Method of forming monodispersed crosslinked polystyrene microspheres

Abstract

The present disclosure relates to a method of forming a crosslinked polystyrene microsphere comprising forming a seed particle by subjecting a composition comprising styrene, a stabilizer, a surfactant, and an alcohol to dispersion polymerization; and forming the crosslinked polystyrene microsphere by subjecting the seed particle to first and second emulsion steps. The first emulsion step can comprise reacting the seed particle with a surfactant, water, and a swelling agent, such as dibutyl phthalate, to form an activated seed particle. The second emulsion step can comprise reacting the seed particle with a surfactant, water, a stabilizer, styrene, a hydrophilic monomer, a crosslinking agent, and an initiator. The method provides crosslinked polystyrene microspheres that are highly uniform with an average diameter that can be about 5.5 µm to about 6 µm.

Description

DESCRIPTIONMETHOD OF FORMING MONODISPERSED CROSSLINKED POLYSTYRENE MICROSPHERESCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to U.S. Provisional Patent Application Serial Number 63 / 616,359 filed December 29, 2023, the entire contents of which are incorporated herein by reference.BACKGROUND

[0001] Multiplex assays use polystyrene bead-based technology to detect and quantitate multiple analytes, including secreted proteins and growth factors. Polystyrene microspheres can be used to retain fluorescent dyes to classify distinct subsets, provide a substrate for biological reactions, and trigger detection of an event. The size of the microsphere affects each of these functions. Variations in size will result in large variations in the fluorescent signals, since larger beads absorb more dye than smaller ones, which has the effect of reducing the total number of distinguishable bead sets. Slight differences in particle diameter also translate to larger differences in surface area, which can lead to differences in the quantities of bound biomolecules. This results in higher coefficients of variation for the reporter signal. Size variation also broadens the signal in the doublet discriminator (DD), making elimination of bead doublets less efficient. Not only uniformity of size, but also of shape and composition of the microspheres are critical parameters. Non-uniformity of shape (e.g., non-spherical beads) can manifest itself much the same as a broad size distribution. In some polymerizations, the styrene, divinylbenzene, and acrylic acid are not randomly distributed throughout the bead, and regions of high divinylbenzene concentration can form that are denser than the rest of the bead, resulting in the scattering of light being different from the rest of the beads, potentially causing similar problems.

[0002] Thus, there is a need for a method to produce uniform monodispersed polystyrene microspheres for consistent DD signal and even dye uptake.BRIEF SUMMARY

[0003] The present disclosure relates to a method of forming a crosslinked polystyrene microsphere comprising forming a seed particle by subjecting a composition comprising styrene, a stabilizer, a surfactant, and an alcohol to dispersion polymerization; and forming the crosslinked polystyrene microsphere by subjecting the seed particle to first and second emulsion steps.

[0004] In some aspects, the seed particle is swelled with a swelling agent prior to the second emulsion step. In some aspects, the first emulsion step comprises reacting the seed particle with a surfactant, water, and a swelling agent to form an activated seed particle. In some aspects, the swelling agent comprises dibutyl phthalate. In some aspects, the first emulsion step does not include an initiator or a carrier.

[0005] In some aspects, the second emulsion step comprises reacting the seed particle with a surfactant, water, a stabilizer, styrene, a hydrophilic monomer, a crosslinking agent, and an initiator.

[0006] In some aspects, the seed particle has an average diameter of about 1 pm to about 1.6 m. In some aspects, the seed particle has a number average molecular weight of about 7500 g / mol to about 9500 g / mol.

[0007] In some aspects, the crosslinked polystyrene microsphere has an average diameter of about 5.5 pm to about 6 pm. In some aspects, the crosslinked polystyrene microsphere has an aspect ratio of about 1.

[0008] In some aspects, the styrene is purified prior to use. In some aspects, the styrene is distilled prior to use.

[0009] In some aspects, the stabilizer comprises a high molecular weight nonionic polymeric material. In some aspects, the stabilizer comprises polyvinyl alcohol, polyacrylic acid, polyvinyl pyrrolidone, or any combination thereof. In some aspects, the stabilizer in the dispersion polymerization comprises polyvinyl pyrrolidone. In some aspects, the stabilizer in the second emulsion step comprises polyacrylic acid.

[0010] In some aspects, the surfactant comprises a low molecular weight non- polymeric moiety that is ionic or nonionic. In some aspects, the surfactant comprises sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, sodium bis(2-ethylhexyl) sulfosuccinate, or any combination thereof. In some aspects, the surfactant in thedispersion polymerization comprises sodium dioctyl sulfosuccinate. In some aspects, the surfactant in the first and second emulsion steps comprises sodium dodecyl sulfate.

[0011] In some aspects, the alcohol comprises a C1-4 alcohol. In some aspects, the alcohol comprises ethanol.

[0012] In some aspects, the crosslinked polystyrene microsphere comprises hydrophilic surface groups. In some aspects, the hydrophilic monomer is acrylic acid, methacrylic acid, or a combination thereof. In some aspects, the hydrophilic monomer is acrylic acid.

[0013] In some aspects, the crosslinked polystyrene microsphere has a crosslink density of about 4-7%. In some aspects, the crosslinked polystyrene microsphere has a crosslink density of about 5-5.5%. In some aspects, the crosslinking agent is divinyl benzene.

[0014] In some aspects, the initiator in the second emulsion step comprises benzoyl peroxide.

[0015] In some aspects, the dispersion polymerization is performed at a temperature of about 55 °C to about 65 °C. In some aspects, the dispersion polymerization is mixed at a sheer rate of about 105 rpm to about 130 rpm.

[0016] In some aspects, the second emulsion step is performed at a temperature of about 68 °C to about 72 °C. In some aspects, the temperature can be ramped at about 10 + 1 °C / min.

[0017] In some aspects, the second emulsion polymerization is performed using sonication.

[0018] In some aspects, the method further comprises washing the crosslinked polystyrene microsphere with water, an alcohol, toluene, tetrahydrofuran, or a combination thereof. In some aspects, the crosslinked polystyrene microsphere is washed sequentially in order with water, methanol, tetrahydrofuran, methanol, and water. In some aspects, the crosslinked polystyrene microsphere is washed sequentially in order with water, isopropanol, tetrahydrofuran, isopropanol, and water.

[0019] In some aspects, the crosslinked polystyrene microsphere is soaked in methanol, isopropanol, or a mixture thereof.

[0020] In some aspects, the method further comprises coating the crosslinked polystyrene microsphere with a polymer. In some aspects, the coating comprises polyvinyl pyrrolidone.

[0021] In some aspects, the amount of seed particles inversely correlates to the microsphere size.

[0022] In some aspects, the Doublet Discriminator (DD) profile of the microsphere is sharper than the DD profile of microspheres produced by the method in FIG. 2 as measured using a Luminex 200 instrument.

[0023] In some aspects, the method produces a population of microspheres, wherein the population of the microspheres has a coefficient of variation (CV) of dyeing that is 3% or less, 2.5% or less, 2% or less, 1.5% or less, or 1% or less.

[0024] The present disclosure relates to a population of crosslinked polystyrene microspheres produced by the method described herein. In some aspects, the population has a crosslink density of about 4-7%. In some aspects, the population has a crosslink density of about 5-5.5%.

[0025] The present disclosure further relates to a population of crosslinked polystyrene microspheres comprising less than 3% coefficient of variation (CV) of dyeing, wherein each microsphere has a diameter of 5.5 to 6 microns, and a carboxylated surface with a nominal parking area of 50 to 150 sq. angstroms. In some aspects, the population has a CV of dyeing less than 2.5%, less than 2%, less than 1.5%, or less than 1%.

[0026] The present disclosure further relates to a method of conducting a multiplex assay comprising detecting or quantitating one or more analytes with a population of microspheres, as described herein.

[0027] Additional aspects and advantages of the disclosure will be set forth, in part, in the description that follows, and will flow from the description, or can be learned by practice of the disclosure.

[0028] It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only, and do not restrict the scope of the claims.BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0029] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0030] FIG. 1 is a flow chart of the method of preparing a crosslinked polystyrene microsphere in accordance with some aspects of the present disclosure.

[0031] FIG. 2 is a flow chart of conventional methods of preparing a crosslinked polystyrene microsphere.

[0032] FIG. 3 is a scanning electron microscope (SEM) image of a crosslinked polystyrene microsphere prepared with dibutyl phthalate as a swelling agent.

[0033] FIGs. 4A and 4B are graphs of the DD profiles of crosslinked polystyrene microspheres prepared in accordance with the method of FIG. 2 and in accordance with the present disclosure (FIG. 1), respectively.

[0034] FIG. 5 is a graph of the relationship between microsphere diameters (pm) versus concentration of seed particles.DETAILED DESCRIPTION

[0035] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.Definitions

[0036] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular aspects, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification will control.

[0037] The articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0038] As used herein, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e.. the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 10% (e.g., up to 5% or up to 1%) of a given value.

[0039] The term “at least” prior to a number or series of numbers is understood to include the number associated with the term “at least,” and all subsequent numbers or integers that could logically be included, as clear from context. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range. For example, “at least 3” means at least 3, at least 4, at least 5, etc. When at least is present before a component in a method step, then that component is included in the step, whereas additional components are optional.

[0040] As used herein, the terms “comprises,” “comprising,” “having,” “including,” “containing,” and the like are open-ended terms meaning “including, but not limited to.” To the extent a given aspect disclosed herein “comprises” certain elements, it should be understood that present disclosure also specifically contemplates and discloses aspects that “consist essentially of’ those elements and that “consist of’ those elements.

[0041] As used herein the terms “consists essentially of,” “consisting essentially of,” and the like are to be construed as a semi-closed terms, meaning that no other ingredients which materially affect the basic and novel characteristics of an aspect are included.

[0042] As used herein, the terms “consists of,” “consisting of,” and the like are to be construed as closed terms, such that an aspect “consisting of’ a particular set of elements excludes any element, step, or ingredient not specified in the aspect.

[0043] As used herein, the term “crosslinking agent” refers to a molecule that contains at least two (e.g., 2, 3, or 4) reactive groups (e.g., terminal functional groups) that can link at least two molecules together (intermolecular crosslinking) or at least two portions of the same molecule together (intramolecular crosslinking). A crosslinking agent having more than two reactive groups can be capable of both intermolecular and intramolecular crosslinking at the same time.

[0044] As used herein, the term “initiator” refers to a compound that generates a radical, cation, or anion, typically upon exposure to heat or light, to initiate a chain-growth polymerization reaction.

[0045] As used herein, the term “seed particle” refers to a polystyrene particle that can be used as the starting point in subsequent growth steps to grow larger sized crosslinked polystyrene microspheres.

[0046] As used herein, the term “stabilizer” refers to a compound that stabilizes the interface of the growing particle and the reaction medium. In some aspects, the stabilizer can be a high molecular weight (e.g., 1000 g / mol or more), non-ionic polymer that can be added to the polymerization reaction and is capable of being incorporated in the final particle, e.g., mostly surface level.

[0047] The phrase “substantially free of’ means that a composition contains little to none of the specified ingredient / component, such as less than about 5 wt%, less than about 4 wt%, less than about 3 wt%, less than about 2 wt%, less than about 1 wt%, less than about 0.5 wt%, less than about 0.3 wt%, less than about 0.2 wt%, less than about 0.1 wt%, less than about 0.05 wt%, less than about 0.01 wt%, or about 0 wt% of the specified ingredient.

[0048] As used herein, the term “swelling agent” refers to a compound that can swell a seed particle. Typically, a suitable swelling agent will have a low molecular weight (e.g., less than 500 g / mol, less than 300 g / mol) and generally is insoluble in water.Methods of the Disclosure

[0049] The present disclosure relates to a method of forming a crosslinked polystyrene microsphere comprising forming a seed particle by subjecting a composition comprising styrene, a stabilizer, a surfactant, and an alcohol to dispersion polymerization; and forming the crosslinked polystyrene microsphere by subjecting the seed particle to first and second emulsion steps.

