Patent Description:
Flow cytometry is a technique that allows for the rapid separation, counting, and characterization of individual cells and is routinely used in clinical and laboratory settings for a variety of applications. The technology relies on directing a beam of light onto a hydrodynamically-focused stream of liquid. A number of detectors are then aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC). FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (e.g., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). As a result of these correlations, different specific cell types exhibit different FSC and SSC, allowing cell types to be distinguished in flow cytometry.

The ability to identify specific cell types, however, relies on proper calibration of the instrument, a process that has relied on the use of purified cells of the cell type of interest. Obtaining these purified cells can require costly, laborious procedures that are prone to batch-to-batch variation. Therefore, there is a need in the art for synthetic compositions with tunable optical properties that can mimic specific cell types in devices such as flow cytometers.

<CIT> discloses a standard fluid for a flow cytometer which shows, after staining, almost the same fluorescence intensity and scattered light intensity as those of the cells to be assayed. <CIT> discloses a microbead kit and method for quantitative calibration performance monitoring of a fluorescence instrument.

The present invention is defined by the method for calibrating a cytometric device for analysis of a target cell according to claim <NUM>. Preferred aspects are defined in the dependent claims.

In one embodiment of the methods provided herein, the hydrogel particle comprises a biodegradable monomer. In a further embodiment, the biodegradable monomer is a monosaccharide, disaccharide, polysaccharide, peptide, protein, or protein domain. In even a further embodiment, the biodegradable monomer is functionalized with acrylamide or acrylate.

The indefinite articles "a" and "an" and the definite article "the" are intended to include both the singular and the plural, unless the context in which they are used clearly indicates otherwise.

"At least one" and "one or more" are used interchangeably to mean that the article may include one or more than one of the listed elements.

Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth, used in the specification and claims are contemplated to be able to be modified in all instances by the term "about".

Several critical calibration measurements for flow cytometers require precise time resolution, such as setting the offset time between lasers, and calculating the delay time between detection and sorting of an object. Due to the fluidic conditions within the instrument, precise setting of these timing parameters requires the use of calibration particles that are the same size as the cells to be analyzed. Timing calibrations are typically performed using polystyrene beads with variable fluorescent intensities to calibrate the response of an excitation source and to set the inter-laser timing delay and sorting delay. Flow cytometers can also be calibrated using forward and side scatter signals which are general measures of size and granularity or complexity of the target sample. These calibrations are crucial for the accurate performance of the cytometer and for any downstream analysis or sorting of cell populations. The disclosed hydrogel particles exhibit tuned scatter properties and are suitable for use as calibration reagents for a range of mammalian or bacterial cell types. Scattering is a standard metric for distinguishing cell types in heterogeneous mixtures for clinical, food safety, and research purposes.

Although polystyrene particles can be used to set inter-laser and sorting delays for some applications, many eukaryotic cell types fall outside of the size range of commercially available polystyrene particles (<NUM>-<NUM>) making it nearly impossible to accurately calibrate a flow cytometer for these targets. Also, as shown in <FIG>, polystyrene particles are fundamentally limited in the optical properties that can possess such as side scattering, which is a general measure of cellular complexity. Polystyrene particles are therefore limited in the two most important passive optical measurements used in flow cytometry: FSC (forward scattering), and SSC (side scattering) which measure the size and complexity of the target respectively. Due to these limitations of polystyrene, users must rely on purified cell lines to calibrate fluorescent intensity, inter-laser delay, sort delays, size and cellular complexity for experiments. This is a lengthy and labor-intensive process that increases the cost of flow cytometry validation and research pipelines significantly. More importantly, these calibration cell lines introduce biological variation, causing disparities in the interpretation of data.

Moreover, quality control (QC) for calibration of flow cytometers is also a crucial consideration when these instruments are used for clinical applications, for example, to isolate human T-regulatory cells or stem cells for downstream cellular therapies. The FDA mandates that the sterility, identity, purity, and potency of a cell therapy product be demonstrated before administration to patients (<NPL>). Contamination of a cellular population with polystyrene QC particles could therefore be problematic, as polystyrene has been implicated in certain cancers. Additionally, a cellular population that is contaminated with a QC standard that is enzymatically degraded or digested internally after administration to a patient potentially overcomes contamination issues, should they arise.

The present invention addresses these and other needs, as discussed below.

In one aspect, a composition comprising a plurality of hydrogel particles is provided, wherein the individual hydrogel particles of the plurality each has one or more optical properties substantially similar to one or more optical properties of a target cell. Each of the individual hydrogel particles of the plurality independently comprises a hydrogel which is synthesized by polymerizing one or more monomers, i.e., to form a homopolymer or copolymer. As discussed further below, the use of bifunctional monomers allows for the further derivatization of hydrogels, e.g., with fluorescent dyes, cell surface markers or epitope binding fragments thereof, or a combination thereof. An example of hydrogel parameter tuning to meet/match desired cell subpopulation metrics is provided at <FIG>. Methods for tuning the properties of a hydrogel are described herein. The ability to adjust a range of parameters including hydrogel components and concentration of the same allows for the ability to tune a particle to mimic a wide range of cells, for example one of the cell types described herein.

As provided above, in one aspect, the present invention provides individual hydrogel particles each having one or more optical properties substantially similar to one or more optical properties of a target cell. In one embodiment, the one or more optical properties, is a side scatter profile, a forward scatter profile or a secondary marker profile, such as a fluorescence marker profile, for example a fluorescence marker profile of a fluorescently-labeled antibody that binds to the surface of the hydrogel particle. "Substantially similar," as used herein, denotes at least <NUM>% similar, at least <NUM>% similar, at least <NUM>% similar, at least <NUM>% similar, at least <NUM>% similar, at least <NUM>% similar, at least <NUM>% similar, at least <NUM>% similar, at least <NUM>% similar, at least <NUM>% similar or at least <NUM>% similar.

The present invention is based in part on the unexpected discovery that one or more optical properties of a hydrogel particle can be independently modulated by altering the composition of the hydrogel particle, for example, by altering the amount of initial monomer (or co-monomer) in the composition, by altering the surface functionalization, by altering the amount of a polymerization initiator or by altering the amount of crosslinker. For example, side scattering (SSC) can be modulated without substantially affecting forward scattering (FSC), and vice versa. Furthermore, the optical properties (e.g. refractive index) of hydrogel particles can be tuned without having a substantial effect on density of the particle. This is a surprising and useful feature, as hydrogel particles that serve as surrogates for cells in cytometric methods such as flow cytometry or (fluorescence-activated cell sorting) FACS require a minimal density in order to function in those assays.

In another aspect, a method for producing a hydrogel particle is provided, wherein the hydrogel particle has one or more optical properties substantially similar to the optical properties of one or more target cells. In one embodiment, the hydrogel particle has pre-determined optical properties. The optical property, in one embodiment, is SSC, FSC, fluorescence emission, or a combination thereof.

In yet another aspect, a method of calibrating a cytometric device for analysis of a target cell is provided. In one embodiment, the method comprises (a) inserting into the device a hydrogel particle having optical properties substantially similar to the optical properties of the target cell; b) measuring the optical properties of the hydrogel particle using the cytometric device, thereby calibrating the cytometric device for analysis of the target cell. Cytometric devices are known in the art, and include commercially available devices for performing flow cytometry and FACS.

As provided above, in one aspect of the invention, compositions comprising a plurality of hydrogel particles are provided. A hydrogel is a material comprising a macromolecular three-dimensional network that allows it to swell when in the presence of water, to shrink in the absence of (or by reduction of the amount of) water, but not dissolve in water. The swelling, i.e., the absorption of water, is a consequence of the presence of hydrophilic functional groups attached to or dispersed within the macromolecular network. Crosslinks between adjacent macromolecules result in the aqueous insolubility of these hydrogels. The cross-links may be due to chemical (i.e., covalent) or physical (i.e., VanDer Waal forces, hydrogen-bonding, ionic forces, etc.) bonds. Synthetically prepared hydrogels can be prepared by polymerizing a monomeric material to form a backbone and cross-linking the backbone with a crosslinking agent. As referred to herein, the term "hydrogel" refers to the macromolecular material whether dehydrated or in a hydrated state. A characteristic of a hydrogel that is of particular value is that the material retains the general shape, whether dehydrated or hydrated. Thus, if the hydrogel has an approximately spherical shape in the dehydrated condition, it will be spherical in the hydrated condition.

In one embodiment, a hydrogel particle disclosed herein comprises greater than about <NUM>%, greater than about <NUM>%, greater than about <NUM>%, greater than about <NUM>%, greater than about <NUM>%, greater than about <NUM>%, greater than about <NUM>%, greater than about <NUM>%, greater than about <NUM>%, greater than about <NUM>%, greater than about <NUM>%, or greater than about <NUM>% water. In another embodiment, a hydrogel particle has a water content of about <NUM> percent by weight to about <NUM> percent by weight, or about <NUM> percent by weight to about <NUM> percent by weight, or about <NUM> percent by weight to about <NUM> percent by weight, or about <NUM> percent by weight to about <NUM> percent by weight, or about <NUM> percent by weight to about <NUM> percent by weight, or about <NUM> percent by weight to about <NUM> percent by weight, or about <NUM> percent by weight to about <NUM> percent by weight, or about <NUM> percent by weight to about <NUM> percent by weight.

The hydrogels provided herein, in the form of particles, are synthesized by polymerizing one or more of the monomers provided herein. The synthesis is carried out to form individual hydrogel particles. The monomeric material (monomer) in one embodiment is polymerized to form a homopolymer. However, in another embodiment copolymers of different monomeric units (i.e., co-monomers) are synthesized and used in the methods provided herein. The monomer or co-monomers used in the methods and compositions described herein, in one embodiment, is a bifunctional monomer or includes a bifunctional monomer (where co-monomers are employed). In one embodiment, the hydrogel is synthesized in the presence of a crosslinker. In a further embodiment, embodiment, the hydrogel is synthesized in the presence of a polymerization initiator.

The amount of monomer can be varied by the user of the invention, for example to obtain a particular optical property that is substantially similar to that of a target cell. In one embodiment, the monomeric component(s) (i.e., monomer, co-monomer, bifunctional monomer, or a combination thereof, for example, bis/acrylamide in various crosslinking ratios, allyl amine or other co-monomers which provide chemical functionality for secondary labeling/conjugation or alginate is present at about <NUM> percent by weight to about <NUM> percent weight of the hydrogel. In a further embodiment, the monomeric component(s) is present at about <NUM> percent by weight to about <NUM> percent weight of the hydrogel, or about <NUM> percent by weight to about <NUM> percent weight of the hydrogel.

