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WO2025212750A1 - Procédés, kits et systèmes de préparation de biomolécules - Google Patents

Procédés, kits et systèmes de préparation de biomolécules

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Publication number
WO2025212750A1
WO2025212750A1 PCT/US2025/022713 US2025022713W WO2025212750A1 WO 2025212750 A1 WO2025212750 A1 WO 2025212750A1 US 2025022713 W US2025022713 W US 2025022713W WO 2025212750 A1 WO2025212750 A1 WO 2025212750A1
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WO
WIPO (PCT)
Prior art keywords
composition
particle
biomolecules
particles
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/022713
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English (en)
Inventor
Hongwei XIA
Evan O'Brien
Jacob FABER-RICO
Craig STOLARCZYK
Richard Baldwin
Tristan Brown
Asim Siddiqui
Xiaoyan Zhao
Maedeh ZAMANI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Seer Inc
Original Assignee
Seer Inc
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Filing date
Publication date
Application filed by Seer Inc filed Critical Seer Inc
Publication of WO2025212750A1 publication Critical patent/WO2025212750A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L33/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides or nitriles thereof; Compositions of derivatives of such polymers
    • C08L33/04—Homopolymers or copolymers of esters
    • C08L33/06—Homopolymers or copolymers of esters of esters containing only carbon, hydrogen and oxygen, which oxygen atoms are present only as part of the carboxyl radical
    • C08L33/10—Homopolymers or copolymers of methacrylic acid esters
    • C08L33/12—Homopolymers or copolymers of methyl methacrylate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
    • G01N2446/84—Polymer coating, e.g. gelatin

Definitions

  • the methods, kits, and systems provided herein rely on the use of a combination of at least two particles (e.g., comprising macromolecule structures) comprising different surface charges for the isolation, purification, and identification of biomolecules (e.g., proteins or peptides).
  • the first macromolecule structure may have a neutral to negative surface charge, such as measured by zeta potential.
  • the second macromolecule structure may have a highly negative or more negative surface charge compared to that of the first macromolecule structure.
  • this first and second macromolecule structure provides a synergistic relationship that allows for increased ability to isolate, purify, and identify biomolecules, such as proteins in comparison to the use of each of the particles (e.g., comprising the macromolecule structures) on their own.
  • Increases in ability to isolate and identify biomolecules present advantages over generally used methods such as various forms of chromatography or suspension trapping, since the methods provided herein are widely applicable and lead to high yields of desired biomolecules, high reproducibility in the identification of biomolecules, and significant number of identifiable biomolecules.
  • the methods, kits, and systems provided herein provide for the preparation of biomolecules for further identification or analysis.
  • compositions comprising two or more particles comprising: (a) a first particle comprising a first macromolecule structure, the first particle comprising a neutral to negative surface charge; and (b) a second particle comprising a second macromolecule structure, the second particle comprising a greater negative surface charge than the first particle.
  • the first particle has a surface charge of greater than -20 mV.
  • the second macromolecule structure comprises a polymer comprising one or more units represented by Formula (A): Formula (A) wherein, R is hydrogen R 1 is hydrogen or hydroxyl; q is an integer from 1 to 6; R 2 is C1-C8 diamine, N-substituted with one or more R 3 ; WSGR Docket No.53344-792.601 R 3 is each independently hydrogen or C1-C8 alkyl optionally substituted with one or more oxo, hydroxyl, C1-C8 alkyl, C1-C8 alkenyl, and C1-C8 alkynyl; or R 1 and R 2 are taken together to form C 2 -C 6 heterocycloalkyl; and R 4 is hydrogen or C 1 -C 6 alkyl.
  • the second macromolecule structure comprises a copolymer comprising two or more units represented by Formula (A) and Formula (B): wherein, R is hydrogen R 1 is hydrogen or hydroxyl; q is an integer from 1 to 6; m is an integer from 1 to 6; R 2 is C1-C8 diamine, N-substituted with one or more R 3 ; R 3 is each independently hydrogen or C1-C8 alkyl optionally substituted with one or more oxo, hydroxyl, C 1 -C 8 alkyl, C 1 -C 8 alkenyl, and C 1 -C 8 alkynyl; or R 1 and R 2 are taken together to form C 2 -C 6 heterocycloalkyl; R 4 is each independently hydrogen or C1-C6 alkyl; is a single bond or a double bond; and * is an attachment point to another unit of Formula (A) or Formula (B) when is a single bond.