[0050] In some aspects, the method of forming a crosslinked polystyrene microsphere can be in accordance with the steps set forth in FIG. 1. The method of the present disclosure differs from prior methods, such as the method set forth in FIG. 2. For example, the method of the present disclosure includes a dispersion polymerization, a first emulsion step that does not require an initiator or carrier, and a second emulsion step that includes initiator. It was discovered that the method of the present disclosure can provide larger seed particles (e.g., 1 pm or greater) compared to prior methods (e.g., less than 1 pm, such as about 500 nm).

[0051] The dispersion polymerization requires reacting styrene, a stabilizer, a surfactant, and an alcohol to form a seed particle. Dispersion polymerization is similar toemulsion polymerization, except that monomer is dissolved in a medium that acts as a precipitant for the polymer.

[0052] It was discovered that the concentration of styrene effects particle size and that increasing the concentration leads to larger particles. In some aspects, the concentration of styrene in the dispersion polymerization can be from about about 5 wt% to about 50 wt% relative to the total weight of the components of the dispersion polymerization. For example, the styrene content can be from about 5 wt% to about 45 wt%, about 5 wt% to about 40 wt%, about 5 wt% to about 35 wt%, about 5 wt% to about 30 wt%, about 5 wt% to about 25 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 15 wt%, about 5 wt% to about 10 wt%, about 8 wt% to about 50 wt%, about 8 wt% to about 45 wt%, about 8 wt% to about 40 wt%, about 8 wt% to about 35 wt%, about 8 wt% to about 30 wt%, about 8 wt% to about 25 wt%, about 8 wt% to about 20 wt%, about 8 wt% to about 15 wt%, about 8 wt% to about 10 wt%, about 10 wt% to about 50 wt%, about 10 wt% to about 45 wt%, about 10 wt% to about 40 wt%, about 10 wt% to about 35 wt%, about 10 wt% to about 30 wt%, about 10 wt% to about 25 wt%, about 10 wt% to about 20 wt%, about 10 wt% to about 15 wt%, about 15 wt% to about 50 wt%, about 15 wt% to about 45 wt%, about 15 wt% to about 40 wt%, about 15 wt% to about 35 wt%, about 15 wt% to about 30 wt%, about 15 wt% to about 25 wt%, about 15 wt% to about 20 wt%, about 20 wt% to about 50 wt%, about 20 wt% to about 45 wt%, about 20 wt% to about 40 wt%, about 20 wt% to about 35 wt%, about 20 wt% to about 30 wt%, about 20 wt% to about 25 wt%, about 25 wt% to about 50 wt%, about 25 wt% to about 45 wt%, about 25 wt% to about 40 wt%, about 25 wt% to about 35 wt%, about 25 wt% to about 30 wt%, about 30 wt% to about 50 wt%, about 30 wt% to about 45 wt%, about 30 wt% to about 40 wt%, about 30 wt% to about 35 wt%, about 35 wt% to about 50 wt%, about 35 wt% to about 45 wt%, about 35 wt% to about 40 wt%, about 40 wt% to about 50 wt%, about 40 wt% to about 45 wt%, about 45 wt% to about 50 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, about 25 wt%, about 26 wt%, about 27 wt%, about 28 wt%, about 29 wt%, about 30 wt%, about 31 wt%, about 32 wt%, about 33 wt%, about 34 wt%, about 35 wt%, about 36 wt%, about 37 wt%, about 38 wt%, about 39 wt%, about 40 wt%, about 41 wt%, about 42 wt%, about 43 wt%, about 44 wt%, about 45 wt%, about 46 wt%, about 47 wt%, about 48 wt%, about 49 wt%, or about 50 wt%, of the total weight of the components of the dispersion polymerization. In someaspects, the dispersion polymerization composition comprises from about 5 wt% to about 15 wt% of styrene relative to the total weight of the components of the dispersion polymerization. In some aspects, the dispersion polymerization composition comprises from about 8 wt% to about 10 wt% of styrene relative to the total weight of the components of the dispersion polymerization.

[0053] In some aspects of the method, one or more of the monomers (e.g., styrene; a hydrophilic monomer, such as acrylic acid or methacrylic acid; divinyl benzene) can be purified prior to use. In some aspects, purification can include filtering, distillation, extraction, chromatography, drying, recrystallization, or any combination thereof. In some aspects, one or more of the monomers is distilled prior to use. In some aspects, the styrene is purified prior to use, such as being distilled prior to use. Commercially purchased styrene almost always contains a radical scavenger, such as t-butylcatechol, to prevent polymerization during storage. In some aspects, the method is improved by removing any impurities, such as polystyrene and a radical scavenger, to achieve a reproducible polymerization. Polymer can form in the distilled styrene, even when stored at low temperature (e.g., about -20 °C), so in some aspects, the styrene is purified (e.g., distilled) within about 4 days (e.g., within about 3 days, within about 2 days, or within about 1 day) of use. In some aspects, acrylic acid and / or methacrylic acid is purified prior to use, such as being distilled prior to use. In some aspects, divinyl benzene is purified prior to use, such as being distilled prior to use.

[0054] In some aspects, the stabilizer in the dispersion polymerization comprises a high molecular weight (e.g., a number average molecular weight of about 1,000 g / mol or more) nonionic polymeric material. In some aspects, the stabilizers can be polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid (PAA), or any combination thereof. In some aspects, PAA is used as the stabilizer because the resulting particles exhibit a sharp DD profile, high yield, and low dyeing %CVs. Moreover, in some aspects, the surfactant can be used well below its critical micelle concentration, resulting in weak electrostatic stabilization. While not wishing to be bound by any theory, it is believed that a charged stabilizer, such as PAA, can provide the electrostatic repulsion required as the seeds swell and polymerize. Other stabilizers, such as polyvinyl alcohol and polyvinylpyrrolidone, are nonionic polymers, and steric stabilization can rely on physical hindrance rather than surface charge. In some aspects, the stabilizer comprises polyvinyl alcohol. In some aspects, the stabilizer comprises polyacrylic acid. In some aspects, the stabilizer comprises polyvinyl pyrrolidone.

[0055] In some aspects, the concentration of stabilizer in the dispersion polymerization can be from about 0.1 wt% to about 10 wt% relative to the total weight of the components of the dispersion polymerization. For example, the stabilizer can be present in an amount of about 0.1 wt% to about 5 wt%, about 0.1 wt% to about 3 wt%, about 0.1 wt% to about2 wt%, about 0.1 wt% to about 1 wt%, about 0.1 wt% to about 0.8 wt%, about 0.1 wt% to about 0.6 wt%, about 0.1 wt% to about 0.4 wt%, about 0.1 wt% to about 0.3 wt%, about 0.2 wt% to about 10 wt%, about 0.2 wt% to about 5 wt%, about 0.2 wt% to about 3 wt%, about 0.2 wt% to about 2 wt%, about 0.2 wt% to about 1 wt%, about 0.2 wt% to about 0.8 wt%, about 0.2 wt% to about 0.6 wt%, about 0.2 wt% to about 0.4 wt%, about 0.2 wt% to about 0.3 wt%, about 0.5 wt% to about 10 wt%, about 0.5 wt% to about 5 wt%, about 0.5 wt% to about 3 wt%, about 0.5 wt% to about 2 wt%, about 0.5 wt% to about 1 wt%, about 0.5 wt% to about 0.8 wt%, about 0.5 wt% to about 0.6 wt%, about 1 wt% to about 10 wt%, about 1 wt% to about 9 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 5 wt%, about 1 wt% to about 3 wt%, about 1.5 wt% to about 10 wt%, about 1.5 wt% to about 8 wt%, about 1.5 wt% to about 5 wt%, about 1.5 wt% to about 3 wt%, about 2 wt% to about 10 wt%, about 2 wt% to about 8 wt%, about 2 wt% to about 5 wt%, about 2 wt% to about3 wt%, about 0.1 wt%, about 0.15 wt%, about 0.2 wt%, about 0.25 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt% relative to the total weight of the components of the dispersion polymerization. In some aspects, the dispersion polymerization composition comprises from about 0.1 wt% to about 2 wt% or about 0.5 wt% to about 1 wt% of a stabilizer relative to the total weight of the components of the dispersion polymerization.

[0056] In some aspects, the surfactant in the dispersion polymerization comprises a low molecular weight (e.g., less than 1,000 g / mol), non-polymeric moiety that can be ionic (e.g., cationic, anionic) or nonionic. For example, the surfactant can be an anionic or cationic surfactant selected from the group consisting of an alkyl sulfonate, an alkyl aryl sulfonate, a condensed naphthalene sulfonate, an alkyl sulfate, an ethoxylated sulfate, a phosphate ester, an ester of sulfosuccinic acid, an alkyl quaternary ammonium salt, an aryl quaternary ammonium salt, an alkylaryl quaternary ammonium salt, a betaine, and any combination thereof. In some aspects, the surfactant can comprise sodium dodecyl sulfate, sodiumdioctyl sulfosuccinate, sodium bis(2-ethylhexyl) sulfosuccinate, or any combination thereof. In some aspects, the surfactant in the dispersion polymerization comprises sodium dioctyl sulfosuccinate.

[0057] In some aspects, the concentration of surfactant in the dispersion polymerization can be from about 0.1 wt% to about 10 wt% relative to the total weight of the components of the dispersion polymerization. For example, the surfactant can be present in an amount of about 0. 1 wt% to about 5 wt%, about 0.1 wt% to about 3 wt%, about 0.1 wt% to about 2 wt%, about 0.1 wt% to about 1 wt%, about 0.1 wt% to about 0.8 wt%, about 0.1 wt% to about 0.6 wt%, about 0.1 wt% to about 0.4 wt%, about 0.1 wt% to about 0.3 wt%, about 0.2 wt% to about 10 wt%, about 0.2 wt% to about 5 wt%, about 0.2 wt% to about 3 wt%, about 0.2 wt% to about 2 wt%, about 0.2 wt% to about 1 wt%, about 0.2 wt% to about 0.8 wt%, about 0.2 wt% to about 0.6 wt%, about 0.2 wt% to about 0.4 wt%, about 0.2 wt% to about 0.3 wt%, about 0.5 wt% to about 10 wt%, about 0.5 wt% to about 5 wt%, about 0.5 wt% to about 3 wt%, about 0.5 wt% to about 2 wt%, about 0.5 wt% to about 1 wt%, about 0.5 wt% to about 0.8 wt%, about 0.5 wt% to about 0.6 wt%, about 1 wt% to about 10 wt%, about 1 wt% to about 9 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 5 wt%, about 1 wt% to about 3 wt%, about 1.5 wt% to about 10 wt%, about 1.5 wt% to about 8 wt%, about 1.5 wt% to about 5 wt%, about 1.5 wt% to about 3 wt%, about 2 wt% to about 10 wt%, about 2 wt% to about 8 wt%, about 2 wt% to about 5 wt%, about 2 wt% to about 3 wt%, about 0. 1 wt%, about 0. 15 wt%, about 0.2 wt%, about 0.25 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt% of the total weight of the components of the dispersion polymerization. In some aspects, the dispersion polymerization composition comprises from about 0. 1 wt% to about 1 wt% or about 0.4 wt% to about 0.5 wt% of a surfactant relative to the total weight of the components of the dispersion polymerization.

