Compositions and methods for passive optical barcoding of multiplexed assays

Abstract

The invention provides a composition comprising a plurality of hydrogel particles having substantially equal diameters, but each sub-group of particles from the plurality of hydrogel particles has a different associated value of one or more passive optical properties, which can be deconvoluted using a cytometer. Each hydrogel particle from the plurality of hydrogel particles can be functionalized with a different biochemical or chemical target from a set of targets. A method of making a hydrogel particle comprises forming a droplet and polymerizing the droplet, optionally with functionalization.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 019,478, filed May 4, 2020, entitled “Compositions and Methods for Passive Optical Barcoding for Multiplexed Assays,” the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to the optical barcoding of beads by engineering the passive optical properties of hydrogel polymers, and their applications. Background Technology

[0004] Flow cytometry and high-throughput cell counting analysis (e.g., high-content imaging) are techniques that allow for the rapid separation, counting, and characterization of individual cells and are commonly used in a variety of clinical and laboratory settings. Cell counting devices are known in the art and include commercially available devices for performing flow cytometry and FACS, hematological analysis, and high-content imaging. Summary of the Invention

[0005] In some embodiments, the composition comprises hydrogel particles having passive optical properties (e.g., FSC and / or SSC), said hydrogel particles being intentionally engineered or “tuned” without altering the size (e.g., diameter) of the particles themselves. The engineered hydrogel can then be used for multiplexing, utilizing the passive optical properties, optionally in combination with one or more additional properties such as fluorescence, for multiplexed determinations (e.g., chemical or biochemical) in a single reaction, which can be deconvolved based on the passive optical properties of individual bead populations.

[0006] In some embodiments, the method for producing hydrogel particles includes forming droplets and polymerizing the droplets, optionally functionalizing them. The method yields hydrogel particles having substantially equal diameters but different associated predetermined optical properties (e.g., passive optical properties), which can be deconvoluted using a cell counting instrument.

[0007] In some embodiments, a method is provided for multiplexing assays. The method includes using a population of multiple hydrogel particles with unique passive optical properties in a single assay. Each hydrogel particle from the plurality of hydrogel particles has one or more uniquely associated biochemical targets. The population of multiple hydrogel particles is assayed, and the hydrogel particles and / or the biochemical targets are separated based on their passive optical properties. The result of the multiplexing assay is then determined based on the passive optical properties. The method described herein allows for high-dimensional (>1) multiplexing assays in a single reaction, for example, using high-throughput cell counting measurements. Attached Figure Description

[0008] It should be understood that the diagrams are primarily for illustrative purposes and are not intended to limit the scope of the topics discussed herein.

[0009] Figure 1 is a flowchart showing a method for preparing hydrogel particles according to some embodiments.

[0010] Figure 2 is a flowchart showing a method for biochemical multiplexing according to some implementation schemes.

[0011] Figure 3A is a simplified diagram illustrating the optical properties of the cells and the disclosed hydrogel particles (A) according to some embodiments.

[0012] Figure 3B is a simplified diagram illustrating the optical properties of polystyrene beads (B, C).

[0013] Figure 4 is a simplified diagram showing the variables that can be tuned to encode specific forward and side-scattering "barcodes" according to some implementation schemes.

[0014] Figure 5 is a simplified diagram showing particle formation in a microfluidic channel according to some implementation schemes.

[0015] Figure 6 is a simplified diagram showing an encoding scheme for generating a group of beads of similar size according to some embodiments, the group being multiplexed using passive optical properties.

[0016] Figures 7A and 7B illustrate how particles of the same size can be deconvolved based on optical scattering characteristics. Figure 7A shows the barcoding of particles of the same size via optical scattering features / coordinates, and Figure 7B shows the fluorescence detection of each bead group. In contrast, these particles cannot be distinguished based solely on fluorescence or biochemical targets. This figure demonstrates the ability to perform biochemical multiplexing and demultiplexing using passive optical properties as the primary deconvolution variable according to some implementations.

[0017] Based on experimental examples of elegant demonstrations of optical tuning of biologically relevant target populations, Figure 8A is a graph of white blood cell counts of an exemplary population of lysed whole blood, and Figure 8B is a graph of the counts of hydrogel particles with tuned passive optical properties.

[0018] Figures 9A and 9B are plots of lateral and forward scattering from hydrogel particles with tunable passive optical properties, based on experimental examples, showing that lateral scattering can be tunable independently of forward scattering. Figure 9A shows that the lateral scattering signal increases with a higher percentage of encapsulated nanoparticles, while Figure 9B shows that the forward scattering signal remains largely unchanged, thus confirming that lateral scattering can be tunable independently of forward scattering.

[0019] Figures 10A-10C are plots of lateral scattering versus forward scattering for multiple monomer:comonomer ratios based on experimental examples, showing that the effect of increased forward scattering on lateral scattering is negligible. In the experimental example shown in Figure 10A: %T = 8, %C = 2.5, %np = 0.06, 15 min at 80°C; in the experimental example shown in Figure 10B: %T = 8, %C = 5, %np = 0.06, 15 min at 80°C; in the experimental example shown in Figure 10C: %T = 8, %C = 5, %np = 0.06, overnight at 45°C. Detailed Implementation

[0020] Flow cytometry and high-throughput cell counting analysis can also be used to determine beads (e.g., for biochemical measurements). In some such implementations, a beam is directed onto a focused stream of liquid containing beads. Multiple detectors are then aimed at points where the stream flows through the beam, with one detector aligned with the beam (e.g., to detect forward scattering (“FSC”)) and several detectors perpendicular to the beam (e.g., to detect side scattering (“SSC”)). FSC and SSC measurements are often referred to as “passive optical properties.” For particles such as cells (e.g., human cells), FSC is typically associated with cell volume, while SSC is typically associated with the internal complexity or particulateness of the particle (e.g., nuclear shape, amount and type of cytoplasmic granules, or membrane roughness). Due to these associations, different specific cell types can exhibit different FSCs and SSCs, allowing cell types to be distinguished from each other in flow cytometry based on their passive optical properties. These measurements (i.e., FSC and SSC) form the basis for cell counting analysis in clinical and research settings. Most synthetic or polymeric products used in such cell analyses are made from (or contain in large quantities) polystyrene or latex (typically opaque polymers with fixed FSC and SSC values ​​based on the diameter of the particles themselves). Therefore, polystyrene particles of equal diameter are generally indistinguishable from each other based solely on passive optical properties (FSC and SSC).