Examples of various monomers and cross-linking chemistries available for use with the present invention are provided in the Thermo Scientific Crosslinking Technical Handbook entitled "<NPL> Here, and in all following instances in this specification, where the expression "the disclosure of which is incorporated by reference [in its entirety for all purposes]" is used, this expression should be ignored, because in all these instances, the reference documents are not considered essential for carrying out the invention. For example, hydrazine (e.g., with an NHS ester compound) or EDC coupling reactions (e.g., with a maleimide compound) can be used to construct the hydrogels of the invention.

In one embodiment, a monomer for use with the hydrogels provided herein is lactic acid, glycolic acid, acrylic acid, <NUM>-hydroxyethyl methacrylate, ethyl methacrylate, <NUM>-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, a derivatized version thereof, or a combination thereof.

In one embodiment, one or more of the following monomers is used herein to form a hydrogel of the present invention: <NUM>-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate or a combination thereof.

In another embodiment, one or more of the following monomers is used herein to form a tunable hydrogel: phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, <NUM>-phenylethyl acrylate, <NUM>-phenylethyl methacrylate, <NUM>-phenoxyethyl acrylate, <NUM>-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, <NUM>,<NUM>,<NUM>-tribromophenyl acrylate, <NUM>,<NUM>,<NUM>-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, <NUM>,<NUM>-dibromopropyl acrylate, <NUM>,<NUM>-dibromopropyl methacrylate, <NUM>-naphthyl acrylate, <NUM>-naphthyl methacrylate, <NUM>-methoxybenzyl acrylate, <NUM>-methoxybenzyl methacrylate, <NUM>-benzyloxyethyl acrylate, <NUM>-benzyloxyethyl methacrylate, <NUM>-chlorophenoxyethyl acrylate, <NUM>-chlorophenoxyethyl methacrylate, <NUM>-phenoxyethoxyethyl acrylate, <NUM>-phenoxyethoxyethyl methacrylate, N-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide N-(<NUM>-methylphenyl)methyl acrylamide, N-<NUM>-naphthyl acrylamide, N-<NUM>-nitrophenyl acrylamide, N-(<NUM>-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(<NUM>-hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(<NUM>-methyl phenyl)methyl methacrylamide, N-<NUM>-naphthyl methacrylamide, N-<NUM>-nitrophenyl methacrylamide, N-(<NUM>-phenylethyl)methacrylamide, N-triphenylmethyl methacrylamide, N-(<NUM>-hydroxyphenyl)methacrylamide, N,N-methylphenyl methacrylamide, N,N'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, <NUM>-vinylpyridine, <NUM>-vinylpyridine, as described in <CIT>, which is incorporated by reference in its entirety herein for all purposes.

Both synthetic monomers and bio-monomers can be used in the hydrogels provided herein, to form synthetic hydrogels, bio-hydrogels, or hybrid hydrogels that comprise a synthetic component and a bio-component (e.g., peptide, protein, monosaccharide, disaccharide, polysaccharide, primary amines sulfhydryls, carbonyls, carbohydrates, carboxylic acids present on a biolmolecule). For example, proteins, peptides or carbohydrates can be used as individual monomers to form a hydrogel that includes or does not include a synthetic monomer (or polymer) and in combination with chemically compatible co-monomers and crosslinking chemistries (see for example, the Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology," available at tools. lifetechnologies. com/content/sfs/brochures/<NUM>-Crosslinking-Reagents-Handbook. pdf, the disclosure of which is incorporated by reference in its entirety for all purposes. Compatible crosslinking chemistries include, but are not limited to, amines, carboxyls, and other reactive chemical side groups. Representative reactive groups amenable for use in the hydrogels and monomers described herein are provided in Table <NUM>, below.

In general, any form of polymerization chemistry/methods commonly known by those skilled in the art, can be employed to form polymers. In some embodiments, polymerization can be catalyzed by ultraviolet light-induced radical formation and reaction progression. In other embodiments, a hydrogel particle of the disclosure is produced by the polymerization of acrylamide or the polymerization of acrylate. For example, the acrylamide in one embodiment is a polymerizable carbohydrate derivatized acrylamide as described in <CIT>, the disclosure of which is incorporated by reference in its entirety for all purposes. As described therein and known to those of ordinary skill in the art, specific attachment of acrylamide groups to sugars is readily adapted to a range of monosaccharides and higher order polysaccharides, e.g., synthetic polysaccharides or polysaccharides derived from natural sources, such as glycoproteins found in serum or tissues.

In one embodiment, an acrylate-functionalized poly(ethylene) glycol monomer is used as a hydrogel monomer. For example, the PEG in one embodiment is an acrylate or acrylamide functionalized PEG.

In some embodiments, a hydrogel particle comprises a monofunctional monomer polymerized with at least one bifunctional monomer. One example includes, but is not limited to, the formation of poly-acrylamide polymers using acrylamide and bis-acrylamide (a bifunctional monomer). In another embodiment, a hydrogel particle provided herein comprises a bifunctional monomer polymerized with a second bifunctional monomer. One example include, but is not limited to, the formation of polymers with mixed composition containing compatible chemistries such as acrylamide, bis-acrylamide, and bis-acrylamide structural congeners containing a wide range of additional chemistries. The range of chemically compatible monomers, bifunctional monomers, and mixed compositions is obvious to those skilled in the art and follows chemical reactivity principles know to those skilled in the art. (reference Thermo handbook and acrylamide polymerization handbook). See, for example, the Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology," (available at tools. lifetechnologies. com/content/sfs/brochures/<NUM>-Crosslinking-Reagents-Handbook. pdf) and the Polyacrylamide Emulsions Handbook (SNF Floerger, available at snf. au/downloads/Emulsion_Handbook_E. pdf), the disclosure of each of which is incorporated by reference in its entirety for all purposes.

In one embodiment, a hydrogel particle provided herein comprises a polymerizable monofunctional monomer and is a monofunctional acrylic monomer. Non-limiting examples of monofunctional acrylic monomers for use herein are acrylamide; methacrylamide; N-alkylacrylamides such as N-ethylacrylamide, N-isopropylacrylamide or N-tertbutylacrylamide; N-alkylmethacrylamides such as N-ethylmethacrylamide or Nisopropylmethacrylamide; N,N-dialkylacrylamides such as N,N-dimethylacrylamide and N ,N -diethyl-acrylamide; N-[( dialkylamino)alkyl] acrylamides such as N-[3dimethylamino) propyl]acrylamide or N-[<NUM>-(diethylamino)propyl] acrylamide; N-[(dialkylamino) alkyl] methacrylamides such as N-[<NUM>-dimethylamino)propyl] methacrylamide or N-[<NUM>-(diethylamino) propyl] methacrylamide; (dialkylamino)alkyl acrylates such as <NUM>-(dimethylamino )ethyl acrylate, <NUM>-( dimethylamino)propyl acrylate, or <NUM>-(diethylamino )ethyl acrylates; and (dialkylamino) alkyl methacrylates such as <NUM>-(dimethylamino) ethyl methacrylate.

A bifunctional monomer is any monomer that can polymerize with a monofunctional monomer of the disclosure to form a hydrogel as described herein that further contains a second functional group that can participate in a second reaction, e.g., conjugation of a fluorophore or cell surface receptor (or domain thereof).

In some embodiments, a bifunctional monomer is selected from the group consisting of: allyl amine, allyl alcohol, allyl isothiocyanate, allyl chloride, and allyl maleimide.

A bifunctional monomer can be a bifunctional acrylic monomer. Non-limiting examples of bifunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'methylene bismethacrylamide, N ,N'-ethylene bisacrylamide, N ,N' -ethylene bismethacrylamide, N,N'propylenebisacrylamide and N,N'-(<NUM>,<NUM>- dihydroxyethylene) bisacrylamide.

Higher-order branched chain and linear co-monomers can be substituted in the polymer mix to adjust the refractive index while maintaining polymer density, as described in <CIT>, incorporated herein by reference in its entirety for all purposes.

In some embodiments, a hydrogel comprises a molecule that modulates the optical properties of the hydrogel. Molecules capable of altering optical properties of a hydrogel are discussed further below.

In one embodiment, an individual hydrogel particle or a plurality thereof comprises a biodegradable polymer as a hydrogel monomer. In one embodiment, the biodegradable polymer is a poly(esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and their copolymers. In one embodiment, the biodegradable polymer is a carbohydrate or a protein, or a combination thereof. For example, in one embodiment, a monosaccharide, disaccharide or polysaccharide, (e.g., glucose, sucrose, or maltodextrin) peptide, protein (or domain thereof) is used as a hydrogel monomer. Other biodegradable polymers include poly(hydroxyalkanoate)s of the PHB-PHV class, additional poly(ester)s, and natural polymers, for example, modified poly(saccharide)s, e.g., starch, cellulose, and chitosan. In another embodiment, the biocompatible polymer is an adhesion protein, cellulose, a carbohydrate, a starch (e.g., maltodextrin, <NUM>-hydroxyethyl starch, alginic acid), a dextran, a lignin, a polyaminoacid, an amino acid, or chitin. Such biodegradable polymers are available commercially, for example, from Sigma Aldrich (St. Louis, MO).

The protein in one embodiment comprises only natural amino acids. However, the invention is not limited thereto. For example, self-assembling artificial proteins and proteins with non-natural amino acids (e.g., those incorporated into non-ribosomal peptides or synthetically introduced via synthetic approaches, see for example, <NPL>, the disclosure of which is incorporated by reference in its entirety for all purposes), or protein domains thereof, can also be used as hydrogel monomers. The range of non-natural (unnatural) amino acids that can be incorporated into such compositions is well known to those skilled in the art (<NPL>; incorporated by reference in its entirety for all purposes). The biodegradable polymer in one embodiment, is used as a co-monomer, i.e., in a mixture of monomers. The biodegradable polymer in one embodiment is a bifunctional monomer.

The biomonomer, in one embodiment, is functionalized with acrylamide or acrylate. For example, in one embodiment, the polymerizable acrylamide functionalized biomolecule is an acrylamide or acrylate functionalized protein (for example, an acrylamide functionalized collagen or functionalized collagen domain), an acrylamide or acrylate functionalized peptide, or an acrylamide or acrylate functionalized monosaccharide, disaccharide or polysaccharide.

Any monosaccharide, disaccharide or polysaccharide (functionalized or otherwise) can be used as a hydrogel monomer. In one embodiment, an acrylamide or acrylate functionalized monosaccharide, disaccharide or polysaccharide is used as a polymerizable hydrogel monomer. In one embodiment, a structural polysaccharide is used as a polymerizable hydrogel monomer. In a further embodiment, the structural polysaccharide is an arabinoxylan, cellulose, chitin or a pectin. In another embodiment, alginic acid (alginate) is used as a polymerizable hydrogel monomer. In yet another embodiment, a glycosaminoglycan (GAG) is used as a polymerizable monomer in the hydrogels provided herein. In a further embodiment, the GAG is chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate or hyaluronic acid (also referred to in the art as hyaluron or hyaluronate) is used as a polymerizable hydrogel monomer. The additional range of compatible biomonomers and their reactive chemistries are known be individuals skilled in the art and follow general chemical reactivity principles.