[0058] It was discovered that the weight ratio of surfactant to stabilizer can impact the monodispersity and / or the sharpness of the DD profile. In some aspects, the weight ratio of surfactant to stabilizer can be about 1:1 to about 1:3 (e.g., about 1:1, about 1: 1.1, about 1: 1.2, about 1: 1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1 :1.9, about 1:2, about 1:2.1, about 1:2.2, about 1:2.3, about 1 :2.4, about 1:2.5, about 1:2.6,about 1:2.7, about 1:2.8, about 1:2.9, or about 1:3). In some aspects, the weight ratio of surfactant to stabilizer can be about 1:2.

[0059] In some aspects, the alcohol in the dispersion polymerization comprises a Ci-4 alcohol (e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, t-butanol, or any combination thereof). In some aspects, the alcohol comprises ethanol.

[0060] In some aspects, the concentration of alcohol in the dispersion polymerization can be from about about 65 wt% to about 95 wt% relative to the total weight of the components of the dispersion polymerization. For example, the alcohol can be present in an amount from about 65 wt% to about 94 wt%, about 65 wt% to about 93 wt%, about 65 wt% to about 90 wt%, about 65 wt% to about 85 wt%, about 65 wt% to about 80 wt%, about 65 wt% to about 75 wt%, about 65 wt% to about 70 wt%, about 70 wt% to about 95 wt%, about 70 wt% to about 94 wt%, about 70 wt% to about 93 wt%, about 70 wt% to about 90 wt%, about 70 wt% to about 85 wt%, about 70 wt% to about 80 wt%, about 70 wt% to about 75 wt%, about 75 wt% to about 95 wt%, about 75 wt% to about 94 wt%, about 75 wt% to about 93 wt%, about 75 wt% to about 90 wt%, about 75 wt% to about 85 wt%, about 75 wt% to about 80 wt%, about 80 wt% to about 95 wt%, about 80 wt% to about 94 wt%, about 80 wt% to about 93 wt%, about 80 wt% to about 92 wt%, about 80 wt% to about 91 wt%, about 80 wt% to about 90 wt%, about 85 wt% to about 95 wt%, about 85 wt% to about 94 wt%, about 85 wt% to about 93 wt%, about 85 wt% to about 92 wt%, about 85 wt% to about 91 wt%, about 85 wt% to about 90 wt%, about 90 wt% to about 95 wt%, about 90 wt% to about 94 wt%, about 90 wt% to about 93 wt%, about 90 wt% to about 93 wt%, about 90 wt% to about 92 wt%, about 90 wt% to about 91 wt%, about 65 wt%, about 66 wt%, about 67 wt%, about 68 wt%, about 69 wt%, about 70 wt%, about 71 wt%, about 72 wt%, about 73 wt%, about 74 wt%, about 75 wt%, about 76 wt%, about 77 wt%, about 78 wt%, about 79 wt%, about 80 wt%, about 81 wt%, about 82 wt%, about 83 wt%, about 84 wt%, about 85 wt%, about 86 wt%, about 87 wt%, about 88 wt%, about 89 wt%, about 90 wt%, about 91 wt%, about 92 wt%, about 93 wt%, about 94 wt%, or about 95 wt%, relative to the total weight of the components of the dispersion polymerization. In some aspects, the dispersion polymerization composition comprises from about 80 wt% to about 95 wt% or about 88 wt% to about 92 wt% alcohol relative to the total weight of the components of the dispersion polymerization.

[0061] In some aspects of the method, the dispersion polymerization can further include an initiator. In some aspects, the initiator is not water soluble (i.e., the initiator isonly soluble in an organic medium). The initiator can be any suitable photopolymerization initiator or thermal polymerization initiator. The initiator can form a radical, cation, or anion. In an example, the initiator can be a peroxide (e.g., benzoyl peroxide, dicumyl peroxide, di-t-butyl peroxide, cumene hydroperoxide, 2,2’-azobis[2-(2-imidazolin-2-yl)- propane] dihydrochloride, t-butyl hydroperoxide (TBHP)), ammonium persulfate (APS), dicyandiamide, cyclohexyl tosylate, (4-hydroxyphenyl)-dimethylsulfonicum hexafluorophosphate, diphenyl(methyl)sulfonium tetrafluoroborate, triphenylsulphonium nonaflate, a phenone (e.g., acetophenone, 4'-hydroxyacetophenone, 3'- hydroxyacetophenone, benzophenone, 3 -methylbenzophenone, 2-methylphenone, 2- isonitrosopropiophenone, 3,4-dimethylbenzophenone, 3-hydroxybenzophenone, 4- hydroxybenzophenone, 4,4'-dihydroxybenzophenone, 4-(dimethylamino)-benzophenone, 4,4'-bis(dimethylamino)benzophenone, 4,4'-dichlorobenzophenone, 4- phenylbenzophenone, 4-(p-tolylthio)-benzophennone), 4-benzoylbenzoic acid, 2- benzoylbenzoic acid, methyl 2-benzoylbenzoate, 1,4-dibenzoylbenzene, dibenzosuberenone, benzil, p-anisil, methyl benzoylformate, 9,10-phenanthrenequinone, benzoin, anisoin, benzoin methyl ether, benzoin ethyl ether, benzil dimethylketal, 2,7- dimethoxythioxanthone, diphenyliodonium trifluoromethanesulfonate, diphenyliodonium hexafluorophosphate, diphenyliodonium tetrefluoroborate, a triphenylsulfonium salt (e.g., triphenylsulfonium chloride, triphenylsulfonium bromide), 4-nitrobenzenediazonium tetrafluoroborate, acetophenone O-benzoyloxime, 2-nitrobenzyl cyclohexylcarbamate, nifedipine, l-(2-formylbenzyol)piperidine, or any combination thereof.

[0062] In some aspects, the concentration of initiator in the dispersion polymerization can be from about 0.01 wt% to about 5 wt% relative to the total weight of the components of the dispersion polymerization. For example, the initiator can be present in an amount from about 0.01 wt% to about 3 wt%, about 0.01 wt% to about 2 wt%, about 0.01 wt% to about 1 wt%, about 0.05 wt% to about 5 wt%, about 0.05 wt% to about 3 wt%, about 0.05 wt% to about 2 wt%, about 0.05 wt% to about 1 wt%, about 0. 1 wt% to about 5 wt%, about 0.1 wt% to about 3 wt%, about 0.1 wt% to about 2 wt%, about 0.1 wt% to about 1 wt%, about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, about 0.08 wt%, about 0.09 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 1 .1 wt%, about 1 .2 wt%, about 1 .3 wt%, about 1.4 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about4 wt%, about 4.5 wt%, or about 5 wt% of the total weight of the components of the dispersion polymerization. In some aspects, the dispersion polymerization composition comprises from about 0.1 wt% to about 2 wt% or about 0.25 wt% to about 1 wt% of initiator relative to the total weight of the components of the dispersion polymerization.

[0063] In some aspects, the dispersion polymerization reaction can be initiated by heat alone. In such aspects, the dispersion polymerization does not comprise or is substantially free of an initiator.

[0064] In some aspects, the seed particle formed in the dispersion polymerization can have an average diameter of about 1 pm to about 10 pm (e.g., about 1 pm to about 9 pm, about 1 pm to about 8 pm, about 1 pm to about 7 pm, about 1 pm to about 6 pm, about 1 pm to about 5 pm, about 1 pm to about 4 pm, about 1 pm to about 3 pm, about 1 pm to about 2 pm, about 2 pm to about 10 pm, about 2 pm to about 9 pm, about 2 pm to about 8 pm, about 2 pm to about 7 pm, about 2 pm to about 6 pm, about 2 pm to about 5 pm, about 2 pm to about 4 pm, about 2 pm to about 3 pm, about 3 pm to about 10 pm, about 3 pm to about 9 pm, about 3 pm to about 8 pm, about 3 pm to about 7 pm, about 3 pm to about 6 pm, about 3 pm to about 5 pm, about 3 pm to about 4 pm, about 4 pm to about 10 pm, about 4 pm to about 9 pm, about 4 pm to about 8 pm, about 4 pm to about 7 pm, about 4 pm to about 6 pm, about 4 pm to about 5 pm, about 5 pm to about 10 pm, about 5 pm to about 9 pm, about 5 pm to about 8 pm, about 5 pm to about 7 pm, about 5 pm to about 6 pm, about 6 pm to about 10 pm, about 6 pm to about 9 pm, about 6 pm to about 8 pm, about 6 pm to about 7 pm, about 7 pm to about 10 pm, about 7 pm to about 9 pm, about 7 pm to about 8 pm, about 8 pm to about 10 pm, about 8 pm to about 9 pm, about 9 pm to about 10 pm, about 1 pm, about 2 pm, about 3 pm, about 4 pm, about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, or about 10 pm,. In some aspects, the seed particle formed in the dispersion polymerization can have an average diameter of about 1 pm to about 1.6 pm (e.g., about 1 pm, about 1.1 pm, about 1.2 pm, about 1.3 pm, about 1.4 pm, about 1.5 pm, or about 1.6 pm). The average particle diameter can be measured using any technique, including a Coulter counter.

[0065] In some aspects, the seed particle formed in the dispersion polymerization can have a number average molecular weight (Mn) of about 7500 g / mol to about 9500 g / mol. In some aspects, the seed particle formed in the dispersion polymerization can have a number average molecular weight (Mn) of about 7500 g / mol or more (e.g., about 7600 g / mol or more, about 7700 g / mol or more, about 7800 g / mol or more, about 7900 g / mol or more, about 8000 g / mol or more, about 8100 g / mol or more, about 8200 g / mol or more,about 8300 g / mol or more, about 8400 g / mol or more, about 8500 g / mol or more, about 8600 g / mol or more, about 8700 g / mol or more, about 8800 g / mol or more, about 8900 g / mol or more, about 9000 g / mol or more, about 9100 g / mol or more, about 9200 g / mol or more, about 9300 g / mol or more, or about 9400 g / mol or more). In some aspects of these lower end of the molecular weight range, the seed particle formed in the dispersion polymerization can have a number average molecular weight (Mn) of about 9500 g / mol or less (e.g., about 9400 g / mol or less, about 9300 g / mol or less, about 9200 g / mol or less, about 9100 g / mol or less, about 9000 g / mol or less, about 8900 g / mol or less, about 8800 g / mol or less, about 8700 g / mol or less, about 8600 g / mol or less, about 8500 g / mol or less, about 8400 g / mol or less, about 8300 g / mol or less, about 8200 g / mol or less, about 8100 g / mol or less, about 8000 g / mol or less, about 7900 g / mol or less, about 7800 g / mol or less, about 7700 g / mol or less, or about 7600 g / mol or less). In some aspects, the seed particle formed in the dispersion polymerization can have a number average molecular weight (Mn) of about 7500 g / mol to about 9000 g / mol, about 7800 g / mol to about 8200 g / mol, or about 8000 g / mol. The number average molecular weight (Mn) can be measured using any technique, including gel permeation chromatography.

[0066] In some aspects, the seed particle formed in the dispersion polymerization can have a polydispersity index of about 0.3 or less (e.g., about 0.2 or less, about 0.1 or less, or about 0).