  • R is hydrogen
  • R 1 is hydrogen or hydroxyl
  • q is
  • R is .
  • R 1 is hydroxyl.
  • R 2 is a C2 diamine, N-substituted with one or more R 3 .
  • At least one R 3 is C 1 -C 8 alkyl optionally substituted with one or more oxo, hydroxyl, C1-C8 alkyl, C1-C8 alkenyl, and C1-C8 alkynyl. In some embodiments, at least one R 3 is C1-C8 alkyl substituted with at least one of oxo, hydroxyl, and C 1 -C 8 alkenyl. In some embodiments, the structure of Formula (A) is represented by Formula (A-A): Formula (A-A) wherein, R 5 is C 1 -C 10 alkyl, C 1 -C 10 alkenyl, or C 1 -C 10 alkynyl.
  • the structure of Formula (A) is represented by Formula (A-B): Formula (A-B) wherein, WSGR Docket No.53344-792.601 R 5 is C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl.
  • the structure of Formula (A) is Formula (A-C): Formula (A-C) wherein, R 5 is each independently C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl.
  • the structure of Formula (A-C) is: .
  • the structure of Formula (A) is represented by Formula (A-D): Formula (A-D)
  • q is an integer from 1 to 3.
  • q is an integer of 1.
  • R 5 is C1-C10 alkenyl.
  • R is hydrogen.
  • the structure of Formula (A) is: .
  • R 1 and R 2 are taken together to form C 2 heterocycloalkyl.
  • m is an integer of 1.
  • R 4 is C 1 -C 6 alkyl.
  • the structure of Formula (B) is: WSGR Docket No.53344-792.601 .
  • the copolymer comprises the structure: .
  • the first macromolecule structure comprises a copolymer comprising two or more units represented by Formula (D) and Formula (E): wherein, R 4 is each independently hydrogen or C 1 -C 6 alkyl; R 6 is hydrogen or (CH2)pOR 7 ; R 7 is hydrogen or C 1 -C 6 alkyl; m is an integer from 1 to 6; p is an integer from 1 to 6; is a single bond or a double bond; and * is an attachment point to another unit of Formula (D) or Formula (E) when is a single bond.
  • R 6 is (CH 2 ) p OR 7 .
  • the first macromolecule structure comprises a copolymer comprising three or more units represented by: WSGR Docket No.53344-792.601
  • the unit represented by is present in the first macromolecule structure in an amount of less than 5 wt%.
  • the two or more particles are nanoparticles.
  • the two or more particles are microparticles.
  • the two or more particles each individually have a diameter of from about 100 nm to about 750 nm. In some embodiments, the two or more particles each individually have a diameter of from about 100 nm to about 500 nm.
  • the two or more particles each individually have a polydispersity index (PDI) of from about 0.01 to about 0.2. In some embodiments, the two or more particles each individually have a PDI of from about 0.1 to about 0.2. In some embodiments, the two or more particles comprises iron oxide. In some embodiments, the two or more particles comprises a superparamagnetic iron oxide nanoparticle. In some embodiments, the two or more particles comprises a core-shell structure. In some embodiments, the core of the core-shell structure is paramagnetic. In some embodiments, the two or more particles comprise an iron oxide core and a silica shell. In some embodiments, the particle comprises iron oxide crystals embedded in a polystyrene core.
  • PDI polydispersity index
  • the polymer or the copolymer is covalently coupled to a surface of the particle. In some embodiments, the polymer is non-covalently coupled to a surface of the particle. In some embodiments, the polymer or copolymer is covalently coupled to a surface of the particle via a linker.
  • the first particle comprises the structure: wherein, is a surface of the first particle; L is a linker; and A is a copolymer described herein.
  • the second particle comprises the structure: WSGR Docket No.53344-792.601 wherein, is a surface of the second particle; L is a linker; and B is the polymer or copolymer described herein.