An additional range of biocompatible monomers that can be incorporated are known in the art, see, for example the non-degradable biocompatible monomers disclosed in <NPL>, incorporated by reference herein in its entirety for all purposes. Other monomers are provided in de <NPL>; <NPL>; <NPL>, the disclosure of each of which are hereby incorporated by reference in their entirety.

Biocompatible monomers for use with the hydrogels described herein include in one embodiment, ethyleglycol dimethacrylate (EGDMA), <NUM>-hydroxyethyl methacrylate (HEMA), methylmethacrylte (MMA), methacryloxymethyltrimethylsilane (TMS-MA), N-vinyl-<NUM>-pyrrolidon (N-VP), styrene, or a combination thereof.

Naturally occurring hydrogels useful in this invention include various polysaccharides available from natural sources such as plants, algae, fungi, yeasts, marine invertebrates and arthropods. Non-limiting examples include agarose, dextrans, chitin, cellulose-based compounds, starch, derivatized starch, and the like. These generally will have repeating glucose units as a major portion of the polysaccharide backbone. Cross-linking chemistries for such polysaccharides are known in the art, see for example Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology," (available at tools. lifetechnologies. com/content/sfs/brochures/<NUM>-Crosslinking-Reagents-Handbook.

Hyaluronan in one embodiment is used as a hydrogel monomer (either as a single monomer or as a co-monomer). Hyaluronan in one embodiment, is functionalized, for example with acrylate or acrylamide. Hyaluronan is a high molecular weight GAG composed of disaccharide repeating units of N-acetylglucosamine and glucuronic acid linked together through alternating β-<NUM>,<NUM> and β-<NUM>,<NUM> glycosidic bonds. In the human body, hyaluronate is found in several soft connective tissues, including skin, umbilical cord, synovial fluid, and vitreous humor. Accordingly, in one embodiment, where one or more optical properties of a skin cell, umbilical cord cell or vitreous humor cell is desired to be mimicked, in one embodiment, hyaluronan is used as a hydrogel monomer. Methods for fabricating hydrogel particles are described in <NPL>, the disclosure of which is incorporated herein in its entirety for all purposes. As described therein, hyaluronan can be derivatized with various reactive handles depending on the desired cross-linking chemistry and other monomers used to form a hydrogel particle.

In yet other embodiments, chitosan, a linear polysaccharide composed of randomly distributed β-(<NUM>-<NUM>)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit), is used as a hydrogel monomer (either as a single monomer or as a co-monomer).

Other polysaccharides for use as a hydrogel monomer or co-monomer include but are not limited to, agar, agarose, alginic acid, alguronic acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta-glucan, callose, capsullan, carrageenan polysaccharides (e.g., kappa, iota or lambda class), cellodextrin, cellulin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminoogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof. As described throughout, depending on the desired cross-linking chemistry and/or additional co-monomers employed in the hydrogel, the polysaccharide can be further functionalized. For example, one or more of the polysaccharides described herein in one embodiment is functionalized with acrylate or acrylamide.

In one embodiment, an individual hydrogel particle or a plurality thereof comprises a peptide, protein, a protein domain, or a combination thereof as a hydrogel monomer or plurality thereof. In a further embodiment, the protein is a structural protein, or a domain thereof, for example, such as silk, elastin, titin or collagen, or a domain thereof. In one embodiment, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycan). In even a further embodiment, the structural protein is collagen. In yet a further embodiment, the collagen is collagen type I, collagen type II or collagen type III or a combination thereof. In another embodiment, the hydrogel monomer comprises a proteoglycan. In a further embodiment, the proteoglycan is decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.

In another embodiment, an acrylate-functionalized structural protein hydrogel monomer is used as a component of the hydrogel provided herein (e.g., an acrylate functionalized protein or protein domain, for example, silk, elastin, titin, collagen, proteoglycan, or a functionalized domain thereof). In a further embodiment, the acrylate functionalized structural protein hydrogel monomer comprises a proteoglycan, e.g., decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.

In one embodiment PEG monomers and oligopeptides can be that mimic extracellular matrix proteins are used in the hydrogels provided herein, for example, with vinyl sulfone-functionalized multiarm PEG, integrin binding peptides and bis-cysteine matrix metalloproteinase peptides as described by <NPL>, incorporated by reference in its entirety for all purposes. In this particular embodiment, hydrogels are formed by a Michael-type addition reaction between the di-thiolated oligopeptides and vinyl sulfone groups on the PEG. The range of additional compatible chemistries that can be incorporated here are obvious to those skilled in the art and follow general chemical reactivity principles, see for example Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology," (available at tools. lifetechnologies. com/content/sfs/brochures/<NUM>-Crosslinking-Reagents-Handbook.

Other bioactive domains in natural proteins can also be used as a hydrogel monomer or portion thereof. For example, a cell-adhesive integrin binding domain, a controlled release affinity binding domain or a transglutaminase cross-linking domain can be used in the hydrogels provided herein. Details for producing such hydrogels can be found in <NPL>; <NPL>; <NPL>, each of which is incorporated by reference in its entirety for all purposes.

In one embodiment, recombinant DNA methods are used to create proteins, designed to gel in response to changes in pH or temperature, for example, by the methods described by <NPL>, incorporated by reference in its entirety for all purposes. Briefly, the proteins consist of terminal leucine zipper domains flanking a water-soluble polyelectrolyte segment. In near-neutral aqueous solutions, coiled-coil aggregates of the terminal domains form a three-dimensional hydrogel polymer network.

Common cross linking agents that can be used to crosslink the hydrogels provided herein include but are not limited to ethylene glycol dimethacrylate (EGDMA), tetraethylene glycol dimethacrylate, and N,N'-<NUM> methylenebisacrylamide. The range of additional crosslinking chemistries which can be used are obvious to those skilled in the art and follow general chemical reactivity principles, see for example<NPL>).

In one embodiment, polymerization of a hydrogel is initiated by a persulfate or an equivalent initiator that catalyzes radical formation. The range of compatible initiators are known to those skilled in the art and follow general chemical reactivity principles, see for example<NPL>). The 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 embodiments, the persulfate is ammonium persulfate or potassium persulfate. In a further embodiment, polymerization of the hydrogel provided herein is initiated by ammonium persulfate.

Polymerization of a hydrogel can be accelerated by an accelerant which can catalyze the formation of polymerization-labile chemical side groups. The range of possible accelerants is known to those skilled in the art and follow general chemical reactivity principles see for example <NPL>). The accelerant in one embodiment, is a tertiary amine. The tertiary amine can be any water-soluble tertiary amine. In one embodiment, an accelerant is used in the polymerization reaction and is N,N,N',N'tetramethylethylenediamine, <NUM>-dimethylamino) propionitrile, or N,N,N',N'tetramethylethylenediamine (TEMED). In another embodiment, an accelerant is used in the polymerization reaction and isazobis (isobutyronitrile) (AIBN).

As discussed above, the hydrogel for use in the compositions and methods described herein can include any of the monomeric units and crosslinkers as described herein, and in one aspect, are produced as hydrogel particles by polymerizing droplets (see, e.g., <FIG>). Microfluidic methods of producing a plurality of droplets, including fluidic and rigidified droplets, are known to those of ordinary skill in the art, and described in <CIT> and <CIT>, each incorporated herein by reference in their entireties for all purposes. Such methods provide for a plurality of droplets containing a first fluid and being substantially surrounded by a second fluid, where the first fluid and the second fluid are substantially immiscible (e.g., droplets containing an aqueous-based liquid being substantially surrounded by an oil based liquid).

A plurality of fluidic droplets (e.g., prepared using a microfluidic device) may be polydisperse (e.g., having a range of different sizes), or in some cases, the fluidic droplets may be monodisperse or substantially monodisperse, e.g., having a homogenous distribution of diameters, for instance, such that no more than about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, or about <NUM>% of the droplets have an average diameter greater than about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, or about <NUM>% of the average diameter. The average diameter of a population of droplets, as used herein, refers to the arithmetic average of the diameters of the droplets. Average diameters of the particles can be measured, for example, by light scattering techniques. Average diameters of hydrogel particles in one embodiment, are tailored, for example by varying flow rates of the fluid streams of the first and second fluids within the channel(s) of a microfluidic device, or by varying the volume of the channel(s) of the microfluidic device.

Accordingly, the disclosure provides population of hydrogel particles comprising a plurality of hydrogel particles, wherein the population of hydrogel particles is substantially monodisperse.

The term microfluidic refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than <NUM>, and a ratio of length to largest cross-sectional dimension perpendicular to the channel of at least about <NUM>:<NUM>. A micro fluidic device comprising a micro fluidic channel is especially well suited to preparing a plurality of mono disperse droplets.

Non-limiting examples of microfluidic systems that may be used with the present invention are disclosed in <CIT>; <CIT>; <CIT>; International Patent Application Publication No. <CIT>; <CIT>; <CIT>; and International Patent Application Publication No. <CIT>, each incorporated herein by reference in their entireties for all purposes.

Droplet size is related to microfluidic channel size. The micro fluidic channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about <NUM> or <NUM>, or less than about <NUM>, or less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, or less than about <NUM>.

Droplet size can be tuned by adjusting the relative flow rates. In some embodiments, drop diameters are equivalent to the width of the channel, or within about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% the width of the channel.

The dimensions of a hydrogel particle of the disclosure are substantially similar to the droplet from which it was formed. Therefore, in some embodiments, a hydrogel particle has a diameter of less than about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or less than <NUM> in diameter. In some embodiments, a hydrogel particle has a diameter of more than about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or greater than <NUM> in diameter. In one embodiment, a hydrogel particle has a diameter in the range of <NUM> to <NUM>.

In some embodiments, a hydrogel particle of the disclosure is spherical in shape.

In some embodiments, a hydrogel particle of the disclosure does not comprise agarose.

Hydrogel particles in one embodiment, is carried by suspension polymerization, which is also referred to in the art as pearl, bead or granular polymerization (see <NPL>, incorporated by reference herein in its entirety for all purposes). In suspension polymerization, the monomer is insoluble in the continuous phase, for example an aqueous monomer solution in a continuous oil phase. In suspension polymerization, polymerization initiation occurs within the monomer-rich droplets and with greater than one radical per droplet at any time. The monomer phase in one embodiment includes a monomer which can be a bifunctional monomer or a plurality of monomer species (co-monomers, which can be a plurality of bifunctional monomers. The monomer phase in one embodiment, includes an initiator and/or a crosslinking agent.

Emulsion polymerization can also be used to form the hydrogel particles described herein. In emulsion polymerization, the monomer has poor solubility in the continuous phase, similar to suspension polymerization, however, polymerization initiation occurs outside the monomer droplets (see <NPL>, incorporated by reference herein in its entirety for all purposes). In emulsion polymerization embodiments, the initiator causes chain growth of the monomer (or co-monomers) dissolved in the continuous phase or monomer contained in micelles if surfactants are present.