[0021] To distinguish subgroups of polystyrene particles of similar size from one another, fluorophores can be added to the particles, allowing for multiplexing (e.g., multicolor) assays. By combining different fluorophores at different concentrations into a single bead, unique identifiers can be generated that allow the beads to be distinguished from groups of differently stained beads. When combined with unique assay targets, fluorescently barcoded bead groups can facilitate the simultaneous assay of multiple targets (referred to herein as "biochemical multiplexing"). However, known products such as Luminex beads are limited in fluorescence multiplexing because they are made of polystyrene, which, as described above, has fixed passive optical properties. In addition to the material limitations of existing products made of polystyrene, instruments used to measure such beads typically have a fixed and limited number of fluorescence detectors, thus limiting the number of size / targets that fluorescence-driven multiplexing strategies can address. Modern biochemical assays have benefited from additional sizes of multiplexing, but have been limited by the availability of instrument detectors. Therefore, there is a need for additional and orthogonal sizes of products that allow for multiplexing using passive optical properties. The embodiments described herein meet this need through passive optical barcoding of the hydrogel substrate.

[0022] Some known products, such as LEGENDplex (BioLegend), use particles of different sizes for >1-channel assays. Although the bead groups in such products may be distinguishable by their passive optical properties (LEGENDplex, BioLegend), they inherently possess different surface areas, hydrodynamic and biochemical properties (e.g., analyte concentrations) due to size differences, leading to poor assay performance and non-quantitative measurements.

[0023] Overview

[0024] Given the fluid conditions within flow cytometry and high-content imaging systems, particles used for biochemical assays or calibration typically fall within a finite size range to avoid fluid dynamic particle settling and related clogging (which can occur with larger particles) and / or to prevent particles from floating to the surface of the liquid suspension (which can occur with smaller particles, making effective sampling difficult). This size constraint limits the range of forward scattering that polystyrene particles can induce. Unlike polystyrene particles, the hydrogel particles disclosed herein can exhibit a variety of different optical scattering properties while maintaining a fixed diameter, thus facilitating the optimization of fluid properties and the introduction of additional sizes for multiplexing.

[0025] Figure 1 is a flowchart illustrating a method for preparing hydrogel particles according to some embodiments. As shown in Figure 1, method 100 includes droplet formation at 110 (e.g., to generate multiple polydisperse or monodisperse droplets, as described herein). At 112, one or more surfactants are optionally added to the droplets, and at 114, one or more comonomers are added to the liquid. The droplets are then polymerized at 116 to form hydrogel particles, followed by optional functionalization of the hydrogel particles at 118 (e.g., with one or more chemical side groups or fluorescent dyes, as discussed further below).

[0026] Figure 2 is a flowchart illustrating a method for biochemical multiplexing according to some embodiments. As shown in Figure 2, method 200 includes providing a plurality of engineered hydrogel particles at 220, each engineered hydrogel particle having its own unique passive optical properties. The engineered hydrogel particles may be transparent or translucent. At 222, a determination is prepared, including the engineered hydrogel particles and at least one biochemical target. At 224, one or more passive optical properties of the engineered hydrogel particles are measured. Based on these measurements, the engineered hydrogel particles and / or at least one biochemical target can be separated at 226, and / or the determination result can be determined at 228.

[0027] Figure 3A is a simplified diagram illustrating the optical properties of cells and the disclosed hydrogel particles according to some embodiments. As shown in Figure 3A, the engineered hydrogel described herein is translucent, allowing the use of a side-scattering (SSC) detector to resolve its internal features (i.e., cell complexity). In contrast, Figure 3B is a simplified diagram illustrating the optical properties of polystyrene beads. Compared to Figure 3A, the polystyrene beads in Figure 3B are opaque and have a fixed SSC, which depends on their diameter and is unaffected by their internal features (i.e., does not vary based on their internal features). Therefore, polystyrene particles have limited practicality in the two most important passive optical measurements used in flow cytometry (FSC and SSC, which measure target size and complexity, respectively). Due to these limitations of polystyrene, users must typically rely solely on fluorescence for multiplexed immunophenotyping experiments.

[0028] In some embodiments described herein, the composition comprises hydrogel particles engineered to possess passive optical properties that can be distinguished from the optical properties of other particles (e.g., hydrogel particles) of equal diameter using only FSC and SSC. The inventors unexpectedly discovered that the optical properties of the hydrogel particles can be independently tuned by changing the composition of the hydrogel particles. For example, the SSC can be tuned without significantly affecting the FSC, and vice versa (i.e., the FSC can be tuned without significantly affecting the SSC). Furthermore, the optical properties (e.g., refractive index) of the hydrogel particles can be tuned without significantly affecting the density or size of the particles themselves. This is a surprising and useful feature because these properties allow multiple particles of the same size to be “encoded” with a specific FSC / SSC ratio, which are then deconvoluted using detectors such as those found on all cell counting instruments, including low-cost instruments without fluorescence measurement capabilities.