[0067] In general, the dispersion polymerization is performed at a relatively mild temperature. For example, the dispersion polymerization can be performed at a temperature of about 40 °C to about 75 °C (e.g., about 40 °C to about 72 °C, about 40 °C to about 70 °C, about 40 °C to about 68 °C, about 40 °C to about 65 °C, about 40 °C to about 62 °C, about 40 °C to about 60 °C, about 40 °C to about 58 °C, about 45 °C to about 75 °C, about 45 °C to about 72 °C, about 45 °C to about 70 °C, about 45 °C to about 68 °C, about 45 °C to about 65 °C, about 45 °C to about 62 °C, about 45 °C to about 60 °C, about 45 °C to about 58 °C, about 50 °C to about 75 °C, about 50 °C to about 72 °C, about 50 °C to about 70 °C, about 50 °C to about 68 °C, about 50 °C to about 65 °C, about 50 °C to about 62 °C, about 50 °C to about 60 °C, about 50 °C to about 58 °C, about 55 °C to about 75 °C, about 55 °C to about 72 °C, about 55 °C to about 70 °C, about 55 °C to about 68 °C, about 55 °C to about 65 °C, about 55 °C to about 65 °C, about 55 °C to about 60 °C, or about 55 °C to about 58 °C). In some aspects, the dispersion polymerization can be performed at a temperature of about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C, about51 °C, about 52 °C, about 53 °C, about 54 °C, about 55 °C, about 56 °C, about 57 °C, about58 °C, about 59 °C, about 60 °C, about 61 °C, about 62 °C, about 63 °C, about 64 °C, about65 °C, about 66 °C, about 67 °C, about 68 °C, about 69 °C, about 70 °C, about 71 °C, about72 °C, about 73 °C, about 74 °C, or about 75 °C. In some aspects, the dispersion polymerization can be performed at a temperature of about 50 °C to about 70 °C, about 55 °C to about 65 °C, about 50 °C, about 60 °C, or about 70 °C. In some aspects, the dispersion polymerization can be performed at a temperature of about 60 °C. In some aspects, the dispersion polymerization can be performed at a temperature of about 65 °C. In some aspects, a water bath can be used to control the temperature rather than a conventional heating mantle.

[0068] The dispersion polymerization can take place for any suitable reaction time, such as at least 1 min, at least 30 min, at least 1 hour, at least 2 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 18 hours, or at least 24 hours. In some aspects, the dispersion polymerization can be about 24 hours or each temperature stage of the dispersion polymerization can be about 24 hours.

[0069] In some aspects of the dispersion polymerization, the temperature can be ramped, which optionally includes ramping in stages. In some aspects, the temperature can be ramped at about 1 °C / min, about 2 + 1 °C / min, about 5 + 1 °C / min, about 8 + 1 °C / min, about 10 + 1 °C / min, about 12 + 1 °C / min or more slowly, such as about 5 °C / hour, about 10 °C / hour, or about 15 °C / hour. In an example, seed particles can form by heating to 50 °C for one day (i.e., about 24 hours) followed by heating to about 70 °C and maintaining this for another day (i.e., about 24 hours). In another example, seed particles can be formed by heating to 70 °C and maintaining for one day (i.e., about 24 hours).

[0070] In an aspect, the vessel containing the dispersion polymerization composition can be immersed in a water bath that has been pre-heated to about 50 °C. In some aspects, the vessel can remain in the 50 °C water bath until near completion of the reaction (e.g., at least about 90% of the styrene has reacted), which typically takes about one day (i.e., about 24 hours). At this point, the temperature of the water bath can be increased to about 70 °C to complete the polymerization reaction.

[0071] The dispersion polymerization can be mixed using any suitable technique, such as agitation, sheer mixing, or sonication. In some aspects, the dispersion polymerization can be mixed at suitable sheer rate, which is any rate that does not agglomerate the mircospheres. In some aspects, the dispersion polymerization can be mixed at a sheer rate of about 95 rpm to about 130 rpm (e.g., 95 rprn to about 128 rprn, 95 rpm to about 125 rpm,95 rpm to about 122 rpm, 95 rpm to about 120 rpm, 95 rpm to about 118 rpm, 95 rpm to about 115, rpm 95 rpm to about 112 rpm, 95 rpm to about 110 rpm, 95 rpm to about 108 rpm, 95 rpm to about 105 rpm, 95 rpm to about 102 rpm, 95 rpm to about 100 rpm, 95 rpm to about 98 rpm, 98 rpm to about 130 rpm, 98 rpm to about 128 rpm, 98 rpm to about 125 rpm, 98 rpm to about 122 rpm, 98 rpm to about 120 rpm, 98 rpm to about 118 rpm, 98 rpm to about 115, rpm 98 rpm to about 112 rpm, 98 rpm to about 110 rpm, 98 rpm to about 108 rpm, 98 rpm to about 105 rpm, 98 rpm to about 102 rpm, 98 rpm to about 100 rpm, 100 rpm to about 130 rpm, 100 rpm to about 128 rpm, 100 rpm to about 125 rpm, 100 rpm to about 122 rpm, 100 rpm to about 120 rpm, 100 rpm to about 118 rpm, 100 rpm to about 115, rpm 100 rpm to about 112 rpm, 100 rpm to about 110 rpm, 100 rpm to about 108 rpm, 100 rpm to about 105 rpm, 100 rpm to about 102 rpm, about 105 rpm to about 128 rpm, about 105 rpm to about 125 rpm, about 105 rpm to about 122 rpm, about 105 rpm to about 120 rpm, about 105 rpm to about 118 rpm, about 105 rpm to about 115 rpm, about 105 rpm to about 112 rpm, about 105 rpm to about 110 rpm, about 105 rpm to about 108 rpm, about 108 rpm to about 130 rpm, about 108 rpm to about 128 rpm, about 108 rpm to about 125 rpm, about 108 rpm to about 122 rpm, about 108 rpm to about 120 rpm, about 108 rpm to about 118 rpm, about 108 rpm to about 115 rpm, about 108 rpm to about 112 rpm, about 108 rpm to about 110 rpm, about 110 rpm to about 130 rpm, about 110 rpm to about 128 rpm, about 110 rpm to about 125 rpm, about 110 rpm to about 122 rpm, about 110 rpm to about 120 rpm, about 110 rpm to about 118 rpm, about 110 rpm to about 115 rpm, about 110 rpm to about 112 rpm, about 112 rpm to about 130 rpm, about 112 rpm to about 128 rpm, about 112 rpm to about 125 rpm, about 112 rpm to about 122 rpm, about 112 rpm to about 120 rpm, about 112 rpm to about 118 rpm, about 112 rpm to about 115 rpm, about 115 rpm to about 130 rpm, about 115 rpm to about 128 rpm, about 115 rpm to about 125 rpm, about 115 rpm to about 122 rpm, about 115 rpm to about 120 rpm, about 115 rpm to about 118 rpm, about 118 rpm to about 130 rpm, about 118 rpm to about 128 rpm, about 118 rpm to about 125 rpm, about 118 rpm to about 122 rpm, about 118 rpm to about 120 rpm, about 120 rpm to about 130 rpm, about 120 rpm to about 128 rpm, about 120 rpm to about 125 rpm, about 120 rpm to about 122 rpm, about 122 rpm to about 130 rpm, about 122 rpm to about 128 rpm, about 122 rpm to about 125 rpm, about 125 rpm to about 130 rpm, about 125 rpm to about 128 rpm, about 128 rpm to about 130 rpm, about 105 rpm, about 106 rpm, about 107 rpm, about 108 rpm, about 109 rpm, about 110 rpm, about 111 rpm, about 112 rpm, about 113 rpm, about 114 rpm, about 115 rpm, about 116 rpm, about 117 rpm, about 118 rpm, about 119 rpm, about 120 rpm, about 121 rpm, about 122 rpm,about 123 rpm, about 124 rpm, about 125 rpm, about 126 rpm, about 127 rpm, about 128 rpm, about 129 rpm, or about 130 rpm. In some aspects, the dispersion polymerization can be mixed at suitable sheer rate. In some aspects, the dispersion polymerization can be mixed at a sheer rate of about 111 rpm to about 121 rpm or about 116 rpm. In some aspects, the dispersion polymerization can be mixed at a sheer rate of about 95 rpm to about 105 rpm or about 100 rpm.

[0072] Once the seed particle is formed, the crosslinked polystyrene microsphere can be formed by subjecting the seed particle to first and second emulsion steps. In some aspects, the seed particle can be initially swelled with a swelling agent in the first emulsion step prior to the second emulsion step to form an activated seed particle. It was found that a swollen (i.e., activated) seed particle can absorb hundreds of times its weight in monomer in the subsequent emulsion step (e.g., emulsion polymerization). In some aspects, the swelling agent can comprise dibutyl phthalate, hexadecane, chlorohexadecane, or any combination thereof. In some aspects, the swelling agent can be dibutyl phthalate. The swelling agent can be used with or without a carrier (e.g., acetone) to disperse the swelling agent within the seed particle. In some aspects, the first emulsion step does not include an initiator, a carrier, or neither an initiator nor carrier.

[0073] In some aspects, the concentration of swelling agent in the first emulsion step can be from about 1 wt% to about 10 wt% relative to the total weight of the components of the first emulsion step. For example, the swelling agent can be present in an amount from about 1 wt% to about 9 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 5 wt%, about 1 wt% to about 3 wt%, about 1.5 wt% to about 10 wt%, about 1.5 wt% to about 8 wt%, about 1.5 wt% to about 5 wt%, about 1.5 wt% to about 3 wt%, about 2 wt% to about 10 wt%, about 2 wt% to about 8 wt%, about 2 wt% to about 5 wt%, about 2 wt% to about3 wt%, about 3 wt% to about 10 wt%, about 3 wt% to about 8 wt%, about 3 wt% to about5 wt%, about 5 wt% to about 10 wt%, about 5 wt% to about 8 wt%, about 5 wt% to about7 wt%, about 6 wt% to about 10 wt%, about 6 wt% to about 8 wt%, about 7 wt% to about10 wt%, about 8 wt% to about 10 wt%, about 1 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about9.5 wt%, or about 10 wt% relative to the total weight of the components of the first emulsion step. In some aspects, the first emulsion step composition comprises from about4 wt% to about 10 wt% or about 5 wt% to about 7 wt% of a stabilizer relative to the total weight of the components of the first emulsion step.

[0074] The swelling step can take place at any suitable temperature. In some aspects, the swelling step can take place at about room temperature (RT) to about 75 °C (e.g., about RT to about 70 °C, about RT to about 65 °C, about RT to about 60 °C, about RT to about55 °C, about RT to about 50 °C, about RT to about 45 °C, about RT to about 40 °C, aboutRT to about 35 °C, about RT to about 30 °C, or about RT to about 25 °C). In some aspects, the dispersion polymerization can be performed at a temperature of about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about49 °C, about 50 °C, about 51 °C, about 52 °C, about 53 °C, about 54 °C, about 55 °C, about56 °C, about 57 °C, about 58 °C, about 59 °C, about 60 °C, about 61 °C, about 62 °C, about63 °C, about 64 °C, about 65 °C, about 66 °C, about 67 °C, about 68 °C, about 69 °C, about70 °C, about 71 °C, about 72 °C, about 73 °C, about 74 °C, or about 75 °C. In some aspects, the swelling step takes place at about RT to about 30 °C, about room temperature (about 20 °C) or about 30 °C. In some aspects, a water bath can be used to control the temperature rather than a conventional heating mantle.

[0075] The swelling step can take place over any suitable time period. In some aspects, the swelling step can be about 5 minutes or more, about 10 minutes or more, about 20 minutes or more, about 30 minutes or more, about 45 minutes or more, about 1 hour or more, about 1.5 hours or more, about 2 hours or more, about 2.5 hours or more, about 3 hours or more, about 3.5 hours or more, about 4 hours or more, about 5 hours or more, about 6 hours or more, about 8 hours or more, about 10 hours or more, about 12 hours or more, about 18 hours or more, or about 24 hours or more. In general, the swelling step can be 1 day or less (e.g., about 5 min to about 24 hr, about 1 hr to about 5 hrs). In some aspects, the swelling step can be about 4 hours, since it was surprisingly discovered that a shorter duration (e.g., less than about 4 hours) decreased the monodispersity. Longer durations (e.g., more than about 4 hours) did not provide any additional uniformity benefits.