In another embodiment, hydrogel particles are formed by precipitation polymerization, for example as described in <NPL>, incorporated by reference herein in its entirety for all purposes. Precipitation polymerization is a technique that takes advantage of the differences in the solubility of monomer and polymer to produce microparticles. Specifically, it is known that larger polymer chains generally have lower solubility than smaller ones. Accordingly, above a specific molecular weight, phase separation may be favored. Precipitation polymerization initially begins as solution polymerizations in a single phase, homogenous system. Shortly after the start of the polymerization, in one embodiment, a relatively high concentration of polymer chains is present, favoring phase separation by nucleation. As polymerization proceeds, the concentration of polymer chains is low and existing particles capture the chains before nucleation of new particles can occur. Thus, nucleation of particles occurs only for a brief period of time shortly after the start of the reaction, which in one embodiment, results in a narrow size distribution of particles. Additional methods include but are not limited to lithographic particle formation (<NPL>, incorporated by reference herein in its entirety for all puposes) membrane emulsification (e.g., by the micosieve emulsification technology techniques described by Nanomi B. (Netherlands)) and microchannel emulsification (Sugiura et al.

(<NPL>, incorporated by reference herein in its entirety) and bulk emulsification (SNF Floerger, available at snf. au/downloads/Emulsion_Handbook E. pdf, incorporated by reference herein in its entirety).

In one embodiment, hydrogel particles are formed within a microfluidic device having two oil channels that focus on a central stream of aqueous monomer solution. In this embodiment, droplets form at the interface of the two channels and central stream to break off droplets in water-in-oil emulsion. Once droplets are formed, in one embodiment, they are stabilized prior to polymerization, for example, by adding a surfactant to the oil phase. However, in another embodiment, droplets are not stabilized prior to polymerization. Polymerization of the monomer in one embodiment is triggered by adding an accelerator (e.g., N,N,N',N'tetramethylethylenediamine) to one or both of the oil channels after initial droplets are formed.

The aqueous monomer solution as provided above can include a single monomer species or a plurality of monomer species. The aqueous monomer solution can include co-monomers, a bifunctional monomer or a combination thereof. In one embodiment, the monomer or plurality of monomers can includes a bifunctional monomer, for example, one of the monomers described above. As described below, co-monomers can be used to modulate forward scatter or side scatter, for example, by adjusting the refractive index of the hydrogel particle.

In one embodiment, the central stream of aqueous monomer solution comprises a cross-linker, for example, N,N'-bisacrylamide. In a further embodiment, the central stream of aqueous monomer solution comprises a cross-linker and an accelerator, in addition to the monomer. In yet a further embodiment, the aqueous monomer solution comprises an initiator, for example an oxidizing agent such as ammonium persulfate.

Forward scatter was modulated by adjusting the refractive index of the gel by adding co-monomers allyl acrylate and allyl methacrylate (see also <FIG> and <FIG>). Forward scatter can also be modulated with side scattering nanoparticles containing sufficient optical resolution/size/density including, but not limited to, higher density colloidal suspensions of silica and/or PMMA particles. Side scattering of the droplets was tuned by adding a colloidal suspension of silica nanoparticles and/or PMMA (poly( methyl methacrylate)) particles ( ~ <NUM>) to the central aqueous phase prior to polymerization (<FIG> and <FIG>).

In one embodiment, a bead, plurality of beads, biomolecule, or plurality of biomolecules is embedded (encapsulated) within the hydrogel particle. An encapsulated bead or biomolecule, in one embodiment, is employed to mimic one or more intracellular organelles of a target cell, or a cell after it engulfs a particle. In one embodiment, encapsulating or embedding a bead or biomolecule is accomplished at the time of hydrogel particle formation. For example, beads can be suspended in the appropriate concentration to allow for an average of one bead to be embedded/encapsulated in a single hydrogel particle. The bead suspension can be included, for example, within the aqueous solution of monomer. Similarly, a biomolecule or mixture of biomolecules can be incorporated into the aqueous solution of monomer to encapsulate the biomolecule or biomolecules.

Alternatively, once a hydrogel particle is formed, for example by the methods described above, in one embodiment, it can be further manipulated, for example, by embedding a bead, plurality of beads, biomolecule or plurality of biomolecules within the hydrogel particle.

Accordingly, in one aspect of the invention, a hydrogel comprising an embedded substance is provided.

In one embodiment, the embedded substance is an embedded molecule, for example a biomolecule. The biomolecule can be a single species or a plurality of different species. For example, a protein, peptide, carbohydrate, nucleic acid or combination thereof can be encapsulated within a hydrogel particle of the invention. Moreover, different nucleic acid molecules (e.g., of varying sequences or nucleic acid type such as genomic DNA, messenger RNA or DNA-RNA hybrids) can be encapsulated by the hydrogel particle of the invention. These can be comprised of any protein or nucleic acid as both forms of biological material contain labile chemical side-groups (or can be modified by commercial vendors (e.g., Integrated DNA Technology chemical side group modifications). Such side-groups are compatible with reaction chemistries commonly found in co-monomer compositions (e.g. acrylate chemistry, NHS-ester, primary amines, copper catalyzed click chemistry (Sharpless)). The range of possible embedded molecules which contain compatible chemistries is understood by those skilled in the art.

In one embodiment, different subpopulations of hydrogel particles are fabricated, each with a different concentration of biomolecule. In a further embodiment, the biomolecule is a nucleic acid, a protein, an intracellular ion such as calcium acid (or other biomolecule of the user's choosing, for example, calcium). In another embodiment, different subpopulations of hydrogel particles are fabricated, each with a different concentration of a drug substance. The drug substance in one embodiment is a biomolecule (i.e., a biologic, antibody, antibody drug conjugate, protein/enzyme, peptide, non-ribosomal peptide, or related molecule) or a small molecule synthetic drug (e.g., Type I/II/III polyketide, non-ribosomal peptide with bioactive properties, or other small molecule entity as generally classified by those skilled in the art).

In this regard, the present invention is particularly useful for determining assay resolution where cells are stained for their respective nucleic acid or protein content. In one embodiment, different populations of the hydrogel particles provided herein are encapsulated with known, differing amounts of an intracellular substance, e.g., nucleic acid or protein. Individual hydrogel particles are stained for the intracellular substance and fluorescence is measured via a cytometric device for the individual hydrogels of the various populations. This allows for a generation of a standard curve to establish the sensitivity and dynamic range of the intracellular assay. Once established, a sample can be run through the cytometer to detect target cell(s) if present, and to quantify the amount of intracellular substance in the respective target cell(s). In one embodiment, the embedded substance is an infectious disease biomarker, for example one of the infectious disease biomarkers in the Infectious Disease Biomarker Database (IDBD, see <NPL>, incorporated by reference in its entirety for all purposes). In a further embodiment, the infectious disease biomarker is a biomarker of gastrointestinal infection, respiratory infection, neurological infection, urogenital infection, viral infection, hemorrhagic fever, zoonosis, arbovirus, antibiotics resistance or bioterrorism. In a further embodiment, the viral infection is an Ebola infection.

In one embodiment, the methods provided herein are used to determine the sensitivity and/or dynamic range of a cellular nucleic acid quantification assay. In this embodiment, a sample is interrogated for cell types within the sample (if present), and amount of cellular nucleic acid within the cell.

In another embodiment, the present invention provides a means for determining the resolution and/or sensitivity of an intracellular protein quantification assay. Hydrogel particles, in one embodiment, encapsulate known amounts of protein, at various concentrations, and subsequently stained with the appropriate protein antibody. Fluorescence is measured for the various particles to determine the sensitivity and/or dynamic range of the assay. The fluorescence values can then be compared to the values obtained from cells in a sample, to determine whether a target cell is present and whether it contains the intracellular protein, and the amount of the protein.

In one embodiment, individual hydrogel particles are tuned to have at least one optical property substantially similar to a circulating tumor cell or a fetal cell, present in maternal blood. The individual particles are embedded with known quantities of a biomolecule of interest. The particles are used to generate a standard curve for a biomolecule detection assay for the particular cell type.

As provided above, in one aspect of the invention, a hydrogel comprising an embedded substance is provided. In one embodiment, the embedded substance is a bead or plurality of beads. In one embodiment, a hydrogel particle is embedded with a single bead. In another embodiment, individual hydrogels the average number of embedded beads in a plurality of hydrogel particles is one.

In the case where a bead or plurality of beads are embedded into a hydrogel particle, in one embodiment, the optical properties of the bead or plurality of beads are used in combination with the FSC and SSC properties of the hydrogel particle for quality control of a flow cytometry assay. For example, the embedded bead in one embodiment is used as a control to calibrate the flow cytometer system, including the laser source, optics, and stream flow. In another embodiment, the embedded bead is used as a means for quantitating the amount of fluorescence in a sample, e.g., a particular cell. In this regard, embedded beads of various intensities can be used to generate a standard curve of fluorescence to determine whether a cell expresses a certain marker and at what level of expression.

In one embodiment, a bead with the diameter of about <NUM> to about <NUM>, about <NUM> to about <NUM> or about <NUM> to about <NUM> is embedded in a hydrogel provided herein. For example, in one embodiment, the bead has a diameter of about <NUM> to about <NUM>. In a further embodiment, the bead is a fluorescent bead. In another embodiment, the bead has a diameter of about <NUM> to about <NUM> or about <NUM> to about <NUM>. In a further embodiment, the bead is a fluorescent bead and can be stained either internally or at its surface. In even a further embodiment, the fluorescent bead is stained internally. Without wishing to be bound by theory, it is thought that internal staining insulates the fluorophores from environmental interactions that could cause variable fluorescence output.