  • the linker comprises an alkylene, esteralkylene, or aralkylene.
  • the polymer or copolymer is covalently coupled to the surface via a base polymer.
  • the composition further comprises a stabilizing agent.
  • the stabilizing agent comprises a metal salt.
  • the metal salt comprises aluminum chloride.
  • the copolymer is a random copolymer.
  • the copolymer is a block copolymer.
  • the composition comprises at least 1 wt% of the polymer or copolymer. In some embodiments, the composition comprises from about 1 wt% to about 30 wt% of the polymer or copolymer.
  • a method of isolating one or more biomolecules from a biological sample comprising: (a) contacting the biological sample comprising one or more biomolecules with a composition provided herein to bind the one or more biomolecules to the at least two particles, thereby forming at least two biomolecule corona; and (b) eluting the one or more biomolecules from the at least two particles, thereby providing one or more isolated biomolecules.
  • assaying detects at least 10% more unique biomolecules than a method comprising contacting the biological sample with a composition comprising the second particle in absence of the first particle. In some embodiments, assaying detects at least 60% more unique biomolecules than a method comprising contacting the biological sample with a composition comprising the first particle in absence of the second particle. In some embodiments, the method further comprises separating the one or more biomolecules and the at least two particles from the biological sample. In some embodiments, the method further comprises optionally digesting, alkylating, and/or lysing the one or more biomolecules to provide one or more digested biomolecules. In some embodiments, the one or more biomolecules comprises proteins, peptides, or a combination thereof.
  • the biological sample comprises plasma, serum, or blood. In some embodiments, the biological sample comprises biofluid. In some embodiments, the biofluid is a cell-free biofluid. In some embodiments, the method further comprises diluting the one or more biomolecules and at least two particles. In some embodiments, the one or more biomolecules and at least two particles are diluted in a buffer. In some embodiments, the methods further comprise eluting the one or more biomolecules from the two or more particles. In some embodiments, eluting is in the presence of buffer or an aqueous solution. In some embodiments, the contacting is in the presence of a buffer.
  • the buffer comprise a pH of from about 7 to about 8 (e.g., pH of about 8.5). In some embodiments, the buffer comprises a pH of from about 7.4 to 7.6 (e.g., pH of about 7.5). In some embodiments, the buffer comprises HEPES. In some embodiments, one of the first particle or second particle is contacted with the one or more biomolecules at a concentration of 0.1 mg/mL to 0.2 mg/mL. In some embodiments, one of the first particle or second particle is contacted with the one or more biomolecules at a concentration of 0.5 mg/mL to 0.6 mg/mL.
  • the first particle and second particle are contacted with the one or more biomolecules at a concentration of 0.6 mg/mL to 0.8 mg/mL.
  • the method further comprises contacting the one or more biomolecules with the at least two particles in the presence of an organic solvent.
  • the method comprises purifying the one or more digested biomolecules.
  • purifying the one or more digested biomolecules comprises contacting the one or more digested biomolecules with a third particle in an organic solvent to form a biomolecule WSGR Docket No.53344-792.601 corona.
  • the aqueous solution comprises an organic solvent. In some embodiments, the aqueous solution comprises an organic solvent in an amount of no more than 50 wt% (e.g., 40 wt%, 30 wt%, 20 wt%, 10 wt%, or no more than 5 wt%). In some embodiments, the aqueous solution comprises an organic solvent in an amount of no more than 50 wt% (e.g., 40 wt%, 30 wt%, 20 wt%, 10 wt%, or no more than 5 wt%). In some embodiments, the method is capable of isolating from about 100 to about 20,000 biomolecules.
  • the method further comprises identifying the one or more biomolecules. In some embodiments, the method further comprises assaying the one or more digested biomolecules to identify the one or more biomolecules. In some embodiments, the assaying or identifying comprises performing mass spectrometry (MS), liquid chromatography- mass spectrometry (LC-MS), protein sequencing, or a combination thereof. In some embodiments, the method is capable of assaying or identifying from about 1 to about 20,000 biomolecules. In some embodiments, when repeated, the assaying yields a percent quantile normalized coefficient (QNCV) of variation of 30% or less. In some embodiments, when repeated, the assaying yields a percent quantile normalized coefficient (QNCV) of variation of 20% or less.