[0029] In some embodiments, the method for producing hydrogel particles yields hydrogel particles with predetermined optical properties. In some embodiments, the multiplexing assay method includes using multiple (or "populations") of hydrogel particles with unique passive optical properties in a single assay, each hydrogel particle having a unique biochemical target. One or more passive optical properties of the population are measured, and the population and / or the biochemical target are separated based on the measured passive optical properties. The results of the multiplexing assay can be generated based on the measured passive optical properties. The foregoing procedures and the engineered properties of the hydrogel particles facilitate high-dimensional multiplexing assays in a single reaction, as well as the separation of hydrogel particles and / or biochemical targets using high-throughput cell counting measurements.

[0030] hydrogel

[0031] The hydrogel particles described herein comprise hydrogels. Hydrogels are materials comprising a three-dimensional network of macromolecules that allows them to swell in the presence of water and shrink in the absence of water (or by reducing the amount of water), but are insoluble in water. Swelling (i.e., water absorption) is a result of the presence of hydrophilic functional groups attached to or dispersed within the macromolecular network. Crosslinking between adjacent macromolecules results in the water insolubility of these hydrogels. Crosslinking can be due to chemical bonding (i.e., covalent bonds) or physical bonding (i.e., van der Waals forces, hydrogen bonding, ionic forces, etc.). Although some in the polymer industry may refer to one or more macromolecular materials described herein as “xerogel” in the dry state and “hydrogel” in the hydrated state, for the purposes of this disclosure, the term “hydrogel” will refer to either dehydrated or hydrated macromolecular materials. Hydrogels of particular value are characterized by their ability to retain their general shape / morphology, whether dehydrated or hydrated. Thus, if a hydrogel has a generally spherical shape under dehydrated conditions, it will be spherical under hydrated conditions.

[0032] The hydrogels described herein may contain more than about 30%, more than about 40%, more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, or more than about 85% water.

[0033] In some embodiments, the synthetic hydrogel can be prepared by polymerizing monomeric materials (“hydrogel monomers”) to form a backbone, and then crosslinking the backbone with a crosslinking agent. Suitable hydrogel monomers include (but are not limited to) the following: lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone, methyl methacrylate, glycidyl methacrylate, glycol methacrylate, ethylene glycol, fumaric acid, etc. Suitable crosslinking agents include (but are not limited to) tetraethylene glycol dimethacrylate and N,N'-15-methylenebisacrylamide. In some embodiments, the hydrogel particles are produced by the polymerization of acrylamide.

[0034] In some embodiments, the hydrogel comprises a mixture of at least one monofunctional monomer and at least one difunctional monomer.

[0035] Monofunctional monomers can be monofunctional acrylic monomers. Non-limiting examples of monofunctional acrylic monomers are acrylamides; methacrylamides; N-alkylacrylamides (such as N-ethylacrylamides, N-isopropylacrylamides, or N-tert-butylacrylamides); N-alkylmethylacrylamides (such as N-ethylmethylacrylamides or N-isopropylmethylacrylamides); N,N-dialkylacrylamides (such as N,N-dimethylacrylamides and N,N-diethylacrylamides; N-[(dialkylamino)alkyl]acrylamides (such as N-[3-dimethylamino)propyl]acrylamides or N-[ 3-(diethylamino)propyl]acrylamide; N-[(dialkylamino)alkyl]methacrylamide (such as N-[3-dimethylamino)propyl]methacrylamide or N-[3-(diethylamino)propyl]methacrylamide); (dialkylamino)alkyl acrylates (such as 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)propyl acrylate or 2-(diethylamino)ethyl acrylate); and (dialkylamino)alkyl methacrylates (such as 2-(dimethylamino)ethyl methacrylate).

[0036] A bifunctional monomer is any monomer that can be polymerized with a monofunctional monomer of the present disclosure to form a hydrogel as described herein, the hydrogel further containing a second functional group that can participate in a second reaction (e.g., fluorophore conjugation).

[0037] In some embodiments, the bifunctional monomer is selected from allyl alcohol, allyl isothiocyanate, allyl chloride, and allyl maleimide.

[0038] The bifunctional monomer can be a bifunctional acrylic monomer. Non-limiting examples of bifunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'-methylenebisacrylamide, N,N'-ethylenebisacrylamide, N,N'-ethylenebismethylacrylamide, N,N'-propylenebisacrylamide, and N,N'-(1,2-dihydroxyethylene)bisacrylamide.

[0039] Higher-order branched and linear comonomers in polymer blends can be replaced to adjust the refractive index while maintaining polymer density, as described in U.S. Patent No. 6,657,030 entitled "High Refractive Index Hydrogel Compositions for Ophthalmic Implants," the contents of which are incorporated herein by reference in their entirety for all purposes.

[0040] In some embodiments, the hydrogel contains molecules that modulate the optical properties of the hydrogel. These molecules, capable of altering the optical properties of the hydrogel, are further discussed below.

[0041] Naturally occurring hydrogels that can be used in the embodiments described herein include a variety of polysaccharides that are obtained from or derived from natural sources, such as plants, algae, fungi, yeast, marine invertebrates, and arthropods. Non-limiting examples of polysaccharides suitable for use in the embodiments described herein include (but are not limited to) agarose, dextran, chitosan, cellulose-based compounds, starch, derived starches, etc. Such polysaccharides may include multiple repeating glucose units as the main component of the polysaccharide backbone.

[0042] The polymerization of hydrogels can be initiated by persulfate. Persulfate can be any water-soluble persulfate. Non-limiting examples of water-soluble persulfates are ammonium persulfate and alkali metal persulfates. Alkali metals include lithium, sodium, and potassium. In some preferred embodiments, the persulfate is ammonium persulfate or potassium persulfate.