[0076] In some aspects, the surfactant in the first emulsion step comprises a low molecular weight (e.g., less than 1,000 g / mol), non-polymeric moiety that can be ionic (e.g., cationic, anionic) or nonionic. For example, the surfactant can be an anionic or cationic surfactant selected from the group consisting of an alkyl sulfonate, an alkyl aryl sulfonate,a condensed naphthalene sulfonate, an alkyl sulfate, an ethoxylated sulfate, a phosphate ester, an ester of sulfosuccinic acid, an alkyl quaternary ammonium salt, an aryl quaternary ammonium salt, an alkylaryl quaternary ammonium salt, a betaine, and any combination thereof. In some aspects, the surfactant can comprise sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, sodium bis(2-ethylhexyl) sulfosuccinate, or any combination thereof. In some aspects, the surfactant in the first emulsion step can comprise sodium dodecyl sulfate.

[0077] In some aspects, the concentration of surfactant in the first emulsion step can be from about 0.2 to about 0.8 wt% relative to the total weight of the components of the first emulsion step. For example, the surfactant can be present in an amount of about 0.2 wt% to about 0.7 wt%, about 0.2 wt% to about 0.6 wt%, about 0.2 wt% to about 0.5 wt%, about 0.2 wt% to about 0.4 wt%, about 0.2 wt% to about 0.3 wt%, about 0.3 wt% to about 0.8 wt%, about 0.3 wt% to about 0.7 wt%, about 0.3 wt% to about 0.6 wt%, about 0.3 wt% to about 0.5 wt%, about 0.3 wt% to about 0.4 wt%, 0.4 wt% to about 0.8 wt%, about 0.4 wt% to about 0.7 wt%, about 0.4 wt% to about 0.6 wt%, about 0.4 wt% to about 0.5 wt%, 0.5 wt% to about 0.8 wt%, about 0.5 wt% to about 0.7 wt%, about 0.5 wt% to about 0.6 wt%, 0.6 wt% to about 0.8 wt%, about 0.6 wt% to about 0.7 wt%, about 0.7 wt% to about 0.8 wt%, about 0.2 wt%, about 0.25 wt%, about 0.3 wt%, about 0.35 wt%, about 0.4 wt%, about 0.45 wt%, about 0.5 wt%, about 0.55 wt%, about 0.6 wt%, about 0.65 wt%, about 0.7 wt%, about 0.75 wt%, or about 0.8 wt%, relative to the total weight of the components of the first emulsion step composition. In some aspects, the first emulsion step composition comprises from about 0.3 wt% to about 0.4 wt% of surfactant relative to the total weight of the components of the first emulsion step.

[0078] The first emulsion step can be mixed using any suitable technique, such as agitation, sheer mixing, or sonication. In some aspects, the first emulsion polymerization can be performed using sonication.

[0079] In some aspects, sonication can take place over two separate steps. In an aspect, an initial step involves seed activation with a low amplitude (e.g., less than 50%) for a suitable amount of time (e.g., on the order of minutes, such as about 30 min or less, about 20 min or less, about 15 min or less, or about 10 min or less). For example, an amplitude of 40% can be applied for about 10 minutes using a sonicator (e.g., a Branson 550 sonicator). This initial, low amplitude step can provide sufficient energy to emulsify the components into droplets smaller than the seed particles. In an aspect, the subsequent (second) step can comprise sonicating the monomer used to swell the activated seedparticles at higher application (50% or more) for a suitable amount of time (e.g., on the order of minutes, such as about 30 min or less, about 20 min or less, about 15 min or less, or about 10 min or less). For example, using a sonicator (e.g., a Q2000 sonicator), an amplitude of 50% can be applied for about 20 minutes. Experimental results evidence that these conditions, i.e., amplitude and duration, can reduce the droplets to a submicron size range with an adequate level of uniformity in droplet size distribution.

[0080] The activated seed particle is then subjected to a second emulsion step, which is an emulsion polymerization. In some aspects, the second emulsion step comprises reacting the seed particle (e.g., activated seed particle) with a surfactant, water, a stabilizer, styrene, a hydrophilic monomer, a crosslinking agent, and an initiator.

[0081] In some aspects, the surfactant in the second emulsion step comprises a low molecular weight (e.g., less than 1,000 g / mol), non-polymeric moiety that can be ionic (e.g., cationic, anionic) or nonionic. For example, the surfactant can be an anionic or cationic surfactant selected from the group consisting of an alkyl sulfonate, an alkyl aryl sulfonate, a condensed naphthalene sulfonate, an alkyl sulfate, an ethoxylated sulfate, a phosphate ester, an ester of sulfosuccinic acid, an alkyl quaternary ammonium salt, an aryl quaternary ammonium salt, an alkylaryl quaternary ammonium salt, a betaine, and any combination thereof. In some aspects, the surfactant can comprise sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, sodium bis(2-ethylhexyl) sulfosuccinate, or any combination thereof. In some aspects, the surfactant in the second emulsion step can comprise sodium dodecyl sulfate.

[0082] In some aspects, the concentration of surfactant in the second emulsion step can be from about 0.01 to about 0.2 wt% relative to the total weight of the components of the second emulsion step. For example, the total surfactant content can be from about 0.01 wt% to about 0.15 wt%, about 0.01 wt% to about 0.1 wt%, about 0.02 wt% to about 0.2 wt%, about 0.02 wt% to about 0.15 wt%, about 0.02 wt% to about 0.1 wt%, about 0.05 wt% to about 0.2 wt%, about 0.05 wt% to about 0.15 wt%, about 0.05 wt% to about 0.1 wt%, about 0.08 wt% to about 0.2 wt%, about 0.08 wt% to about 0.15 wt%, about 0.08 wt% to about 0.1 wt%, about 0.1 wt% to about 0.2 wt%, about 0.1 wt% to about 0.15 wt%, about 0.12 wt% to about 0.2 wt%, about 0.12 wt% to about 0.15 wt%, about 0.15 wt% to about 0.2 wt%, about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, about 0.08 wt%, about 0.09 wt%, about 0.1 wt%, about 0.12 wt%, about 0.15 wt%, about 0.18 wt%, or about 0.2 wt%, relative the total weight of the components of the second emulsion step. In some aspects, the secondemulsion composition comprises a total from about 0.05 wt% to about 1 wt% surfactant relative to the total weight of the components of the second emulsion step.

[0083] In some aspects, the stabilizer in the second emulsion step (e.g., emulsion polymerization) can comprise a high molecular weight (e.g., a number average molecular weight of about 1,000 g / mol or more) nonionic polymeric material. In some aspects, the stabilizer can comprise polyvinyl alcohol, polyacrylic acid, polyvinyl pyrrolidone, or any combination thereof. In some aspects, the stabilizer in the second emulsion step can comprise polyacrylic acid.

[0084] In some aspects, the concentration of stabilizer in the second emulsion step can be from about 0.5 wt% to about 5 wt% relative to the total weight of the components of the second emulsion step. For example, the stabilizer content can be from about 0.5 wt% to about 3 wt%, about 0.5 wt% to about 2 wt%, about 0.5 wt% to about 1 wt%, about 0.7 wt% to about 5 wt%, about 0.7 wt% to about 3 wt%, about 0.7 wt% to about 2 wt%, about 0.7 wt% to about 1 wt%, about 0.8 wt% to about 5 wt%, about 0.8 wt% to about 3 wt%, about 0.8 wt% to about 2 wt%, about 0.8 wt% to about 1 wt%, about 1 wt% to about 5 wt%, about 1 wt% to about 4 wt%, about 1 wt% to about 3 wt%, about 1 wt% to about 2 wt%, about 1.5 wt% to about 5 wt%, about 1.5 wt% to about 4 wt%, about 1.5 wt% to about 3 wt%, about 1.5 wt% to about 2 wt%, about 2 wt% to about 5 wt%, about 2 wt% to about 4 wt%, about 2 wt% to about 3 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, about 1.6 wt%, about 1.7 wt%, about 1.8 wt%, about 1.9 wt%, about 2 wt%, about 2.1 wt%, about 2.2 wt%, about 2.3 wt%, about 2.4 wt%, about 2.5 wt%, about 2.6 wt%, about 2.7 wt%, about 2.8 wt%, about 2.9 wt%, about 3 wt%, about 3.1 wt%, about 3.2 wt%, about 3.3 wt%, about 3.4 wt%, about 3.5 wt%, about 3.6 wt%, about 3.7 wt%, about 3.8 wt%, about 3.9 wt%, about 4 wt%, about 4.2 wt%, about 4.5 wt%, about 4.8 wt%, or about 5 wt% relative to the total weight of the components of the second emulsion step composition. In some aspects, the second emulsion step composition comprises from about 0.7 wt% to about 1 wt% of stabilizer relative to the total weight of the components of the second emulsion step.

[0085] In some aspects, the crosslinked polystyrene microsphere can comprise hydrophilic surface groups formed from at least one hydrophilic monomer in the second emulsion step. In some aspects, the hydrophilic monomer can comprise a moiety selected from hydroxyl group, a carboxy group, an amido group, a sulfonate (-SOi- counterbalanced by a suitable cation), a cyano, a phenyl ether, and any combination thereof. For example,the hydrophilic monomer can have the structure HR'C=CHC(O)-R, in which R is alkylene (e.g., (CH2)I-4) substituted with hydroxyl, carboxy, sulfonate, cyano, phenyl ether, or any combination thereof and R' is H or methyl. For example, the amide-containing hydrophilic monomer can have the structure H2C=CHC(O)-NR1R2, in which R1and R2are the same or different and each is H or alkyl (e.g., Ci-6 alkyl) that is optionally substituted with hydroxyl or R1and R2together with the nitrogen form a N-heterocycle that can be optionally substituted in the ring with O (e.g., morpholino), S (e.g., thiomorpholine), or S(=O) (e.g., thiomorpholine 1 -oxide). In some aspects, the hydrophilic monomer can be acrylic acid, methacrylic acid, 2-hydroxyethyl methacrylate, poly(ethylene glycol) methacrylate, betacarboxyethyl acrylate, polyethoxy ethyl methacrylate (e.g., HEMA, such as HEMA-10), hydroxypolyethoxy allyl ether, N,N-dimethylacrylamide, ethylene glycol dimethacrylate, l-allyloxy-2 hydroxypropyl sulfonate (e.g., sodium l-allyloxy-2 hydroxypropyl sulfonate), diallyl maleate, 2-cyanoethyl acrylate, allyl phenyl ether, or any combination thereof. In some aspects, the hydrophilic monomer can be acrylic acid, methacrylic acid, or a combination thereof. In some aspects, the hydrophilic monomer can be acrylic acid. In some aspects, the hydrophilic monomer can be methacrylic acid.

[0086] In some aspects, the concentration of hydrophilic monomer in the second emulsion step can be from about 1 wt% to about 20 wt% relative to the total weight of styrene monomer. For example, the hydrophilic monomer (e.g., acrylic acid) content can be from about 1 wt% to about 18 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 12 wt%, about 1 wt% to about 10 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 5 wt%, about 1 wt% to about 3 wt%, about 2 wt% to about 20 wt%, about 2 wt% to about 18 wt%, about 2 wt% to about 15 wt%, about 2 wt% to about 12 wt%, about 2 wt% to about 10 wt%, about 2 wt% to about 8 wt%, about 2 wt% to about 5 wt%, about 2 wt% to about3 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 18 wt%, about 5 wt% to about 15 wt%, about 5 wt% to about 12 wt%, about 5 wt% to about 10 wt%, about 5 wt% to about 8 wt%, about 10 wt% to about 20 wt%, about 10 wt% to about 18 wt%, about 10 wt% to about 15 wt%, about 10 wt% to about 12 wt%, about 12 wt% to about 20 wt%, about 12 wt% to about 18 wt%, about 12 wt% to about 15 wt%, about 15 wt% to about 20 wt%, about 15 wt% to about 18 wt%, about 20 wt%, about 19 wt%, about 18 wt%, about 17 wt%, about 16 wt%, about 15 wt%, about 14 wt%, about 13 wt%, about 12 wt%, about 11 wt%, about 10 wt%, about 9 wt%, about 8 wt%, about 7 wt%, about 6 wt%, about 5 wt%, about4 wt%, about 3 wt%, about 2 wt%, or about 1 wt% relative to the total weight of styrene monomer. In some aspects, the second emulsion step composition comprises about 1 wt%to about 8 wt% or about 5 wt% of hydrophilic monomer (e.g., acrylic acid) relative to the total weight of styrene monomer.