As provided above, in one embodiment, the embedded bead is a fluorescence bead and in a further embodiment, the fluorescent bead is stained internally. It is within the skill in the art to select the appropriate fluorophore for use in conjunction with an embedded bead. In one embodiment, the bead is derivatized with one or more of the following fluorescent dyes: <NUM>-carboxy-<NUM>', <NUM>'-dichloro- <NUM>', <NUM>'-dimethoxyfluorescein succinimidylester; <NUM>-( and-<NUM>)-carboxyeosin; <NUM>- carboxyfluorescein;<NUM> carboxyfluorescein; <NUM>-(and-<NUM>)-carboxyfluorescein; S-carboxyfluorescein-bis-(<NUM>-carboxymethoxy-<NUM>-nitrobenzyl)ether,-alanine-carboxamide, or succinimidyl ester; <NUM>-carboxy fluorescein succinimidyl ester; <NUM>-carboxyfluorescein succinimidyl ester;<NUM>-( and-<NUM>)-carboxyfluorescein succinimidyl ester; <NUM>-(<NUM>,<NUM>-dichlorotriazinyl) amino fluorescein; <NUM> ', <NUM>' -difluoro fluorescein; eosin-<NUM>-isothiocyanate; erythrosin5-isothiocyanate;<NUM>-( fluorescein-<NUM>-carboxamido) hexanoic acid or succinimidyl ester;<NUM>- (fluorescein-<NUM>-( and-<NUM>)-carboxamido) hexanoic acid or succinimidylester; fluorescein-S-EX succinimidyl ester; fluorescein-<NUM>-isothiocyanate; fluorescein-<NUM>-isothiocyanate; OregonGreen® <NUM> carboxylic acid, or succinimidyl ester; Oregon Green® <NUM> isothiocyanate; Oregon Green® <NUM>-X succinimidyl ester; Oregon Green® <NUM> carboxylic acid; Oregon Green® <NUM> carboxylic acid, succinimidylester or triethylammonium salt; Oregon Green® <NUM> carboxylic acid; Oregon Green® <NUM> carboxylic acid or succinimidyl ester; RhodamineGreen™ carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidylester; Rhodamine Green™-X succinimidyl ester or hydrochloride; RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidylester; bis-(<NUM>- carboxypiperidinyl) sulfonerhodamine or di(succinimidylester); <NUM>-( and-<NUM>)carboxynaphtho fluorescein,<NUM>-( and-<NUM>)carboxynaphthofluorescein succinimidyl ester;<NUM>-carboxyrhodamine <NUM> hydrochloride; <NUM>-carboxyrhodamine6Ghydrochloride, <NUM>-carboxyrhodamine <NUM> succinimidyl ester;<NUM>-carboxyrhodamine <NUM> succinimidyl ester; <NUM>-( and-<NUM>)-carboxyrhodamine6G succinimidyl ester;<NUM>-carboxy-<NUM>',<NUM>',<NUM>',<NUM>'-tetrabromosulfonefluorescein succinimidyl esteror bis-( diisopropylethylammonium) salt; <NUM>-carboxytetramethylrhodamine; <NUM>-carboxytetramethylrhodamine; <NUM>-(and-<NUM>)- carboxytetramethylrhodamine; <NUM>-carboxytetramethylrhodamine succinimidyl ester; <NUM>-carboxytetramethylrhodaminesuccinimidyl ester;<NUM> -(and -<NUM>)-carboxytetramethylrhodamine succinimidyl ester;<NUM>-carboxy-X-rhodamine; <NUM>-carboxy-X-rhodamine succinimidyl ester;<NUM>-carboxy-Xrhodamine succinimidyl ester; <NUM>-( and-<NUM>)-carboxy-Xrhodaminesuccinimidyl ester; <NUM>-carboxy-X-rhodamine triethylammonium salt;LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; NANOGOLD® mono(sulfosuccinimidyl ester); QSY® 21carboxylic acid or succinimidyl ester; QSY® <NUM> carboxylic acid or succinimidyl ester; Rhodamine RedTM-X succinimidyl ester;<NUM>-(tetramethylrhodamine-<NUM>-(and-<NUM>)-carboxamido) hexanoic acid; succinimidyl ester; tetramethylrhodamine-<NUM>-isothiocyanate;tetramethylrhodamine-<NUM>-isothiocyanate; tetramethylrhodamine-<NUM>-(and-<NUM>)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; andX-rhodamine-<NUM>-(and-<NUM>) isothiocyanate, BODIPY® dyes commercially available from Invitrogen, including, but not limited to BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® <NUM>/<NUM>-X STPester; BODIPY® <NUM>/<NUM>-X STP ester;<NUM>-dibromo-<NUM>, <NUM>-difluoro-<NUM>, <NUM> -dimethyl-<NUM>-bora-<NUM> a, 4a-diaza-s-indacene-<NUM>-propionic acid succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>,<NUM>-dipropionic acid;<NUM>,<NUM>- difluoro-<NUM>,<NUM>-dimethyl-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-pentanoicacid; <NUM>,<NUM>-difluoro-<NUM>,<NUM>-dimethyl-<NUM>-bora3a,4a-diaza-s-indacene-<NUM>-pentanoicacid succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>,<NUM>-dimethyl-<NUM>-bora-<NUM> a, 4a-diaza-s-indacene-3propionicacid; <NUM>, <NUM>-difluoro-<NUM>, <NUM> -dimethyl-<NUM>-bora- <NUM> a, 4adiaza-s-indacene-<NUM>-propionicacid succinimidyl ester;<NUM>, 4difluoro-<NUM>, <NUM> -dimefhyl-<NUM>-bora-3a,4a-diaza-s-indacene-3propionic acid; sulfosuccinimidyl ester or sodium salt; <NUM>-(( <NUM>,<NUM>-difluoro-<NUM>, <NUM> -dimethyl-<NUM>-bora-3a,4a-diaza-s-indacene-3propionyl)amino )hexanoicacid; <NUM>-( ( <NUM>,<NUM>-difluoro-<NUM>, <NUM> dimethyl-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-propionyl)amino )hexanoic acid or succinimidyl ester; N-(<NUM>, <NUM>-difluoro <NUM>, <NUM> -dimethyl-<NUM>-bora-<NUM> a, 4a-diaza-s-indacene-<NUM>-propionyl) cysteic acid, succinimidyl ester or triethylammonium salt; <NUM>-<NUM>,<NUM>-difluoro-<NUM>,<NUM>- dimethyl-<NUM>-( <NUM>-methoxyphenyl)-<NUM>-bora3a, 4a4, <NUM>-difluoro-<NUM>, <NUM>-diphenyl-<NUM>-bora-3a,4a-diaza-sindacene-<NUM>-propionicacid; <NUM>, <NUM>-difluoro-<NUM>, <NUM> -diphenyl-<NUM>-bora3 a, 4a-diaza-s-indacene-<NUM>- propionicacid succinimidyl ester;<NUM>, <NUM>-difluoro-<NUM>-phenyl-<NUM>-bora-<NUM> a, 4a-diaza-s-indacene-<NUM>- propionic acid; succinimidyl ester;<NUM>-(( <NUM>, <NUM>-difluoro-<NUM>-phenyl-<NUM> bora-<NUM> a, 4a-diaza-s-indacene-<NUM>-propionyl)amino) hexanoicacid or succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>-(<NUM>-phenyl-<NUM>,3butadienyl)- <NUM>-bora-<NUM> a, 4a-diaza-s-indacene-<NUM>-propionicacid succinimidyl ester; <NUM>, <NUM>-difluoro-<NUM>-(<NUM>- pyrrolyl)-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-propionic acid succinimidyl ester;<NUM>-(((<NUM>,<NUM>- difluoro-<NUM>-(<NUM>-pyrrolyl)-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-yl)styryloxy)acetyl)aminohexanoicacid or succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>-styryl-<NUM>-bora-3a, 4a-diaza-s-indacene-<NUM>-propionic acid; <NUM>, <NUM>-difluoro-<NUM> -styryl-<NUM>-bora-<NUM> a, 4a-diaza-sindacene- <NUM>-propionic acid; succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>,<NUM>,<NUM>,<NUM>-tetramethyl-<NUM>-bora-3a,4adiaza- s-indacene-<NUM>-propionicacid; <NUM>,<NUM>-difluoro-<NUM>,<NUM>,<NUM>,<NUM>-tetramethyl-4bora-3a,4a-diaza-sindacene- <NUM>-propionic acid succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>-(<NUM>-thienyl)-<NUM>-bora-3a,4a-diaza-sindacene- <NUM>-propionic acid succinimidyl ester;<NUM>-( ( ( <NUM>-( <NUM>, <NUM>-difluoro-<NUM> -(<NUM>-thienyl)-<NUM>-bora-<NUM> a, 4adiazas- indacene-<NUM>-yl)phenoxy)acetyl)amino )hexanoic acid or succinimidyl ester; and <NUM>-(((<NUM>,<NUM>-difluoro-<NUM>-(<NUM>-thienyl)-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-yl)styryloxy)acetyl) aminohexanoic acid or succinimidyl ester, Alexa fluor dyes commercially available from Invitrogen, including but not limited to Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM><NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; and Alexa Fluor® <NUM> carboxylic acid, cyanine dyes commercially available from Amersham-Pharmacia Biotech, including, but not limited to Cy3 NHS ester; Cy <NUM> NHS ester; Cy5. <NUM> NHSester; and Cy7 NHS ester.

Other Fluorophores amenable for use with the present invention are provided in Table <NUM> below.

Commercially available beads including, but not limited to, those sold by Bangs Laboratories, Inc, Sperhotech Inc. , Thermo Scientific, Inc. and equivalent suppliers) can be used in combination with the hydrogel particles described herein. Depending on the assay, it is within the ordinary skill in the art to select a bead with the proper bead diameter, fluorescent emission and/or excitation spectrum and/or fluorescent intensity. For example, a quality control bead used in conjunction with a blue, red or UV laser can be embedded into one or more hydrogel particles provided herein. For example, an Alignflow™ flow cytometry alignment bead for blue lasers (catalog no. A-<NUM> (<NUM>), A-<NUM> (<NUM>)), red lasers (catalog no. A-<NUM> (<NUM>), A-<NUM> (<NUM>)) or UV lasers (catalog no. A-<NUM> (<NUM>), A-<NUM> (<NUM>)) can be embedded in on or more of the hydrogel particles provided herein.

In one embodiment, a fluorescent bead that can be excited at any wavelength from <NUM> - <NUM> is embedded in a hydrogel particle. In one embodiment, the bead is a "rainbow particle" that contains a mixture of fluorophores, for example <NUM> fluorophores, <NUM> fluorophores, <NUM> fluorophores, seven fluorophores or eight fluorophores. In this regard, the user selects which wavelength to excite the particle, depending on the fluorophore being interrogated. Rainbow particles are commercially available, for example, from BD Biosciences (catalog nos. <NUM> (mid range FL1 fluorescence), <NUM> (<NUM> color, <NUM>-<NUM>), <NUM> (<NUM> color, <NUM>-<NUM>), <NUM> (<NUM> color)) and Spherotech in various diameters (e.g., catalog nos. RCP20-<NUM> (<NUM> color), RCP-<NUM>-<NUM> (<NUM> peaks), RCP-<NUM>-5A (<NUM> peaks).

A cell sorting set-up bead can be embedded in one or more of the hydrogel particles provided herein. In one embodiment, a cell sorting set-up beads approximates the size, emission wavelength, and intensity of a biological sample, and can be used to calibrate a flow cytometer's cell sorting system, including laser source, optics, and stream flow. In one embodiment, a cell sorting set-up beads is embedded in one or more hydrogel particles and is amenable for use with a UV, blue, green/yellow or red laser. Where a green laser is used, in one embodiment, the embedded bead is excited at <NUM> with emission of <NUM>, but may also be exited at <NUM>. Commercially available cell sorting set-up beads are available, for example, from Life Technologies (catalog nos. C-<NUM> (UV laser), C-<NUM> (blue laser), C-<NUM> (green-yellow laser), C-<NUM> (red laser)).