[0043] The polymerization of hydrogels can be accelerated by an accelerator. The accelerator can be a tertiary amine. The tertiary amine can be any water-soluble tertiary amine. Preferably, the tertiary amine is N,N,N',N'-tetramethylethylenediamine (TEMED) or 3-dimethylaminopropionitrile.

[0044] Figure 4 is a simplified diagram showing variables that can be tuned to encode specific passive optical (e.g., FSC and / or SSC) “barcodes” on / in groups of hydrogel particles according to some embodiments. As shown in the top row (1) of Figure 4, adjustments to the monomer / comonomer ratio and crosslinking density can result in changes in the refractive index of the hydrogel particles (e.g., from n to 2*n, to 3*n as the monomer::comonomer ratio increases). The middle row (2) of Figure 4 shows that adjustments to the nanoparticle composition and concentration can adjust the SSC of the hydrogel particles (e.g., increasing the SSC as the nanoparticle concentration increases), and the bottom row (3) of Figure 4 shows that functionalizing the hydrogel particles with chemical side groups can yield accurate stoichiometric ratios of secondary markers (e.g., fluorophores, proteins, antigens, antibodies) on the hydrogel particles. This feature enables the control of quantitative mean fluorescence intensity (MFI) on the particles, a unique characteristic of the particles described herein.

[0045] Figure 5 is a simplified diagram showing particle formation in an oil-filled microfluidic channel according to some implementation schemes.

[0046] hydrogel particles

[0047] In some embodiments, the hydrogel particles comprise a hydrogel and are generated by polymerizing droplets (see the discussion of “droplet formation” related to Figure 5). Microfluidic methods for generating multiple droplets (including fluid droplets and rigid droplets) may include one or more methods described in the following documents: U.S. Patent Application Publication No. 2011 / 0218123 entitled “Creation of Libraries of Droplets and Related Species” and U.S. Patent No. 7,294,503 entitled “Microfabricated Crossflow Devices and Methods,” the contents of which are incorporated herein by reference in their entirety for all purposes. Such methods provide the generation of multiple droplets, each droplet comprising a first fluid substantially surrounded by a second fluid, wherein the first and second fluids are substantially immiscible (e.g., a droplet containing an aqueous liquid is substantially surrounded by an oil-based liquid). In other embodiments, particles may be generated by precipitation polymerization or membrane emulsification.

[0048] Multiple fluid droplets (e.g., prepared using a microfluidic device) may be polydisperse (e.g., having a range of different sizes), or in some cases, the fluid droplets may be monodisperse or substantially monodisperse, for example, having a uniform diameter distribution, such that no more than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the droplets have an average diameter greater than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the average diameter. As used herein, the average diameter of a population of droplets refers to the arithmetic mean of the droplet diameters.

[0049] In some implementations, the population of hydrogel particles comprises multiple hydrogel particles, and the population of hydrogel particles is substantially monodisperse.

[0050] The term "microfluidic" refers to a device, apparatus, or system comprising at least one fluid channel with a cross-sectional dimension less than 1 mm and a length-to-maximum cross-sectional dimension perpendicular to the channel in a ratio of at least about 3:1. Microfluidic devices comprising microfluidic channels are particularly suitable for preparing multiple monodisperse droplets. Cross-flow film emulsification and precipitation polymerization are other suitable methods for generating multiple monodisperse droplets.

[0051] Non-limiting examples of microfluidic systems that can be used with the present invention include those disclosed in the following documents: U.S. Patent Application Publication No. 2006 / 0163385 (“Forming and Control of Fluidic Species”), U.S. Patent Application Publication No. 2005 / 0172476 (“Method and Apparatus for Fluid Dispersion”), U.S. Patent Application Publication No. 2007 / 000342 (“Electronic Control of Fluidic Species”), International Patent Application Publication No. WO 2006 / 096571 (“Method and Apparatus for Forming Multiple Emulsions”), U.S. Patent Application Publication No. 2007 / 0054119 (“Systems and Methods of Forming Particles”), and International Patent Application Publication No. WO The entire contents of the respective documents 2008 / 121342 (“Emulsions and Techniques for Formation”) and International Patent Application Publication No. WO 2006 / 078841 (“Systems and Methods for Forming Fluidic Droplets Encapsulated in Particles Such as Colloidal Particles”) are incorporated herein by reference in their entirety for all purposes.

[0052] Droplet size may be related to the size of the microfluidic channel, pore size (in the case of membrane emulsification), and / or flow rate. Microfluidic channels can have any of a variety of sizes, for example, having the following maximum dimensions perpendicular to the fluid flow: less than about 5 mm, or less than about 2 mm, or less than about 1 mm, or less than about 500 μm, less than about 200 μm, less than about 100 μm, less than about 60 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm, less than about 25 μm, less than about 10 μm, less than about 3 μm, less than about 1 μm, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm.

[0053] The droplet size can be tuned by adjusting the relative flow rate. In some implementations, the droplet diameter is equal to the width of the channel, or within approximately 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the width of the channel.