[0087] In general, the hydrophilic monomer is added in an amount to provide a crosslinked polystyrene microsphere with a desired level of hydrophilic functional groups at the surface. In some aspects, about 1% to 20% (e.g., about 1% to 15%, about 1% to 12%, about 1% to 10%, about 1% to 8%, about 1% to 5%, about 1% to 4%, about 1% to 3%, about 1% to 2%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%) of the surface of the crosslinked polystyrene microsphere has hydrophilic functional groups. In some aspects, about 1% to 8% or about 5% of the surface of the crosslinked polystyrene microsphere has hydrophilic functional groups.

[0088] The second emulsion step requires a crosslinking agent to form a crosslinked polystyrene microsphere. The crosslinking agent is any suitable multifunctional monomer, such as divinyl benzene, divinyl naphthalene, ethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, 1 ,6-hexanediol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, tetramethylolpropane (meth)acrylate, or any combination thereof. In some aspects, the crosslinking agent can be divinyl benzene.

[0089] In some aspects, the concentration of crosslinking agent in the second emulsion step can be from about 1 wt% to about 10 wt% relative to the total weight of styrene monomer. For example, the crosslinking agent can be present in an amount of about 1 wt% to about 9 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 5 wt%, about 1 wt% to about 3 wt%, about 1.5 wt% to about 10 wt%, about 1.5 wt% to about 8 wt%, about 1.5 wt% to about 5 wt%, about 1.5 wt% to about 3 wt%, about 2 wt% to about 10 wt%, about 2 wt% to about 8 wt%, about 2 wt% to about 5 wt%, about 2 wt% to about 3 wt%, about 0.1 wt%, about 0.15 wt%, about 0.2 wt%, about 0.25 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, about 5 wt%, about 5.5 wt%, about 6 wt%, about 6.5 wt%, about 7 wt%, about 7.5 wt%, about 8 wt%, about 8.5 wt%, about 9 wt%, about 9.5 wt%, or about 10 wt% relative to the total weight of styrene monomer. In some aspects, the second emulsion step composition comprises from about 4 wt% to about 8 wt% or about 5 wt% to about 6 wt% of a crosslinking agent relative to the total weight of styrene monomer.

[0090] In some aspects, the crosslinked polystyrene microsphere can have a low crosslink density, i.e., a crosslink density that is less than about 10%. In some aspects, the crosslinked polystyrene microsphere can have a crosslink density of about 4-7% (e.g., about 6-7%, about 5-7%, about 5-6%, about 5-5.5%, about 4-6%, or about 4-5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, or about 7%). In some aspects, the crosslinked polystyrene microsphere can have a crosslink density of about 5- 5.5%. The crosslink density (%) is based on the proportion in wt% of the crosslinking agent, e.g., divinylbenzene, by comparison to the crosslinked monomer, e.g., styrene. For example, a reaction mixture of 100 g of styrene and 5 g of divinylbenzene would result in a polystyrene microsphere having a crosslink density of 5%.

[0091] The second emulsion step requires an initiator for the emulsion polymerization. The initiator can be any suitable photopolymerization initiator or thermal polymerization initiator. The initiator can form a radical, cation, or anion. In an example, the initiator can be a peroxide (e.g., benzoyl peroxide, dicumyl peroxide, di-t-butyl peroxide, cumene hydroperoxide, 2,2’-azobis[2-(2-imidazolin-2-yl)-propane] dihydrochloride, t-butyl hydroperoxide (TBHP)), ammonium persulfate (APS), dicyandiamide, cyclohexyl tosylate, (4-hydroxyphenyl)-dimethylsulfonicum hexafluorophosphate , diphenyl(methyl)sulfonium tetrafluoroborate, triphenylsulphonium nonaflate, a phenone (e.g., acetophenone, 4'- hydroxyacetophenone, 3'-hydroxyacetophenone, benzophenone, 3-methylbenzophenone, 2-methylphenone, 2-isonitrosopropiophenone, 3,4-dimethylbenzophenone, 3- hydroxybenzophenone, 4-hydroxybenzophenone, 4,4'-dihydroxybenzophenone, 4- (dimethylamino)-benzophenone, 4,4'-bis(dimethylamino)benzophenone, 4,4'- dichlorobenzophenone, 4-phenylbenzophenone, 4-(p-tolylthio)-benzophennone), 4- benzoylbenzoic acid, 2-benzoylbenzoic acid, methyl 2-benzoylbenzoate, 1,4- dibenzoylbenzene, dibenzosuberenone, benzil, p-anisil, methyl benzoylformate, 9,10- phenanthrenequinone, benzoin, anisoin, benzoin methyl ether, benzoin ethyl ether, benzil dimethylketal, 2,7-dimethoxythioxanthone, diphenyliodonium trifluoromethanesulfonate, diphenyliodonium hexafluorophosphate, diphenyliodonium tetrefluoroborate, a triphenyls ulfonium salt (e.g., triphenylsulfonium chloride, triphenylsulfonium bromide), 4- nitrobenzenediazonium tetrafluoroborate, acetophenone O-benzoyloxime, 2-nitrobenzyl cyclohexylcarbamate, nifedipine, l-(2-formylbenzyol)piperidine, or any combination thereof. In some aspects, the initiator in the second emulsion step can comprise benzoyl peroxide.

[0092] In some aspects, the concentration of initiator in the second emulsion step can be from about 1 to about 20 wt% relative to the total weight of styrene monomer. For example, the initiator content can be from about 1 wt% to about 18 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 12 wt%, about 1 wt% to about 10 wt%, about 1 wt% to about 8 wt%, about 1 wt% to about 5 wt%, about 1 wt% to about 3 wt%, about 2 wt% to about 20 wt%, about 2 wt% to about 18 wt%, about 2 wt% to about 15 wt%, about 2 wt% to about 12 wt%, about 2 wt% to about 10 wt%, about 2 wt% to about 8 wt%, about 2 wt% to about 5 wt%, about 2 wt% to about 3 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 18 wt%, about 5 wt% to about 15 wt%, about 5 wt% to about 12 wt%, about 5 wt% to about 10 wt%, about 5 wt% to about 8 wt%, about 10 wt% to about 20 wt%, about10 wt% to about 18 wt%, about 10 wt% to about 15 wt%, about 10 wt% to about 12 wt%, about 12 wt% to about 20 wt%, about 12 wt% to about 18 wt%, about 12 wt% to about 15 wt%, about 15 wt% to about 20 wt%, about 15 wt% to about 18 wt%, about 20 wt%, about 19 wt%, about 18 wt%, about 17 wt%, about 16 wt%, about 15 wt%, about 14 wt%, about 13 wt%, about 12 wt%, about 11 wt%, about 10 wt%, about 9 wt%, about 8 wt%, about 7 wt%, about 6 wt%, about 5 wt%, about 4 wt%, about 3 wt%, about 2 wt%, or about 1 wt% relative to the total weight of the styrene monomer. In some aspects, the second emulsion step composition comprises from about 5 wt% to about 15 wt% or about 9 wt% to about11 wt% of an initiator relative to the weight of styrene monomer. In some aspects, the initiator in the second emulsion step can be dissolved in the monomer mixture.

[0093] In some aspects, the crosslinked polystyrene microsphere can have an average diameter of about 4 pm to about 7 pm (e.g., about 4 pm to about 6.8 pm, about 4 pm to about 6.6 pm, about 4 pm to about 6.4 pm, about 4 pm to about 6.2 pm, about 4 pm to about 6 pm, about 4 pm to about 5.8 pm, about 4 pm to about 5.6 pm, about 4 pm to about 5.4 pm, about 4.5 pm to about 7 pm about 4.5 pm to about 6.8 pm, about 4.5 pm to about 6.6 pm, about 4.5 pm to about 6.4 pm, about 4.5 pm to about 6.2 pm, about 4.5 pm to about 6 pm, about 4.5 pm to about 5.8 pm, about 4.5 pm to about 5.6 pm, about 4.5 pm to about 5.4 pm, about 5 pm to about 7 pm, about 5 pm to about 6.8 pm, about 5 pm to about 6.6 pm, about 5 pm to about 6.4 pm, about 5 pm to about 6.2 pm, about 5 pm to about 6 pm, about 5 pm to about 5.8 pm, about 5 pm to about 5.6 pm, about 5 pm to about 5.4 pm, about 5.3 pm to about 7 pm about 5.3 pm to about 6.8 pm, about 5.3 pm to about 6.6 pm, about 5.3 pm to about 6.4 pm, about 5.3 pm to about 6.2 pm, about 5.3 pm to about 6 pm, about 5.3 pm to about 5.8 pm, about 5.3 pm to about 5.6 pm, about 5.3 pm to about 5.4 pm, about 5.5 pm to about 7 pm about 5.5 pm to about 6.8 pm, about 5.5 pm to about 6.6pm, about 5.5 pm to about 6.4 pm, about 5.5 pm to about 6.2 pm, about 5.5 pm to about 6 pm, about 5.5 pm to about 5.8 pm, about 5.5 pm to about 5.6 pm, about 5.8 pm to about 7 pm about 5.8 pm to about 6.8 pm, about 5.8 pm to about 6.6 pm, about 5.8 pm to about6.4 pim, about 5.8 pm to about 6.2 pm, about 5.8 pm to about 6 pm, about 6 pm to about 7 pm about 6 pm to about 6.8 pm, about 6 pm to about 6.6 pun, about 6 pm to about 6.4 pun, about 6 pun to about 6.2 pun, about 4 pun, about 4. 1 pun, about 4.2 pun, about 4.3 pun, about4.4 pun, about 4.5 pun, about 4.6 pun, about 4.7 pm, about 4.8 pun, about 4.9 pun, about 5 pun, about 5.1 pun, about 5.1 pun, about 5.2 pun, about 5.3 pun, about 5.4 pun, about 5.5 pun, about 5.6 pun, about 5.7 pun, about 5.8 pun, about 5.9 pun, about 6 pun, about 6.1 pun, about 6.2 pun, about 6.3 pun, about 6.4 pun, about 6.5 pun, about 6.6 pun, about 6.7 pun, about 6.8 pun, about 6.9 pun, or about 7 pun. In some aspects, the amount of seed particles inversely correlates to the microsphere size. In some aspects, the crosslinked polystyrene microsphere can have an average diameter of about 5.5 pm to about 6 pm. In another aspect, the crosslinked polystyrene microsphere can have an average diameter of about 5.4 pm to about 5.8 pm. The average particle diameter can be measured using any technique, including a Coulter counter.

[0094] The method described herein is designed to provide a plurality (i.e., a population) of crosslinked polystyrene microspheres that are monodisperse. In some aspects, the plurality (i.e., the population) of crosslinked polystyrene microspheres can have a polydispersity index of about 0.3 or less (e.g., about 0.2 or less, about 0.1 or less, or about 0). In some aspects, a Doublet Discriminator (DD) profile of microspheres produced by the method described herein is sharper than, and / or lacks the “shoulder of, the DD profile of microspheres produced by the method in FIG. 2. Polydispersity can be measured, for example, by using a 90-degree light scattering technique, by using for example a Luminex 200 instrument.

