises: incubating the nucleic acid under conditions to express the enzyme in the at least one droplet; and adding the labeled particle to the at least one droplet.

51. The method of any one of claims 46-50, further comprising, prior to (b): incubating the enzyme and the labeled particle under degradation conditions.

52. The method of claim 51, wherein the degradation conditions comprise one or more conditions selected from the group consisting of a temperature of between about 30 °C and about 90 °C, a pH below about 7.0, and an increased plastic recalcitrance.

53. The method of any one of claims 46-52, wherein (b) comprises sorting the plurality of droplets using fluorescence-activated droplet sorting (FADS) or fluorescence-activated cell sorting (FACS).

54. The method of any one of claims 46-53, wherein the organic polymer comprises a polyether-containing polymer, a polyester-containing polymer, a polypropylene-containing polymer, or a combination thereof.

55. The method of any one of claims 46-54, wherein the organic polymer comprises polyethylene glycol, polyethylene terephthalate, polyester-polyurethane, polytrimethylene terephthalate, polybutylene terephthalate, polyethylene isosorbide terephthalate, polylactic acid, polyhydroxyalkanoate, polybutylene succinate, polybutylene succinate adipate, polybutylene adipate terephthalate, polyethylene furanoate, polycaprolactone, poly(ethylene adipate), polyethylene naphthalate, or a combination thereof.

56. The method of any one of claims 46-55, wherein the organic polymer comprises polyethylene terephthalate.

57. The method of any one of claims 46-56, wherein the one or more luminescent labels comprise one or more fluorescent dyes.

58. The method of any one of claims 46-57, wherein the labeled particle is encapsulated in a polymeric bead.

59. The method of claim 58, wherein the polymeric bead comprises polyacrylamide, agarose, polyethylene glycol, alginate, and/or polystyrene.

60. The method of claim 58 or 59, wherein the polymeric bead comprises polyacrylamide.

61. The method of any one of claims 58-60, wherein the polymeric bead has a diameter of less than about 250 pm.

62. The method of any one of claims 58-61, wherein at least one oligonucleotide tag is attached to the polymeric bead.

63. The method of claim 62, wherein the at least one oligonucleotide tag comprises one or more of a sequence encoding the enzyme, a restriction site, a poly-T sequence, a molecular barcode sequence, a sample index sequence, a primer site sequence, and a promoter sequence.

64. The method of claim 62 or 63, further comprising, prior to (b): attaching the at least one oligonucleotide tag to a nucleic acid encoding the enzyme in the at least one droplet.