[0054] In some embodiments, the size of the hydrogel particles is substantially similar to the size of the droplets that form them. For example, in some such embodiments, the diameter of the hydrogel particles is less than about 1 μm, less than about 2 μm, less than about 5 μm, less than about 10 μm, less than about 15 μm, less than about 20 μm, less than about 25 μm, less than about 30 μm, less than about 35 μm, less than about 40 μm, less than about 45 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, less than about 100 μm, less than about 120 μm, less than about 150 μm, less than about 200 μm, less than about 250 μm, less than about 300 μm, less than about 350 μm, less than about 400 μm, less than about 450 μm, less than about 500 μm, less than about 600 μm, less than about 800 μm, or less than 1000 μm. In some embodiments, the diameter of the hydrogel particles is greater than about 1 μm, greater than about 2 μm, greater than about 5 μm, greater than about 10 μm, greater than about 15 μm, greater than about 20 μm, greater than about 25 μm, greater than about 30 μm, greater than about 35 μm, greater than about 40 μm, greater than about 45 μm, greater than about 50 μm, greater than about 60 μm, greater than about 70 μm, greater than about 80 μm, greater than about 90 μm, greater than about 100 μm, greater than about 120 μm, greater than about 150 μm, greater than about 200 μm, greater than about 250 μm, greater than about 300 μm, greater than about 350 μm, greater than about 400 μm, greater than about 450 μm, greater than about 500 μm, greater than about 600 μm, greater than about 800 μm, or greater than 1000 μm. In typical embodiments, the diameter of the hydrogel particles is in the range of 5 μm to 100 μm.

[0055] In some implementations, one or more hydrogel particles are spherical in shape.

[0056] In some implementations, the material modulus properties (e.g., elasticity) of one or more hydrogel particles are more closely similar to the corresponding material modulus properties of target cells (e.g., human target cells) compared to the corresponding material modulus properties of polystyrene beads having the same diameter as the hydrogel particles.

[0057] In some implementations, one or more hydrogel particles do not contain agarose.

[0058] Optical properties

[0059] Passive optical properties

[0060] The three main modes of deconvolution in flow cytometry are two passive optical properties of the particles (forward scattering, FSC, corresponding to refractive index or RI; and side scattering, SSC), and biomarkers present on the surface of a given cell type (typically measured by fluorescence). The compositions described herein, which allow for the rational engineering of these properties, enable assay multiplexing via deconvolution, or the simultaneous measurement of more than one target (e.g., cellular, molecular, biochemical targets, etc.).

[0061] In some embodiments, the refractive index (RI) of one or more hydrogel particles is greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.10, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0062] In some embodiments, the RI of one or more hydrogel particles is less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.

[0063] In some implementations, the SSC of one or more hydrogel particles may be any value within the full range of possible values, such as those measured by a cell counting device.

[0064] In some implementations, the FSC of one or more hydrogel particles may be any value within the full range of possible values, such as those measured by a cell counting device.

[0065] In some embodiments, the FSC of one or more hydrogel particles can be tuned by incorporating high-refractive-index molecules into the hydrogel. Preferred high-refractive-index molecules include (but are not limited to) colloidal silica, alkyl acrylates, and alkyl methacrylates. Thus, in some embodiments, one or more hydrogel particles comprise alkyl acrylates and / or alkyl methacrylates. The alkyl acrylates or alkyl methacrylates may contain 1 to 18, 1 to 8, or 2 to 8 carbon atoms in an alkyl group (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, 2-ethylhexyl, heptyl, or octyl). The alkyl group may be branched or linear. The high-refractive-index molecules may also include ethylene groups, such as vinyl aromatics like styrene and methylstyrene, optionally substituted with an alkyl group (such as methyl, ethyl, or tert-butyl) or halogenated (such as chlorostyrene) on the aromatic ring.

[0066] In some embodiments, the FSC of one or more hydrogel particles is adjusted by modifying the water content present during hydrogel formation. In other embodiments, the FSC of one or more hydrogel particles is adjusted by modifying the crosslinking density of the hydrogel. Alternatively or additionally, the FSC of one or more hydrogel particles may be related to particle volume and can therefore be adjusted by changing the particle diameter, as described herein.

[0067] In some embodiments, the SSC of one or more hydrogel particles can be engineered by encapsulating nanoparticles within the hydrogel. In some embodiments, the hydrogel particles comprise one or more types of nanoparticles, such as those selected from polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles.

[0068] Functionalization of hydrogel particles

[0069] In some embodiments, in addition to having specific and engineered passive optical properties, the hydrogel particles described herein can be functionalized, thereby allowing them to mimic the fluorescence properties of labeled cells. In some embodiments, the hydrogel particles comprise a bifunctional monomer, and the functionalization of the hydrogel particles is performed via said bifunctional monomer. In some embodiments, the functionalized hydrogel particles comprise free amine groups. In other embodiments, the hydrogel can be functionalized with proteins or peptides, thereby allowing secondary labeling using reagents (including, but not limited to, antibodies).

[0070] Hydrogel particles can be functionalized with any fluorescent dye, including any of the fluorescent dyes listed in the following literature: The Handbook—A Guide to Fluorescent Probes and Labeling Technologies, the contents of which are incorporated herein by reference in their entirety for all purposes. Functionalization can be mediated by compounds containing free amine groups (e.g., allylamine), which can be incorporated into hydrogel particles during the formation process.