[0095] In some aspects, a plurality of crosslinked polystyrene microspheres are highly uniform in size and shape. In some aspects, at least about 80% or more (e.g., at least about 85% or more, at least about 90% or more, at least about 95% or more, at least about 97% or more, at least about 98% or more, or at least about 90% or more) of the crosslinked polystyrene microspheres have a 1 : 1 relationship between the height and width of each microsphere (i.e., an aspect ratio of about 1). In some aspects, the crosslinked polystyrene microsphere has an aspect ratio of about 1 such that the crosslinked polystyrene microsphere is about spherical in shape. The aspect ratio can be measured using any technique, including scanning electron microscope of individual microspheres.

[0096] In general, the second emulsion step (e.g., emulsion polymerization) is performed at a relatively mild temperature. In some aspects, once the seed particle has been activated, the system can be heated. For example, the second emulsion step can be performed at a temperature of about 40 °C to about 75 °C (e.g., about 40 °C to about 72 °C, about 40 °C to about 70 °C, about 40 °C to about 68 °C, about 40 °C to about 65 °C, about 40 °C to about 62 °C, about 40 °C to about 60 °C, about 40 °C to about 58 °C, about 45 °C to about 75 °C, about 45 °C to about 72 °C, about 45 °C to about 70 °C, about 45 °C to about 68 °C, about 45 °C to about 65 °C, about 45 °C to about 62 °C, about 45 °C to about 60 °C, about 45 °C to about 58 °C, about 50 °C to about 75 °C, about 50 °C to about 72 °C, about 50 °C to about 70 °C, about 50 °C to about 68 °C, about 50 °C to about 65 °C, about 50 °C to about 62 °C, about 50 °C to about 60 °C, about 50 °C to about 58 °C, about 55 °C to about 75 °C, about 55 °C to about 72 °C, about 55 °C to about 70 °C, about 55 °C to about 68 °C, about 55 °C to about 65 °C, about 55 °C to about 65 °C, about 55 °C to about 60 °C, about 55 °C to about 58 °C, about 58 °C to about 75 °C, about 58 °C to about72 °C, about 58 °C to about 70 °C, about 58 °C to about 68 °C, about 58 °C to about 65 °C, about 58 °C to about 62 °C, about 58 °C to about 60 °C, about 60 °C to about 75 °C, about 60 °C to about 72 °C, about 60 °C to about 70 °C, about 60 °C to about 68 °C, about 60 °C to about 65 °C, about 60 °C to about 62 °C, about 62 °C to about 75 °C, about 62 °C to about 72 °C, about 62 °C to about 70 °C, about 62 °C to about 68 °C, about 62 °C to about65 °C, about 65 °C to about 75 °C, about 65 °C to about 72 °C, about 65 °C to about 70 °C, or about 65 °C to about 68 °C). In some aspects, the second emulsion step can be performed at a temperature of about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C, about 51 °C, about52 °C, about 53 °C, about 54 °C, about 55 °C, about 56 °C, about 57 °C, about 58 °C, about59 °C, about 60 °C, about 61 °C, about 62 °C, about 63 °C, about 64 °C, about 65 °C, about66 °C, about 67 °C, about 68 °C, about 69 °C, about 70 °C, about 71 °C, about 72 °C, about73 °C, about 74 °C, or about 75 °C. In some aspects, the second emulsion step can be performed at a temperature of about 68 °C to about 72 °C. In some aspects, a water bath can be used to control the temperature rather than a conventional heating mantle.

[0097] The second emulsion step can take place for any suitable reaction time, such as at least 1 min, at least 30 min, at least 1 hour, at least 2 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 18 hours, or at least 24 hours. In some aspects, the dispersion polymerization can be about 18 hours.

[0098] In some aspects of the second emulsion step, the temperature can be ramped, which optionally includes ramping in stages. In some aspects, the temperature can be ramped at about 1 °C / min, about 2 ± 1 °C / min, about 5 ± 1 °C / min, about 8 + 1 °C / min, about 10 ± 1 °C / min, or about 12 + 1 °C / min. In some aspects, the temperature can be ramped at about 10 + 1 °C / min. In an example, a ramp to 70 °C for about 18 hours allows for sufficient diffusion and conversion of the monomer in the seed particles.

[0099] The second emulsion step can be mixed using any suitable technique, such as agitation, sheer mixing, or sonication. In some aspects, the second emulsion polymerization can be performed using sonication. In a specific example, the monomers for the second emulsion step can be sonicated with the surfactant / stabilizer / water system at various amplitudes and durations. When the amplitude or duration is too weak and short, the droplets (monomer) are not reduced enough to diffuse into the seed particles and overcome Ostwald’s ripening. If the amplitude is too high and long, heat can be introduced into the system and prematurely initiate the polymerization. Once the emulsion system is created, the activated seed can be added and allowed to swell (e.g., about 4 hours at 30 °C or room temperature for 8 hours or more).

[0100] After the crosslinked polystyrene microsphere has been formed, the microsphere can be purified by washing with one or more solvents (e.g., water, an organic solvent, or a combination thereof). The washing can remove, for example, non-uniform particles and / or any unreacted starting materials (e.g., unreacted styrene, surfactant) or solvent. Thus, in some aspects, the method further comprises washing the crosslinked polystyrene microsphere with water, an alcohol (e.g., a Ci-4 alcohol), toluene, tetrahydrofuran, or a combination thereof. For example, the crosslinked polystyrene microsphere can be washed with a combination of water and methanol or water alone. One or more than one (e.g., 2, 3, 4, 5, 6, 7, or 8) washing steps can be used. In some aspects, the crosslinked polystyrene microsphere can be centrifuged with a washing solvent and any supernatants can be decanted. The washing and centrifugation steps can be alternated for one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) cycles.

[0101] In some aspects of the method, the seed particle does not become incorporated in the final crosslinked polystyrene microsphere, which can be observed with a scanning electron microscope as having a smooth particle surface without any lobes (FIG. 3). Expulsion of the seed particle can be performed by washing in an organic solvent, such as an organic solvent known to swell polystyrene beads, such as toluene or tetrahydrofuran (THF). This washing step can also remove any residual monomer and initiator, the latterof which is known to cause the decomposition of dyes insides the beads. The type of solvents used during the washing affect the structure of the DD profile.

[0102] In some aspects, the order of solvents used to wash the crosslinked polystyrene microsphere has an effect. In some aspects, the method comprises washing the crosslinked polystyrene microsphere with sequential water and alcohol (e.g., a C1-4 alcohol) washes, followed by washing with THF. In some aspects, the THF wash can swell the microspheres to remove, e.g., initiator and non-crosslinked polymer chains. In some aspects, the crosslinked polystyrene microsphere can be washed sequentially in order with water, methanol, tetrahydrofuran, methanol, and water. In another aspect, the crosslinked polystyrene microsphere can be washed sequentially in order with water, isopropanol, tetrahydrofuran, isopropanol, and water.

[0103] In some aspects, after the last wash (e.g., THF solvent wash), the particles can be allowed to undergo a relaxation period to facilitate the necessary relaxation of the microsphere back to its intrinsic state. Thus, in some aspects, the washed crosslinked polystyrene microsphere can be relaxed in an alcohol (e.g., C1-4 alcohol) for a sufficient time, e.g., few hours and up to about a week, to reduce (e.g., shrink) the crosslinked polystyrene microsphere from the swollen state. It was discovered that not performing this relaxation step can result in the microspheres having poly dispersity when subjected to analysis via the DD (dual peaks). In some aspects, the crosslinked polystyrene microsphere can be soaked in methanol, isopropanol, or a mixture thereof. In some aspects, the crosslinked polystyrene microsphere can be soaked in methanol. In some aspects, the crosslinked polystyrene microsphere can be soaked in isopropanol.

[0104] In some aspects, the method further comprises coating the surface of the crosslinked polystyrene microsphere, including a crosslinked polystyrene microsphere that has been washed and relaxed to a non-s welled state, with a polymer. In some aspects, the coating comprises polyvinyl pyrrolidone (PVP). In some aspects, the coating can (i) reduce flocculation of a plurality of the crosslinked polystyrene microsphere during dyeing, (ii) reduce excess foaming when resuspended from storage (e.g., storage in buffer / preservative water), or (iii) both (i) and (ii).

[0105] It was unexpectedly discovered that crosslinked polystyrene microspheres produced by the method described herein have a low dyeing coefficient of variation (CV), and the crosslinked polystyrene microspheres can take up more dye in general compared to microspheres prepared by prior methods. Dye uptake is important as certain verification regions and / or xMAP regions have dye volumes that are reaching saturation and need morevolume (i.e., a bigger microsphere) to match specifications. In some aspects, the method provides a plurality of crosslinked polystyrene microspheres that can provide a reporter signal of about 3% CV or less (e.g., about 2.9% CV or less, about 2.8% CV or less, about 2.7% CV or less, about 2.6% CV or less, about 2.5% CV or less, about 2.4% CV or less, about 2.3% CV or less, about 2.2% CV or less, about 2.1% CV or less, about 2% CV or less, about 1.9% CV or less, about 1.8% CV or less, about 1.7 % CV or less, about 1.6% CV or less, about 1.5% CV or less, about 1.4% CV or less, about 1.3% CV or less, about 1.2% CV or less, about 1.1% CV or less, about 1% CV or less, about 0.9% CV or less, about 0.8% CV or less, or about 0.7% CV or less). In some aspects, the method provides a plurality of crosslinked polystyrene microspheres that can provide a reporter signal of about 0.7-2.5% CV, about 0.7-2% CV, about 0.7-1.5% CV, about 0.7-1% CV, or about 0.7- 0.8% CV. The percent CV of dyeing can be measured using any suitable technique, including for example a Luminex 200 instrument.

[0106] In some aspects, the crosslinked polystyrene microsphere produced by the method described herein has a refractive index of about 1.4 to about 1.6 (e.g., about 1.4, about 1.5, about 1.6). In some aspects, the crosslinked polystyrene microsphere produced by the method described herein has a refractive index of about 1.59.

[0107] In some aspects, the present disclosure relates to a method of forming a crosslinked polystyrene microsphere comprising forming a seed particle by subjecting a composition comprising styrene (e.g., freshly distilled styrene), a stabilizer (e.g., polyvinyl pyrrolidone), a surfactant (e.g., sodium dioctyl sulfosuccinate), and an alcohol (e.g., ethanol) to dispersion polymerization; and subjecting the seed particle to a first emulsion step; swelling the seed particle with a swelling agent (e.g., dibutyl phthalate) to provide a swelled seed particle; and subjecting the swelled seed particle to a second emulsion step to form the crosslinked polystyrene microsphere.

[0108] In some aspects of this method, the first emulsion step comprises reacting the seed particle with a surfactant, water, and a swelling agent (e.g., dibutyl phthalate) to form an activated seed particle. In some aspects of this method, the second emulsion step comprises reacting the activated seed particle with a surfactant (e.g., sodium dodecyl sulfate), water, a stabilizer (e.g., polyacrylic acid), styrene, a hydrophilic monomer (e.g., acrylic acid), a crosslinking agent (e.g., divinyl benzene), and an initiator (e.g., benzoyl peroxide). In some aspects of this method, the crosslinked polystyrene microsphere hasone or more properties selected from an average diameter of about 5.5 pm to about 6 pm, an aspect ratio of about 1, and a crosslink density of about 4-7%. In some aspects of this method, the method further comprises washing the crosslinked polystyrene microsphere sequentially in order with water, either methanol or isopropanol, tetrahydrofuran, either methanol or isopropanol, and then water.

[0109] In some aspects, the present disclosure relates to a population of crosslinked polystyrene microspheres produced by the method described herein. In some aspects, the population has a crosslink density of about 4-7%. In some aspects, the population has a crosslink density of about 5-5.5%.