A compensation control bead can also be embedded in one or more of the hydrogel particles provided herein. Accurate compensation is an important parameter for effective multicolor analysis in flow cytometry. However, cellular-based compensation controls are not completely effective as many antigens are not highly expressed, and dimly stained cells can lead to inaccurate compensation settings.

A compensation control bead, in one embodiment, includes a fluorescent antibody conjugate capture capacity (positive compensation bead) or is inert (negative compensation bead). The compensation bead is mixed with a fluorophore-conjugated human, mouse, rat, hamster, or rabbit antibody; the two components provide a distinct high-signal positive control with an appropriate negative population that can then be used to set compensation properly regardless of the intensity of the cells in the actual experiment. Once the antibody is mixed with the bead, it is embedded in one or more of the hydrogel particles provided herein. Commercially available compensation beads are available, for example, from Life Technologies (catalog nos. A-<NUM>, A-<NUM>, A10497, A10513) and Spherotech (catalog nos. CMIg-P-<NUM>-<NUM>, CMIg-P-<NUM>-<NUM>, CMIg-P-<NUM>-<NUM>, CMIg-P-<NUM>-<NUM>).

In one embodiment, a hydrogel particle with an embedded/encapsulated bead is used as a reference for a cellular assay, for example, a phagocytosis assay cytoxicity assay, motility assay, viability assay, etc. Phagocytosis is the process by which a cell engulfs a solid particle to form an internal vesicle known as a phagosome. In this regard, a hydrogel particle can be tuned to have one or more optical properties substantially similar to a phagocyte, before and after the phagocyte engulfs a particle. Accordingly, in one embodiment, the hydrogel particles provided herein are used as control particles for a phagocytosis assay. In a further embodiment, (i) one or more of the optical properties of a hydrogel particle is substantially similar to a phagocyte prior to particle uptake and (ii) one or more of the optical properties of a second hydrogel particle is substantially similar to a phagocyte after to particle uptake. In this regard, a control is generated for measuring particle uptake by a phagocyte.

In one embodiment, the phagocyte is a professional phagocyte. In another embodiment, the phagocyte is a non-professional phagocyte (i.e., a cell that consumes dying cells and foreign organisms). In a further embodiment, the non-professional phagocyte is an epithelial cell, endothelial cell, fibroblast or mesenchymal cell. Hydrogel particles in one embodiment, are tuned to have one or more optical properties substantially similar to a professional phagocyte set forth in Table <NUM> below (prior to and/or after particle uptake).

In one embodiment, a plurality of hydrogel particles of the invention, embedded with a substance such as nucleic acid or a bead is used as control reagents for a genomic cytometry assay. In this regard, a specific number of copies of a particular chromosome, RNA sequence and/or DNA sequence can be mimicked by the embedded substance. The hydrogel particle can then be used as a control for a sample being probed for genetic information, such as the number of copies of a chromosome, the number of copies of an RNA sequence and/or the number of copies of an RNA sequence.

The three primary modes of deconvolution for flow cytometry are the two passive optical properties of a particle (forward scattering, FSC, corresponding to the refractive index, or RI; and side scattering, SSC) and biomarkers present on the surface of a given cell type. Therefore, compositions that allow hydrogel particles of the disclosure to mimic specific cell types with respect to these three modes are useful for providing synthetic, robust calibrants for flow cytometry.

In one embodiment, the refractive index (RI) of a disclosed hydrogel particle is greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM><NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, greater than about <NUM>, or greater than about <NUM>.

In another embodiment, the refractive index (RI) of a disclosed hydrogel particle is about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>.

In some embodiments, the refractive index (RI) of a disclosed hydrogel particle is less than about <NUM><NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, less than about <NUM>, or less than about <NUM>.

The SSC of a disclosed hydrogel particle is most meaningfully measured in comparison to that of target cell. In some embodiments, a disclosed hydrogel particle has an SSC within <NUM>%, within <NUM>%, within <NUM>%, within <NUM>%, within <NUM>%, within <NUM>%, or within <NUM>% that of a target cell, as measured by a cytometric device.

The SSC of a hydrogel particle in one embodiment, is modulated by incorporating a high-refractive index molecule (or plurality thereof) in the hydrogel. In one embodiment, a high-refractive index molecule is provided in a hydrogel particle, and in a further embodiment, the high-refractive index molecule is colloidal silica, alkyl acrylate, alkyl methacrylate or a combination thereof. Thus in some embodiments, a hydrogel particle of the disclosure comprises alkyl acrylate and/or alkyl methacrylate. Concentration of monomer in one embodiment is adjusted to further adjust the refractive index of the hydrogel particle.

Alkyl acrylates or Alkyl methacrylates can contain <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM>, carbon atoms in the alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tertbutyl, <NUM>-ethylhexyl, heptyl or octyl groups. The alkyl group may be branched or linear.

High-refractive index molecules can also include vinylarenes such as styrene and methylstyrene, optionally substituted on the aromatic ring with an alkyl group, such as methyl, ethyl or tert-butyl, or with a halogen, such as chlorostyrene.

In some embodiments, FSC is modulated by adjusting the percentage of monomer present in the composition thereby altering the water content present during hydrogel formation. In one embodiment, where a monomer and co-monomer are employed, the ratio of monomer and co-monomer is adjusted to change the hydrogel particle's forward scatter properties. This is shown in both <FIG> and <FIG>.

The FSC of a disclosed hydrogel particle is most meaningfully measured in comparison to that of target cell. In some embodiments, a disclosed hydrogel particle has an FSC within <NUM>%, within <NUM>%, within <NUM>%, within <NUM>%, within <NUM>%, within <NUM>%, or within <NUM>% that of a target cell, as measured by a cytometric device.

FSC is related to particle volume, and thus can be modulated by altering particle diameter, as described herein. Generally, it has been observed that large objects refract more light than smaller objects leading to high forward scatter signals (and vice versa). Accordingly, particle diameter in one embodiment is altered to modulate FSC properties of a hydrogel particle. For example, hydrogel particle diameter is increased in one embodiment is altered by harnessing larger microfluidic channels during particle formation.

SSC can be engineered by encapsulating nanoparticles within hydrogels to mimic organelles in a target cell. In some embodiments, a hydrogel particle of the disclosure comprises one or more types of nanoparticles selected from the group consisting of: polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles. See also <FIG> and <FIG> which show that addition of various concentrations of nanoparticles allow for the adjustment of side scatter of a particle. Without wishing to be bound by theory, the ability to selectively tune both forward and side scatter of a hydrogel, as described herein, allows for a robust platform to mimic a vast array of cell types.

In some embodiments, a hydrogel particle of the disclosure has material modulus properties (e.g., elasticity) more closely resembling that of a target cell as compared to a polystyrene bead of the same diameter.

After the hydrogel particle is formed, one or more of the particle's surfaces can be functionalized, for example, to mimic one or more optical properties of a target cell or a labeled target cell. The functionalized hydrogel particle can also include an embedded bead or substance such as a biomolecule, as described above. In one embodiment, one or more hydrogel particles are functionalized with one or more fluorescent dyes, one or more cell surface markers (or epitope binding regions thereof), or a combination thereof. In one embodiment, the hydrogel particle is formed by polymerizing at least one bifunctional monomer and after formation, the hydrogel particle includes one or more functional groups that can be used for further attachment of a cell surface marker, an epitope binding region of a cell surface marker, a fluorescent dye, or combination thereof. The free functional group, in one embodiment, is an amine group, a carboxyl group, a hydroxyl group or a combination thereof. Depending on the functionalization desired, it is to be understood that multiple bifunctional monomers can be used, for example, to functionalize the particle using different chemistries and with different molecules.

A hydrogel particle can be functionalized with any fluorescent dye known in the art, including fluorescent dyes listed in The MolecularProbes® Handbook-A Guide to Fluorescent Probes and Labeling Technologies, incorporated herein by reference in its entirety for all purposes. Functionalization can be mediated by a compound comprising a free amine group, e.g. allylamine, which can be incorporated into a bifunctional monomer used to form the hydrogel, as discussed above.

Non-limiting examples of known fluorescent dyes that can be used to functionalize the surface of a hydrogel particle described herein include: <NUM>-carboxy-<NUM>', <NUM>'-dichloro- <NUM>', <NUM>'-dimethoxyfluorescein succinimidylester; <NUM>-( and-<NUM>)-carboxyeosin; <NUM>-carboxyfluorescein;<NUM> carboxyfluorescein; <NUM>-( and-<NUM>)-carboxyfluorescein; S-carboxyfluorescein-bis-(<NUM>-carboxymethoxy-<NUM>-nitrobenzyl)ether,-alanine-carboxamide, or succinimidyl ester; <NUM>-carboxyfluoresceinsuccinimidyl ester; <NUM>-carboxyfluorescein succinimidyl ester;<NUM>-( and-<NUM>)-carboxyfluorescein succinimidyl ester;<NUM>-(<NUM>,<NUM>-dichlorotriazinyl) amino fluorescein; <NUM> ', <NUM>' - difluoro fluorescein; eosin-<NUM>-isothiocyanate; erythrosin5-isothiocyanate; <NUM>-(fluorescein-<NUM>-carboxamido) hexanoic acid or succinimidyl ester; <NUM>-(fluorescein-<NUM>-(and-<NUM>)-carboxamido )hexanoic acid or succinimidylester; fluorescein-S-EX succinimidyl ester; fluorescein-<NUM>-isothiocyanate; fluorescein-<NUM>-isothiocyanate; OregonGreen® <NUM> carboxylic acid, or succinimidyl ester; Oregon Green® <NUM> isothiocyanate; Oregon Green® <NUM>-X succinimidyl ester; Oregon Green® <NUM> carboxylic acid; Oregon Green® <NUM> carboxylic acid, succinimidylester or triethylammonium salt; Oregon Green® <NUM> carboxylic acid; Oregon Green® <NUM> carboxylic acid or succinimidyl ester; RhodamineGreen™ carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidylester; Rhodamine Green™-X succinimidyl ester or hydrochloride; RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidylester; bis-(<NUM>- carboxypiperidinyl) sulfonerhodamine or di(succinimidylester); <NUM>-(and-<NUM>)carboxynaphtho fluorescein,<NUM>-( and-<NUM>)carboxynaphthofluorescein succinimidyl ester; <NUM>-carboxyrhodamine <NUM> hydrochloride; <NUM>-carboxyrhodamine6Ghydrochloride, <NUM>-carboxyrhodamine <NUM> succinimidyl ester;<NUM>-carboxyrhodamine <NUM> succinimidyl ester; <NUM>-( and-<NUM>)-carboxyrhodamine6G succinimidyl ester;<NUM>-carboxy-<NUM>',<NUM>',<NUM>',<NUM>'-tetrabromosulfonefluorescein succinimidyl esteror bis-( diisopropylethylammonium) salt; <NUM>-carboxytetramethylrhodamine; <NUM>-carboxytetramethylrhodamine; <NUM>-(and-<NUM>)-carboxytetramethylrhodamine;<NUM>-carboxytetramethylrhodamine succinimidyl ester; <NUM>-carboxytetramethylrhodaminesuccinimidyl ester;<NUM> -(and -<NUM>)-carboxytetramethylrhodamine succinimidyl ester;<NUM>-carboxy-X-rhodamine; <NUM>-carboxy-X-rhodamine succinimidyl ester;<NUM>-carboxy-Xrhodamine succinimidyl ester; <NUM>-( and-<NUM>)-carboxy-Xrhodaminesuccinimidyl ester; <NUM>-carboxy-X-rhodamine triethylammonium salt;LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; NANOGOLD® mono(sulfosuccinimidyl ester); QSY® 21carboxylic acid or succinimidyl ester; QSY® <NUM> carboxylic acid or succinimidyl ester; Rhodamine RedTM-X succinimidyl ester;<NUM>-(tetramethylrhodamine-<NUM>-( and-<NUM>)-carboxamido) hexanoic acid; succinimidyl ester; tetramethylrhodamine-<NUM>-isothiocyanate;tetramethylrhodamine-<NUM>-isothiocyanate; tetramethylrhodamine-<NUM>-( and-<NUM>)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; andX-rhodamine-<NUM>-(and-<NUM>) isothiocyanate.