[0071] Non-limiting examples of suitable fluorescent dyes include: 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimide; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; 5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether, alanine-formamide or succinimide; 5-carboxyfluorescein succinimide; 6-carboxyfluorescein succinimide; 5-(and-6)-Carboxyfluorescein succinimide ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formylamino)hexanoic acid or succinimide ester; 6-(fluorescein-5-(and-6)-formylamino)hexanoic acid or succinimide ester; fluorescein-5-EX succinimide ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; 488 carboxylic acid or succinimide ester; Oregon 488 isothiocyanate; Oregon 488-X succinimide ester; Oregon 500 carboxylic acids; Oregon 500 carboxylic acid, succinimide ester, or triethylammonium salt; Oregon 514 Carboxylic Acids; Oregon 514 Carboxylic acid or succinimide ester; RhodamineGreen™ carboxylic acid, succinimide ester, or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide, or succinimide ester; Rhodamine Green™-X succinimide ester or hydrochloride; RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimide ester; bis-(4-carboxypiperidinyl)sulfonylrhodamine or bis(succinimide ester); 5-(and-6)carboxynaphthofluorescein, 5-(and-6)carboxynaphthofluorescein succinimide ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimide ester; 6-carboxyrhodamine 6G succinimide ester; 5-(and-6)-carboxyrhodamine 6G succinimide ester; 5-carboxy-2',4',5',7' -Tetrabromosulfonyl fluorescein succinimide ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; Lissamine™ rhodamine B sulfonyl chloride; Malachite green; Isothiocyanate; Mono(thiosuccinimide); 21. Carboxylic acid or succinimide ester; 7. Carboxylic acid or succinimide ester; Rhodamine Red™-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-formamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas sulfonyl; Texas Sulfonyl chloride; Texas -X STP ester or sodium salt; Texas -X succinimide ester; Texas -X succinimide ester; and X-rhodamine-5-(and -6)-isothiocyanate.

[0072] Other examples of fluorescent dyes include those commercially available from Invitrogen. Dyes, including but not limited to FL; TMR STP ester; TR-X STP ester; 630 / 650-XSTP ester; 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indarsen-3-propionic acid succinimide ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-indarsen-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indarsen-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indarsen-3-pentanoic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indarsen-3-propionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a 4a-diaza-s-indarsen-3-propanoic acid succinimide; 4,4-difluoro-5,7-dimethyl-4-borza-3a,4a-diaza-s-indarsen-3-propanoic acid; thiosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-borza-3a,4a-diaza-s-indarsen-3-propanoyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-borza-3a,4a-diaza-s-indarsen-3-propanoyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-borza-3a,4a-diaza-s-indarsen-3-propanoyl)sulfonylalanine, succinimide ester or triethylammonium salt ; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-boraz3a,4a4,4-difluoro-5,7-diphenyl-4-boraz3a,4a-diaza-s-indarsen-3-propionic acid; succinimide ester of 4,4-difluoro-5,7-diphenyl-4-boraz3a,4a-diaza-s-indarsen-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-boraz3a,4a-diaza-s-indarsen-3-propionic acid) hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1,3-dimethyl-5-(4-methoxyphenyl)-4-boraz3a,4a-diaza-s-indarsen-3-propionic acid) Butadienyl)-4-bora-3a,4a-diaza-s-indarsen-3-propionic acid succinimide; 4,4-difluoro-5-(2-pyrrolithyl)-4-bora-3a,4a-diaza-s-indarsen-3-propionic acid succinimide; 6-(((4,4-difluoro-5-(2-pyrrolithyl)-4-bora-3a,4a-diaza-s-indarsen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indarsen-3-propionic acid; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indarsen-3-propionic acid; succinimide ester;4,4-Difluoro-1,3,5,7-Tetramethyl-4-bora-3a,4a-diaza-s-indarsen-8-propionic acid; 4,4-Difluoro-1,3,5,7-Tetramethyl-4-bora-3a,4a-diaza-s-indarsen-8-propionic acid succinimide ester; 4,4-Difluoro-5-(2-diethyl)-4-bora-3a,4a-diaza-s-indarsen-3-propionic acid succinimide ester ; 6-(((4-(4,4-difluoro-5-(2-diethyl)-4-bora-3a,4a-diaza-s-indarsen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide ester; and 6-(((4,4-difluoro-5-(2-diethyl)-4-bora-3a,4a-diaza-s-indarsen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimide ester;

[0073] Fluorescent dyes may also include, for example, Alexa fluorescent dyes available from Invitrogen, including but not limited to Alexa. 350 carboxylic acids; Alexa 430 carboxylic acid; Alexa 488 carboxylic acid; Alexa 532 carboxylic acid; Alexa 546 carboxylic acid; Alexa 555 carboxylic acid; Alexa 568 carboxylic acid; Alexa 594 carboxylic acid; Alexa 633 carboxylic acid; Alexa 647 carboxylic acid; Alexa 660 carboxylic acid; and Alexa 680 carboxylic acid. Suitable fluorescent dyes may also include, for example, cyan dyes available from Amersham-Pharmacia Biotech, including but not limited to Cy3 NHS esters; Cy5 NHS esters; Cy5.5 NHS esters; and Cy7 NHS esters.

[0074] Example

[0075] Example 1: Formation of hydrogel particles

[0076] The photomask used for UV lithography originated from FineLine Imaging, Inc. and was designed using AutoCad (AutoDesk, Inc.). SU-8 photoresist (Microchem, Inc.) was photocrosslinked on a 4" silicon wafer using a collimated UV light source (OAI, Inc.) to produce a master for microfluidic device fabrication. PDMS (polydimethylsiloxane, SigmaAldrich, Inc.) was prepared and formed using soft lithography and microfluidic device fabrication methods (see, for example, McDonald JC et al., 2000, Electrophoresis 21:27-40, the contents of which are incorporated herein by reference in their entirety for all purposes).

[0077] Droplets were formed using flow-focusing geometry, where two oil channels concentrated the central flow of the aqueous monomer solution to break down the droplets in the water-in-oil emulsion. A fluorocarbon-based oil (Novec 7500 3M, Inc.) was used as the outer continuous phase liquid for droplet formation. To stabilize the droplets prior to polymerization, a surfactant (0.5% w / w of the ammonium carboxylate salt of Krytox 157FSH, DuPont) was added to the oil phase. To prepare the basic polyacrylamide gel particles, a central phase containing an aqueous monomer solution of N-acrylamide (1%–20% w / v), a crosslinking agent (N,N'-bisacrylamide, 0.05%–1% w / v), an accelerator, and ammonium persulfate (1% w / v) was used. An accelerator (N,N,N',N'-tetramethylethylenediamine 2% v / v) was added to the oil phase to initiate the polymerization of the hydrogel particles after droplet formation.