[0110] In some aspects, the present disclosure further relates to a population of crosslinked polystyrene microspheres comprising less than 3% coefficient of variation (CV) of dyeing, wherein each microsphere has a diameter of 5.5 to 6 microns, and a carboxylated surface with a nominal parking area of 50 to 150 sq. angstroms. In some aspects, this population has a CV of dyeing less than 2.5%, less than 2%, less than 1.5%, or less than 1%.

[0111] The present disclosure further relates to a method of conducting a multiplex assay comprising detecting or quantitating one or more analytes with a population of microspheres, as described herein.EXAMPLES

[0112] The example presented below is provided for the purpose of illustration only and the aspects described herein should in no way be construed as being limited to this example. Rather, the aspects should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.Example 1Preparation of Seed Particle

[0113] To prepare a seed particle, 2.8 g of docusate sodium salt (DSS; Dioctyl Sulfosuccinate Sodium Salt) and 5.6 g of polyvinylpyrrolidone (PVP30) in alcohol (about 620 g) were dissolved in a 3-neck round-bottom flask. In a separate container, 3.08 g of benzoyl peroxide (BPO) was dissolved in 61.6 g of distilled styrene. The styrene solution was then added to the round bottom flask, placed in a 65 °C water bath, and allowed to stir continuously under an inert gas for about 18-24 hours.Preparation of Microspheres

[0114] A solution was prepared by dissolving 2.4 g of sodium dodecyl sulfate (SDS) in 600 g of water in a beaker. Next, 36 g of dibutyl phthalate (DBPT) was added into the aqueous solution. The resulting mixture underwent emulsification via sonication. The emulsified solution was then combined with 170 mL of pre-washed seed particles (successive washes of methanol using centrifugation) suspended in water. The composite was transferred to a 1 -liter container, placed on a rotator, and allowed to tumble slowly over an extended period. This solution was referred to as the “activated seed solution.”

[0115] The next day, a vessel was charged with 7230 g of water, 5.1 g of SDS, and 66.6 g of polyacrylic acid. A mixture of distilled styrene (83 wt% of mixture), divinylbenzene (4.6 wt% of mixture), acrylic acid (4.1 wt% of mixture), and BPO (8.3 wt% of mixture) was prepared in a separate container (total weight about 790 g). Once all the solids were dissolved, they were combined and emulsified via sonication. The resulting emulsion was then poured into a 3 -neck round-bottom flask, situated within a water bath set to 30 °C, and equipped with overhead stirring. The activated seed solution was added; the reaction mixture was degassed with inert gas and allowed to stir for 4 hours. The temperature of the water bath was then increased to 70 °C and allowed to stir for an additional 24 hours. The resultant microspheres were washed with water, alcohol, and tetrahydrofuran.

[0116] A doublet discriminator is a channel that measures the amount of light scattering from particles that flow past the red laser. Light scattering is directly proportional to particle size, and the channel is designed to identify particles that are smaller or larger than a single microsphere, including microspheres that are aggregated. Side scatter profiles (Doublet Discriminator) were produced by analyzing 5000 bead events on a Luminex 200 instrument.

[0117] The resultant microspheres were analyzed by DD. FIG. 4A shows the DD profile of crosslinked polystyrene microspheres (undyed) prepared using conventional methods. FIG. 4B shows the DD profile of crosslinked polystyrene microspheres produced using the present methods. FIG. 4A shows a shoulder, which indicates higher polydispersity of the microspheres. This shoulder often falls outside the fixed gates during calibration and verification routes leading to rejected events and skewed results. The DD profile of FIG. 4B is much sharper compared to FIG. 4A.

[0118] The microspheres can be dyed using the procedure set forth in U.S. Patent 7,445,844, the disclosure of which is incorporated herein by reference in its entirety.Example 2

[0119] The seed amount inversely correlates to the resultant size of the microsphere. Adding more or less seed to the seed activation emulsion (first emulsion step) will decrease or increase the target size, respectively. FIG. 5 shows how the volume of seed affects the resulting diameter. For example, to provide a 7 pm diameter bead, less seed volume will be used than for a 3 pm bead. The quantity of seed particles (mL) can be determine using the formula:[((seed size, pm)3(desired microsphere size, pm)3) * 348.67)] + 0.4641A sizing run can be performed to get close to the target size, then the number of seeds can be adjusted to provide a particle with an about 5.6 pm diameter.Example 3

[0120] Determination of the amount of surfactant was calculated based on the number of moles of sodium dodecyl sulfate (SDS) in solution. A large amount of SDS can be carried over from the seed activation emulsion. Using surfactant alone, the population of the resultant particles were polydispersed. Increasing or decreasing the amount of surfactant did not have a positive change in the overall swelling characteristics. It was determined that 5E-4 moles of SDS was sufficient to stabilize the seed swelling emulsion, but a stabilizer was necessary to control the droplets as they began to swell into the seed particles. The amount of stabilizer was determined experimentally and found to be a 2-fold molar increase relative to the SDS (5E-2 moles of polyacrylic acid (PAA)).

[0121] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0122] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0123] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0124] The breadth and scope of the present invention should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0125] The claims in the instant application are different than those of the parent application or other related applications. The Applicant therefore rescinds any disclaimer of claim scope made in the parent application or any predecessor application in relation to the instant application. The Examiner is therefore advised that any such previous disclaimer and the cited references that it was made to avoid, may need to be revisited. Further, the Examiner is also reminded that any disclaimer made in the instant application should not be read into or against the parent application.

Claims

WHAT IS CLAIMED IS:

1. A method of forming a crosslinked polystyrene microsphere comprising forming a seed particle by subjecting a composition comprising styrene, a stabilizer, a surfactant, and an alcohol to dispersion polymerization; and forming the crosslinked polystyrene microsphere by subjecting the seed particle to first and second emulsion steps.

2. The method of claim 1, wherein the seed particle is swelled with a swelling agent prior to the second emulsion step.

3. The method of claim 1 or 2, wherein the first emulsion step comprises reacting the seed particle with a surfactant, water, and a swelling agent to form an activated seed particle.

4. The method of claim 2 or 3 , wherein the swelling agent comprises dibutyl phthalate.

5. The method of any one of claims 1-4, wherein the first emulsion step does not include an initiator or a carrier.

6. The method of any one of claims 1-5, wherein the second emulsion step comprises reacting the seed particle with a surfactant, water, a stabilizer, styrene, a hydrophilic monomer, a crosslinking agent, and an initiator.

7. The method of any one of claims 1-6, wherein the seed particle has an average diameter of about 1 pm to about 1.6 pm.

8. The method of any one of claims 1-7, wherein the seed particle has a number average molecular weight of about 7500 g / mol to about 9500 g / mol.

9. The method of any one of claims 1-8, wherein the crosslinked polystyrene microsphere has an average diameter of about 5.5 pm to about 6 pm.

10. The method of any one of claims 1-9, wherein the crosslinked polystyrene microsphere has an aspect ratio of about 1.

11. The method of any one of claims 1-10, wherein the styrene is purified prior to use.

12. The method of claim 11, wherein the styrene is distilled prior to use.

13. The method of any one of claims 1-12, wherein the stabilizer comprises a high molecular weight nonionic polymeric material.

14. The method of claim 13, wherein the stabilizer comprises polyvinyl alcohol, polyacrylic acid, polyvinyl pyrrolidone, or any combination thereof.

15. The method of claim 14, wherein the stabilizer in the dispersion polymerization comprises polyvinyl pyrrolidone.

16. The method of claim 14 or claim 15, wherein the stabilizer in the second emulsion step comprises polyacrylic acid.

17. The method of any one of claims 1-16, wherein the surfactant comprises a low molecular weight non-polymeric moiety that is ionic or nonionic.

18. The method of claim 17, wherein the surfactant comprises sodium dodecyl sulfate, sodium dioctyl sulfosuccinate, sodium bis(2-ethylhexyl) sulfosuccinate, or any combination thereof.

19. The method of claim 18, wherein the surfactant in the dispersion polymerization comprises sodium dioctyl sulfosuccinate.

20. The method of claim 18 or 19, wherein the surfactant in the first and second emulsion steps comprises sodium dodecyl sulfate.

21. The method of any one of claims 1-20, wherein the alcohol comprises aCi4 alcohol.

22. The method of claim 21, wherein the alcohol comprises ethanol.

23. The method of any one of claims 1-22, wherein the crosslinked polystyrene microsphere comprises hydrophilic surface groups.

24. The method of any one of claims 6-23, wherein the hydrophilic monomer is acrylic acid, methacrylic acid, or a combination thereof.

25. The method of claim 24, wherein the hydrophilic monomer is acrylic acid.

26. The method of any one of claims 1-25, wherein the crosslinked polystyrene microsphere has a crosslink density of about 4-7%.

27. The method of claim 26, wherein the crosslinked polystyrene microsphere has a crosslink density of about 5-5.5%.

28. The method of any one of claims 6-27, wherein the crosslinking agent is divinyl benzene.

29. The method of any one of claims 6-28, wherein the initiator in the second emulsion step comprises benzoyl peroxide.

30. The method of any one of claims 1-29, wherein the dispersion polymerization is performed at a temperature of about 55 °C to about 65 °C.

31. The method of any one of claims 1-30, wherein the dispersion polymerization is mixed at a sheer rate of about 105 rpm to about 130 rpm.

32. The method of any one of claims 1-31, wherein the second emulsion step is performed at a temperature of about 68 °C to about 72 °C.

33. The method of claim 32, wherein the temperature can be ramped at about 10 ± 1 °C / min.

34. The method of any one of claims 1-33, wherein the second emulsion polymerization is performed using sonication.

35. The method of any one of claims 1-34, further comprising washing the crosslinked polystyrene microsphere with water, an alcohol, toluene, tetrahydrofuran, or a combination thereof.

36. The method of claim 35, wherein the crosslinked polystyrene microsphere is washed sequentially in order with water, methanol, tetrahydrofuran, methanol, and water.

37. The method of claim 35, wherein the crosslinked polystyrene microsphere is washed sequentially in order with water, isopropanol, tetrahydrofuran, isopropanol, and water.

38. The method of claim 36 or 37, wherein the crosslinked polystyrene microsphere is soaked in methanol, isopropanol, or a mixture thereof.

39. The method of any one of claims 1-38, further comprising coating the crosslinked polystyrene microsphere with a polymer.

40. The method of claim 39, wherein the coating comprises polyvinyl pyrrolidone.

41. The method of any one of claims 1-40, wherein the amount of seed particles inversely correlates to the microsphere size.

42. The method of any one of claims 1 to 41, wherein a Doublet Discriminator (DD) profile of the microsphere is sharper than the DD profile of microspheres produced by the method in FIG. 2 as measured by 90-degree light scattering techniques.

43. The method of any one of claims 1 to 42, which produces a population of microspheres, wherein the population of the microspheres has a coefficient of variation (CV) of dyeing that is 3% or less, 2.5% or less, 2% or less, 1.5% or less, or 1% or less.

44. A population of crosslinked polystyrene microspheres produced by the method of any one of claims 1 to 43.

45. The population of claim 44, which has a crosslink density of about 4-7%.

46. The population of claim 44, which has a crosslink density of about 5-5.5%.

47. A population of crosslinked polystyrene microspheres comprising less than 3% coefficient of variation (CV) of dyeing, wherein each microsphere has a diameter of 5.5 to 6 microns, and a carboxylated surface with a nominal parking area of 50 to 150 sq. angstroms.

48. The population of microspheres of claim 47, wherein the CV of dyeing is less than 2.5%, less than 2%, less than 1.5%, or less than 1%.

49. A method of conducting a multiplex assay comprising detecting or quantitating one or more analytes with the population of crosslinked polystyrene microspheres of any one of claims 44 to 48.

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