Other examples of fluorescent dyes for use with the hydrogel particles described herein include, but are not limited to, BODIPY® dyes commercially available from Invitrogen, including, but not limited to BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® <NUM>/<NUM>-X STPester; BODIPY® <NUM>/<NUM>-X STP ester;<NUM>-dibromo-<NUM>, <NUM>-difluoro-<NUM>, <NUM> -dimethyl-<NUM>-bora-<NUM> a, 4a-diaza-s-indacene-<NUM>-propionic acid succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>,<NUM>-dipropionic acid;<NUM>,<NUM>- difluoro-<NUM>,<NUM>-dimethyl-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-pentanoicacid; <NUM>,<NUM>-difluoro-<NUM>,<NUM>-dimethyl-<NUM>-bora3a,4a-diaza-sindacene-<NUM>-pentanoicacid succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>,<NUM>- dimefhyl-<NUM>-bora-<NUM> a, 4a-diaza-sindacene-3propionicacid; <NUM>, <NUM>-difluoro-<NUM>, <NUM> -dimethyl-<NUM>-bora- <NUM> a, 4adiaza-s-indacene-<NUM>-propionicacid succinimidyl ester;<NUM>, 4difluoro-<NUM>, <NUM> -dimethyl-<NUM>-bora- 3a,4a-diaza-s-indacene-3propionic acid; sulfosuccinimidyl ester or sodium salt; <NUM>-((<NUM>,<NUM>- difluoro-<NUM>, <NUM> -dimethyl-<NUM>-bora-3a,4a-diaza-s-indacene-3propionyl)amino) hexanoic acid; <NUM>-((<NUM>,<NUM>-difluoro-<NUM>, <NUM> dimethyl-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-propionyl)amino) hexanoic acid or succinimidyl ester; N -( <NUM>, <NUM>-difluoro <NUM>, <NUM> -dimethyl-<NUM>-bora-<NUM> a, 4a-diaza-s-indacene-<NUM>- propionyl) cysteic acid, succinimidyl ester or triethylammonium salt;<NUM>-<NUM>,<NUM>-difluoro-<NUM>,<NUM>- dimethyl-<NUM>-( <NUM>-methoxyphenyl)-<NUM>-bora3a, 4a4,<NUM>-difluoro-<NUM>, <NUM>-diphenyl-<NUM>-bora-3a,4a-diaza-sindacene-<NUM>-propionicacid; <NUM>, <NUM>-difluoro-<NUM>, <NUM> - diphenyl-<NUM>-bora3 a, 4a-diaza-s-indacene-<NUM>- propionic acid succinimidyl ester;<NUM>, <NUM>-difluoro-<NUM>-phenyl-<NUM>-bora-<NUM> a, 4a-diaza-s-indacene-<NUM>- propionic acid; succinimidyl ester;<NUM>-( ( <NUM>, <NUM>-difluoro-<NUM>-phenyl-<NUM> bora-<NUM> a, 4a-diaza-s-indacene-<NUM>- propionyl)amino) hexanoicacid or succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>-( <NUM>-phenyl-<NUM>,3butadienyl)-<NUM>-bora-<NUM> a, 4a-diaza-s-indacene-<NUM>-propionicacid succinimidyl ester; <NUM>, <NUM>-difluoro-<NUM>-(<NUM>- pyrrolyl)-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-propionic acid succinimidyl ester; <NUM>-(((<NUM>,<NUM>-difluoro-<NUM>-(<NUM>-pyrrolyl)-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-yl)styryloxy)acetyl)aminohexanoicacid or succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>-styryl-<NUM>-bora- <NUM> a, 4a-diaza-s-indacene-<NUM>-propionic acid;<NUM>, <NUM>-difluoro-<NUM> -styryl-<NUM>-bora-<NUM> a, 4a-diaza-sindacene- <NUM>-propionic acid; succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>,<NUM>,<NUM>,<NUM>-tetramethyl-<NUM>-bora-3a,4adiaza- sindacene-<NUM>-propionicacid; <NUM>,<NUM>-difluoro-<NUM>,<NUM>,<NUM>,<NUM>-tetramethyl-4bora-3a,4a-diaza-sindacene- <NUM>-propionicacid succinimidyl ester;<NUM>,<NUM>-difluoro-<NUM>-(<NUM>-thienyl)-<NUM>-bora-3a,4a-diaza-sindacene- <NUM>-propionicacid succinimidyl ester;<NUM>-(((<NUM>-( <NUM>, <NUM>-difluoro-<NUM> -(<NUM>-thienyl)-<NUM>-bora-<NUM> a, 4adiazas-indacene-<NUM>-yl)phenoxy)acetyl)amino )hexanoic acid or succinimidyl ester; and <NUM>-(((<NUM>,<NUM>-difluoro-<NUM>-(<NUM>-thienyl)-<NUM>-bora-3a,4a-diaza-s-indacene-<NUM>-yl) styryloxy)acetyl) aminohexanoic acid or succinimidyl ester.

Fluorescent dyes for derivatization of the surface of one or more hydrogel particles in one embodiment, include, but are not limited to, , Alexa fluor dyes commercially available from Invitrogen, including but not limited to Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; Alexa Fluor® <NUM><NUM> carboxylic acid; Alexa Fluor® <NUM> carboxylic acid; and Alexa Fluor® <NUM> carboxylic acid. In another embodiment, fluorescent dyes for use with the hydrogel particles and methods described herein include cyanine dyes commercially available from Amersham-Pharmacia Biotech, including, but not limited to Cy3 NHS ester; Cy <NUM> NHS ester; Cy5. <NUM> NHSester; and Cy7 NHS ester.

It is within the ordinary skill in the art to select a suitable dye or dyes based on the desired spectral excitation and emission properties of the hydrogel particle.

Hydrogel particles, in one embodiment, are functionalized with one or more cell surface markers (see, e.g., Tables <NUM> and <NUM>-<NUM>), or fragments thereof, for example, extracellular portions thereof in the case of transmembrane proteins, for example, by attaching the one or more cell surface markers, extracellular portions or ligand binding regions thereof to the particle via a free amine, free carboxyl and/or free hydroxyl group present on the surface of the hydrogel particle. Functionalization of a hydrogel particle with a dye or cell surface molecule can also occur through a linker, for example a streptavidin/biotin conjugate.

Depending on the target cell, individual hydrogel particles can be derivatized with one or more cell surface markers, or fragments thereof, for example, extracellular portions thereof in the case of transmembrane proteins to further mimic the structural properties of the target cell. Tables <NUM> and <NUM>-<NUM>, provided below, sets forth a non-limiting list of cell surface markers that can be used to derivative hydrogel particles, depending on the target cell. Although the cell surface marker is provided, it is understood that a portion of the cell surface marker, for example, a receptor binding portion, a ligand binding portion, or an extracellular portion of the marker can be used to derivative the hydrogel particle (at the free functional group, as described above). See also <FIG> and <FIG> which show that hydrogel surface modification with for example, a cell surface receptor, together with the selective tuning of FSC and/or SSC, allows for the fabrication of a hydrogel particle with the desired feature(s).

Cell types including but not limited to various cell lines such as CHO, HEK-<NUM>, BHK-<NUM>, NS0, MDCK, VERO, MRC-S, W1-<NUM> and Sp2/<NUM> Mouse Myeloma (hybridomas). Table <NUM> and Table <NUM> each provides other cell types for use with the hydrogel particles described herein.

In one embodiment, a plurality of hydrogel particles is used to determine the dynamic range and/or sensitivity of detection of a particular cell surface marker or combination thereof on a population of target cells. For example, the population of hydrogel particles can be tuned to have the SSC and/or FSC profile of the target cell, and subpopulations of the hydrogel particle are derivatized with a specific number of copies of a cell surface marker, e.g., a cell surface receptor, or a domain thereof, for example, an epitope binding region thereof. For example, individual subpopulations of hydrogel particles can each be derivatized to have a unique number of copies, e.g., one subpopulation will contain <NUM> copies of a cell surface marker, a second subpopulation will contain <NUM>,<NUM> copies of the same cell surface marker, a third subpopulation will contain <NUM>,<NUM> copies of the same cell surface marker, etc. The populations of hydrogel particles are fluorescently stained for the respective cell surface marker and fluorescence is detected for hydrogel particles in each subpopulation. In this regard, the subpopulations of hydrogel particles can be used to generate a standard curve of fluorescence emission for target cells with the respective cell marker. The cell surface marker can be any of the cell surface markers provided thereof, or binding regions thereof, or a cell surface marker known to one of ordinary skill in the art.

Hydrogel particles of the disclosure behave similarly to target cells in procedures such as staining and analysis by flow cytometry or FACS. For example, in one embodiment, a hydrogel particle has one or more optical properties substantially similar to one of the cell types set forth in Table <NUM>, Table <NUM> or Table <NUM>.

In some embodiments, a target cell is an immune cell. Non-limiting examples of immune cells include B lymphocytes, also called B cells, T lymphocytes, also called T cells, natural killer (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stem cells, dendritic cells, peripheral blood mononuclear cells, tumor infiltrating (TIL) cells, gene modified immune cells including hybridomas, drug modified immune cells, and derivatives, precursors or progenitors of any of the cell types listed herein.