[0078] Several comonomers are added to the base gel formulation to increase functionality. Allyl-amine provides primary amine groups for secondary labeling after gel formation. The FSC of the droplets is adjusted by adding the comonomers allyl acrylate and allyl methacrylate to modify the refractive index of the gel. The SSC of the droplets is tuned by adding a colloidal suspension of silica nanoparticles and / or PMMA (poly(methyl methacrylate)) particles (approximately 100 nm) to the central aqueous phase prior to polymerization.

[0079] Stoichiometric labeling of hydrogel particles is achieved by using comonomers containing chemically orthogonal side groups (amine, carboxyl, maleimide, epoxy, acetylene, etc.) for secondary labeling.

[0080] Droplets were formed at an average rate of 5 kHz and collected in a fluorocarbon oil phase. After polymerization was completed at 50 °C for 30 minutes, the resulting hydrogel particles were washed from the oil into an aqueous solution.

[0081] Figure 6 is a simplified diagram showing an encoding scheme for generating a group of beads of similar size, according to some embodiments, which can be multiplexed using passive optical properties. Passive optical barcoding is achieved by tuning the FSC and SSC and by merging particles with unique FSC / SSC ratios.

[0082] Figures 7A and 7B are characterization diagrams showing that particles of the same size can be encoded with different passive optical properties. This allows for deconvolution of multiplexed biochemical assays using only passive optical properties. Figure 7B highlights the inability to distinguish particle populations based solely on fluorescence signals, thus demonstrating, according to some embodiments, the use of passive optical properties as the primary deconvolution variable for biochemical assay multiplexing and splitting. Figure 7A shows multiple synthetic cell populations of the same size but with different passive optical scattering ratios (FSC / SSC). Different surface markers were conjugated to each subpopulation and co-incubated with FITC-conjugated homologous antibodies. Each biomarker-modified bead population exhibited the same fluorescence spectrum but could be deconjugated based on their different optical properties for biochemical assay multiplexing.

[0083] In some embodiments, the composition comprises a plurality of hydrogel particles, wherein each hydrogel particle from the plurality of hydrogel particles has a substantially equal diameter. The plurality of hydrogel particles comprises a plurality of groups of hydrogel particles, and each group of hydrogel particles from the plurality of groups of hydrogel particles has one or more different correlated values ​​of passive optical properties (e.g., forward scattering and / or side scattering).

[0084] The plurality of hydrogel particles may be included in the mixture, and the mixture may be configured to use only passive optical properties for multiplexing.

[0085] In some embodiments, the plurality of hydrogel particles are included in a mixture, and the mixture is configured to use (1) passive optical properties and (2) fluorescence properties for multiplexing.

[0086] In some embodiments, the hydrogel particles from at least one of the plurality of groups of hydrogel particles have a refractive index (e.g., average refractive index or maximum or minimum refractive index) greater than about 1.15.

[0087] In some embodiments, the hydrogel particles from at least one of the plurality of groups of hydrogel particles have a refractive index (e.g., average refractive index or maximum or minimum refractive index) greater than about 1.3.

[0088] In some embodiments, the hydrogel particles from at least one of the plurality of groups have a refractive index greater than about 1.7.

[0089] In some embodiments, the diameter of each of the plurality of hydrogel particles is less than about 1000 μm, or less than about 100 μm, or less than about 10 μm.

[0090] In some implementations, the plurality of hydrogel particles include nanoparticles.

[0091] In some embodiments, at least one hydrogel particle from the plurality of hydrogel particles is chemically functionalized.

[0092] In some embodiments, at least one hydrogel particle from the plurality of hydrogel particles contains a free amine group.

[0093] In some embodiments, at least one hydrogel particle from the plurality of hydrogel particles contains allylamine.

[0094] In some implementations, each of the plurality of hydrogel particles is generated by polymerizing droplets.

[0095] In some implementations, the plurality of hydrogel particles are a substantially monodisperse group of hydrogel particles.

[0096] In some embodiments, the method for performing multiplexing assays includes measuring a sample using multiple optically coded hydrogel particles, using a cell counting device and deconvolving the multiple hydrogel particles based on the passive optical properties of the multiple hydrogel particles, and determining multiple measurements of the sample from a single reaction. Each hydrogel particle from the multiple hydrogel particles can be functionalized with different biochemical or chemical targets from a target set. Alternatively or additionally, each hydrogel particle from the multiple hydrogel particles can be functionalized with at least one of the following: antigen, protein, small molecule, or antibody.

[0097] In some embodiments, the hydrogel particles from each group (from multiple groups) of the plurality of hydrogel particles have different correlated values ​​for passive optical properties (e.g., forward scattering and / or side scattering).

[0098] Example 1: Passive optical tuning of hydrogel particles

[0099] As depicted in Figures 4 and 6, hydrogel particles are tuned in multiple sizes to generate different populations of beads based on their passive optical properties. These beads can be deconvoluted using a combination of FSC and SSC. Exemplary matching of the three main subpopulations of white blood cells (lymphocytes, monocytes, and granulocytes (neutrophils)) by tuning passive optical properties independently of particle size is shown in Figures 8A-8B. For clarity, all particles in the embodiments of Figures 8A-8B have equal diameters. Figure 8A is a graph of white blood cell (WBC) counts for actual lysed blood cell populations (representing the three subpopulations), and Figure 8B shows the counts of hydrogel particles with tuned passive optical properties and how they simulate the behavior of lysed whole blood without changing instrument settings (e.g., gain, voltage) between data acquisitions. Some end-user applications require assay beads that are optically similar to biological cell populations. The core technologies described herein facilitate accurate placement of assay beads onto the target population while extending multiplexing capabilities via optical coding.

[0100] Example 2: Tuning of Lateral Scattering from Hydrogel Particles

[0101] Colloidal silica was added to the aqueous fraction of the polymer mixture at 12.5%, 6.25%, 3.125%, and 0%, and hydrogel particles were formed as described in Example 1. Forward and side scattering data were obtained using flow cytometry. The results showed that the side scattering signal (Figure 9A) increased with a higher percentage of encapsulated nanoparticles, while the forward scattering signal (Figure 9B) remained generally unchanged, thus confirming that side scattering can be tuned independently of forward scattering.

[0102] Example 3: Tuning of forward scattering from hydrogel particles

[0103] In this experiment, the acrylamide:bisacrylamide percentage in the hydrogel composition was varied between 20% and 40% to tune the refractive index of the hydrogel particles, as measured by forward scattering in flow cytometry. As shown in Figures 10A-10C, forward scattering increases with increasing acrylamide:bisacrylamide percentage.

[0104] All publications, patents, patent applications and other documents cited in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent, patent application or other document were individually cited for all purposes.

[0105] Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of the invention.

[0106] While various embodiments of the systems, methods, and apparatus have been described above, it should be understood that these embodiments are presented by way of example only and not by way of limitation. Where the methods and steps described above indicate certain events to be performed in a certain order, those skilled in the art, benefiting from this disclosure, will recognize that the ordering of certain steps can be modified, and such modifications are consistent with variations of the invention. Furthermore, where possible, certain steps can be performed simultaneously in parallel processes, and can be performed sequentially as described above. Although the embodiments have been specifically shown and described, it will be understood that various changes can be made to their form and details. While the various embodiments have been described as combinations having specific features and / or components, other embodiments having combinations of any features and / or components from any of the embodiments discussed above are possible.

[0107] As used herein, the following terms and expressions are intended to have the following meanings:

[0108] The indefinite articles “a” and “an”, as well as the definite article “the”, are intended to include both singular and plural forms unless otherwise explicitly stated in the context in which they are used.

[0109] The terms “at least one” and “one or more” are used interchangeably to mean that an item may include one or more of the listed elements.

[0110] Unless otherwise indicated, it should be understood that all figures intended to represent the quantity, ratio, and numerical characteristics of ingredients, reaction conditions, etc., are in all cases subject to the term “about”.

[0111] As used herein, the terms “about” and “approximately” generally mean the value plus or minus 10%, for example, about 250 μm will include 225 μm to 275 μm, and about 1,000 μm will include 900 μm to 1,100 μm.

[0112] Unless otherwise expressly stated or clearly apparent from the context, references to singular items in this disclosure shall be construed as including plural items, and vice versa. Unless otherwise stated or clearly apparent from the context, grammatical conjunctions are intended to express any and all separate and connecting combinations of clauses, sentences, words, etc. Therefore, the term “or” should generally be understood to mean “and / or”, etc. The use of any and all examples or exemplary language (“e.g.,” “such as,” “including,” etc.) provided herein is intended only to better illustrate the embodiments and does not constitute a limitation on the scope of the embodiments or claims.

Claims

1. A method for performing multiplexing determination, the method comprising: Samples were measured using multiple optically coded hydrogel particles, wherein the hydrogel particles were spherical; The plurality of hydrogel particles are deconvoluted using a cell counting device and based on the passive optical properties of the plurality of hydrogel particles, wherein the passive optical properties are forward scattering or side scattering. as well as Determining multiple measurements of the sample from a single reaction. The plurality of hydrogel particles comprises a group of plurality of hydrogel particles. Each of the plurality of hydrogel particles has the same diameter, and Each group of hydrogel particles from the plurality of groups of hydrogel particles has a different correlation value for passive optical properties.

2. The method of claim 1, wherein each group of the plurality of hydrogel particles is functionalized with different biochemical or chemical targets from a target set.

3. The method of claim 1, wherein each of the group of the plurality of hydrogel particles is functionalized with at least one of the following: antigen, protein, or small molecule.

4. The method of claim 3, wherein the protein is an antibody.

5. The method according to claim 1, wherein the passive optical property is forward scattering.

6. The method according to claim 1, wherein the passive optical property is lateral scattering.

7. The method of claim 1, wherein the hydrogel particles from at least one of the plurality of groups have a refractive index greater than 1.

3.

8. The method of claim 1, wherein the hydrogel particles from at least one of the plurality of groups have a refractive index greater than 1.

7.

9. The method of claim 1, wherein the diameter of each hydrogel particle from the plurality of hydrogel particles is less than 1000 µm.

10. The method of claim 7, wherein the diameter of each hydrogel particle from the plurality of hydrogel particles is less than 100 µm.

11. The method of claim 8, wherein the diameter of each hydrogel particle from the plurality of hydrogel particles is less than 10 µm.

12. The method of claim 1, wherein the plurality of hydrogel particles comprises nanoparticles.

13. The method of claim 1, wherein at least one of the plurality of hydrogel particles is chemically functionalized.

14. The method of claim 1, wherein at least one hydrogel particle from the plurality of hydrogel particles comprises a free amine group.

15. The method of claim 1, wherein at least one hydrogel particle from the plurality of hydrogel particles comprises allylamine.

16. The method of claim 1, wherein each of the plurality of hydrogel particles is generated by polymerizing droplets.

17. The method of claim 1, wherein the plurality of hydrogel particles are a monodisperse group of hydrogel particles.

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