In some embodiments, a target cell encompasses all cells of a particular class of cell with shared properties. For example, a target cell can be a lymphocyte, including NK cells, T cells, and B cells. A target cell can be an activated lymphocyte.

In some embodiments, a target cell is a primary cell, cultured cell, established cell, normal cell, transformed cell, infected cell, stably transfected cell, transiently transfected cell, proliferating cell, or terminally differentiated cells.

In one embodiment, a target cell is a primary neuronal cell. A variety of neurons can be target cells. As non-limiting examples, a target cell can be a primary neuron; established neuron; transformed neuron; stably transfected neuron; or motor or sensory neuron.

In other embodiments, a target cell is selected from the group consisting of: primary lymphocytes, monocytes, and granulocytes.

A target cell can be virtually any type of cell, including prokaryotic and eukaryotic cells.

Suitable prokaryotic target cells include, but are not limited to, bacteria such as E. coli, various Bacillus species, and the extremophile bacteria such as thermophiles.

Suitable eukaryotic target cells include, but are not limited to, fungi such as yeast and filamentous fungi, including species of Saccharomyces, Aspergillus, Trichoderma, and Neurospora; plant cells including those of com, sorghum, tobacco, canola, soybean, cotton, tomato, potato, alfalfa, sunflower, etc.; and animal cells, including fish, birds and mammals. Suitable fish cells include, but are not limited to, those from species of salmon, trout, tilapia, tuna, carp, flounder, halibut, swordfish, cod and zebrafish. Suitable bird cells include, but are not limited to, those of chickens, ducks, quail, pheasants and turkeys, and other jungle foul or game birds. Suitable mammalian cells include, but are not limited to, cells from horses, cows, buffalo, deer, sheep, rabbits, rodents such as mice, rats, hamsters and guinea pigs, goats, pigs, primates, marine mammals including dolphins and whales, as well as cell lines, such as human cell lines of any tissue or stem cell type, and stem cells, including pluripotent and non-pluripotent, and non-human zygotes.

Suitable cells also include those cell types implicated in a wide variety of disease conditions, even while in a non-diseased state. Accordingly, suitable eukaryotic cell types include, but are not limited to, tumor cells of all types (e.g., melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, dendritic cells, endothelial cells, epithelial cells, lymphocytes (T -cell and B cell), mast cells, eosinophils, vascular intimal cells, macrophages, natural killer cells, erythrocytes, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as hematopoietic, neural, skin, lung, kidney, liver and myocyte stem cells (for use in screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes. In certain embodiments, the cells are primary disease state cells, such as primary tumor cells. Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, COS, etc. See the ATCC cell line catalog, hereby expressly incorporated by reference.

In some embodiments, a target cell is a tumor microvesicle or tumor macrovesicle. Tumor microvesicles, also known as tumor-secreted microvesicles or tumor-secreted exosomes, can be found in circulating blood and may have immune-suppressive activities. Tumor microvesicles typically range in size from <NUM>-<NUM> in diameter. Larger tumor micro vesicles may be referred to as tumor macro vesicles, and can range in size from <NUM>-<NUM> in diameter.

The hydrogel particles described herein can be employed in any flow cytometer known to those of ordinary skill in the art. For example, one or more of the flow cytometers provided in Table <NUM> below are amenable for use with the hydrogels and assays described herein.

The present invention is further illustrated by reference to the following Examples. However, it should be noted that these Examples, like the embodiments described above, are illustrative and are not to be construed as restricting the scope of the invention in any way.

Photomasks for UV lithography were sourced from CADart Services Inc. and were designed using AutoCad (AutoDesk, Inc. SU-<NUM> photo resist (Microchem, Inc. ) was photo crosslinked on <NUM>" silicon wafers using a collimated UV light source (OAI, Inc. ) to create masters for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sigma Aldrich, Inc. ) was prepared and formed using standard published methods for soft lithography and microfluidic device fabrication (See, <NPL>).

Droplets were formed using flow-focusing geometry where two oil channels focus a central stream of aqueous monomer solution to break off droplets in a water-in-oil emulsion. A fluorocarbon-oil (Novec <NUM><NUM>, Inc. ) was used as the outer, continuous phase liquid for droplet formation. To stabilize droplets before polymerization, a surfactant was added at <NUM>% w/w to the oil phase (ammonium carboxylate salt of Krytox <NUM> FSH, Dupont). To make the basic polyacrylamide gel particle, a central phase of an aqueous monomer solution containing N-acrylamide (<NUM>-<NUM>% w/v), a cross-linker (N,N'-bisacrylamide, <NUM>-<NUM>% w/v), an accelerator, and ammonium persulfate (<NUM>% w/v) was used. An accelerator, (N,N,N' ,N'tetramethylethylenediamine (<NUM>% vol%) was added to the oil-phase in order to trigger hydrogel particle polymerization after droplet formation.

Several co-monomers were added to the basic gel formulation to add functionality. Allyl-amine provided primary amine groups for secondary labeling after gel formation. We modulated forward scatter by adjusting the refractive index of the gel by adding co-monomers allyl acrylate and allyl methacrylate. Side scattering of the droplets was tuned by adding a colloidal suspension of silica nanoparticles and/or PMMA (poly( methyl methacrylate)) particles ( ~ <NUM>) to the central aqueous phase prior to polymerization.

Stoichiometric multiplexing of the hydrogel particles was achieved by utilizing co-monomers containing chemically orthogonal side groups (amine, carboxyl, maleimide, epoxide, alkyne, etc.) for secondary labeling.

Droplets were formed at an average rate of <NUM> and were collected in the fluorocarbon oil phase. Polymerization was completed at <NUM> for <NUM> minutes, and the resulting hydrogel particles were washed from the oil into an aqueous solution.

Water containing <NUM>% acrylamide, <NUM>% bisacrylamide, <NUM>% allyl amine, and <NUM>% ammonium persulfate was flowed through a center channel and focused by oil containing <NUM>% TEMED through a <NUM> micron nozzle to produce <NUM> hydrogel particles, shown in <FIG>. Following polymerization, the particles were washed in water, shown in <FIG>, and conjugated to dyes of interest. The fluorescent hydrogel particles were visualized with fluorescence microscopy, shown in <FIG>.

As depicted in <FIG>, hydrogel particles are tuned in multiple dimensions to match specific cell types unlike polystyrene beads. Cells are deconvolved using combinations of optical parameters such as FSC and SSC (<FIG>) or secondary markers. Hydrogel particles are tuned to match the SSC and FSC of specific cell types unlike polystyrene beads (brown) which are limited in size (FSC) and side scattering (<FIG>). Hydrogel particles are further functionalized with stoichiometrically tuned ratios of specific chemical side-groups and secondary labels allowing the cell type to be precisely matched without suffering from biological noise as fixed cell lines do (<FIG>).

As shown in <FIG>, the inter-drop delay for a flow cytometer can be precisely correlated to hydrogel particle diameter. Data are shown for hydrogel particles of3, <NUM>, <NUM>, <NUM>, and <NUM> diameters using flow cytometer nozzle sizes of <NUM> and <NUM>.

To form hydrogel particles with encapsulated DNA, <NUM>µg/mL-<NUM>) µg/mL of reconstituted calf thymus DNA was added to a polymer mix containing <NUM>% <NUM>:<NUM>(acrylamide:bis-acrylamide) and <NUM>% allyl amine in water. <NUM>% ammoniumpersulfate was added to the mix prior to droplet formation. Hydrogel particles were formed as described in Example <NUM>. Hydrogel particles with <NUM>µg/mL of encapsulated calf thymus DNA displayed cell-like staining using propidium iodide as visualized using a commercial imaging cytometer and compared to Chinese Hamster Ovary cells stained using the same procedure. Images were obtained using a Nexcelom Cellometer™ (<FIG>).

Cells obtained from a buccal swab were washed in PBS and stained with propidium iodide. In parallel, populations of hydrogel particles containing a range of DNA concentrations were also stained in the same manner. Both the cell and particle suspensions were analyzed on a flow cytometer (<NUM>/<NUM> excitation/emission). Flow cytometry analysis of cheek cells and the same range of encapsulated DNA particles showed that the particles display a range of cell-like fluorescent properties (<FIG>, left panel). The intensity of staining shows a linear correlation with the median intensity as measured by flow cytometry (<FIG>, right panel).

Colloidal silica was added at <NUM>%, <NUM>%, <NUM>% and <NUM>% to the aqueous fraction of the polymer mix and hydrogel particles were formed as described in Example <NUM>. Forward and side scattering data were obtained using a flow cytometer. The results showed that side scatter signal (<FIG>, left panel) increased with higher percentages of encapsulated nanoparticles while forward scatter (<FIG>, right panel) remained generally unchanged, demonstrating the independent tuning of side scatter and forward scatter.

In this experiment, the percentage of acrylamide:bis-acrylamide in the hydrogel composition was varied from between <NUM> and <NUM>% to tune the refractive index of the hydrogel particles as measured by forward scattering in a flow cytometer. As shown in <FIG>, the forward scattering increased with increasing percentages of acrylamide: bisacrylamide as a fraction of water.

An example of tuning hydrogel particles to match optical properties of a desired cell subtype. Co/monomers can be combined with nanoparticles to tune both forward and side scatter properties of the hydrogels using passive optical measurements in a flow cytometer. By combining these properties with chemically labile co-monomers (e.g. allyl amine, acrylic acid), additional fluorophores/proteins/biological side groups can be added and labeled (if desired) in order to match cell subpopulation staining in addition to scattering properties. These are the three primary metric by which cells are identified using flow cytometry. Additional side groups, such as those containing heavy metals, can be used for Cy-TOF (cytometry, time of flight mass spectrometry) calibration for example. Finally, biocompatible material can be encapsulated to mimic subcellular organelle staining.

A <NUM> nanoparticle colloidal suspension was incorporated into the hydrogel matrix to mimic the optical properties of lymphocytes and monocytes (<FIG>). The percent composition of the suspension was altered to match the blood cell subpopulations from the blood sample control (Streck) (<FIG>).

Claim 1:
A method for calibrating a cytometric device for analysis of a target cell, specified by a user, comprising:
providing hydrogel particles each comprising a polymerized monomer and having at least one surface;
functionalizing the surface of each hydrogel particle with at least one cell surface marker to form functionalized hydrogel particles that mimic one or more optical properties of the specified target cell;
wherein the optical property is side scatter (SSC) or forward scatter (FSC) or FSC and SSC;
wherein functionalized hydrogel particles that mimic one or more optical properties of the specified target cell are functionalized hydrogel particles having the one or more optical properties substantially the same to the specified target cell,
inserting the functionalized hydrogel particles into the cytometric device;
measuring the at least one optical property of the functionalized hydrogel particles using the cytometric device, thereby calibrating the cytometric device for analysis of the specified target cell.