  • QNCV percent quantile normalized coefficient
  • the reduction agent comprises TCEP, dithiothreitol, beta-mercaptoethanol, glutathione, cysteine, or any combination thereof.
  • the alkylating agent comprises iodoacetamide, iodoacetic acid, acrylamide, chloroacetamide, or any combination thereof.
  • a system for isolating one or more biomolecules from a biological sample comprising: (a) a composition provided herein; (b) a suspension solution configured to suspend the at least two particles; (c) a biological sample comprising one or more biomolecules; and (d) an automated system comprising a network of units with differentiated functions configured to isolate one or more biomolecules from the biological sample using the at least two particles.
  • the network of units comprises: (a) a first unit comprising a multichannel fluid transfer instrument for transferring fluids between units within the system; (b) a second unit comprising a support for storing a plurality of biological samples; and (c) a third unit comprising a support for an array plate possessing partitions that comprise the two or more particles for binding of the one or more biomolecules with the two or more particles.
  • the network of units further comprises a fourth unit comprising supports for storing a plurality of reagents.
  • the network of units further comprises a fifth unit comprising supports for storing a reagent to be disposed of.
  • the network of units further comprises supports for storing consumables used by a multichannel fluid transfer instrument.
  • the automated system is configured to perform a method provided herein.
  • a kit for isolating one or more biomolecules from a biological sample the kit comprising a composition provided herein.
  • a kit for preparing one or more biomolecules from a biological sample for assaying by mass spectrometry the kit comprising a composition provided herein.
  • comprises a washing agent configured to wash the one or more biomolecules bound to the at least two particles.
  • the kit comprises an elution agent configured to elute the one or more biomolecules from the at least two particles.
  • the kit comprises a denaturing agent.
  • the kit comprises a reducing agent.
  • the kit further comprises an alkylation agent.
  • the kit further comprises at least one buffer.
  • the at least one buffer comprises a digestion buffer, resuspension buffer, denaturation buffer, digestion buffer, or a lysis buffer.
  • the kit further comprises one or more organic solvents.
  • the buffer comprises HEPES.
  • one or more components of the kit are prepackaged into one or more containers.
  • a method of preparing a mixture of at least two particles comprising recurring units of a first monomer and a second monomer or a first monomer and a third monomer, the method comprising: (a) obtaining a first particle comprising a neutral to negative surface charge; (b) obtaining a second particle comprising a greater negative surface charge than the first particles; and (c) forming a mixture comprising the first and second particles.
  • the first particle comprises a surface charge of about 0 mV to about -15 mV.
  • the second particle comprises a surface charge of about -35 mV to about -60 mV.
  • the surface charge is characterized by a zeta potential.
  • the first particle is a first particle described herein.
  • the second particle is a second particle described herein.
  • a method of preparing a macromolecule structure comprising recurring units of a first component and a second component, the method comprising: (a) providing a mixture of monomers in a solvent comprising a first monomer and a second monomer, wherein the first monomer comprises: wherein q is an integer from 1 to 6; and WSGR Docket No.53344-792.601 the second monomer comprises: wherein m is an integer from 1 to 6; (b) contacting a surface and the mixture of monomers, thereby producing a reaction mixture; (c) polymerizing the mixture of monomers to produce a macromolecule immobilized to the surface of a particle; (d) contacting the macromolecule immobilized to the surface and an amine, thereby producing an
  • the particle is a nanoparticle. In some embodiments, the particle is a microparticle. In some embodiments, the particle has a diameter of from about 100 nm to about 750 nm. In some embodiments, the particle has a diameter of from about 100 nm to about 500 nm. In some embodiments, the particle has a polydispersity index (PDI) of about 0.01 to about 0.2. In some embodiments, the particle has a PDI of about 0.1 to about 0.2. In some embodiments, the particle comprises iron oxide. In some embodiments, the particle comprises is a superparamagnetic iron oxide nanoparticle. In some embodiments, the particle comprises a core- shell structure.
  • the particle comprises an iron oxide core and a silica shell. In some embodiments, the particle comprises iron oxide crystals embedded in a polystyrene core. In some embodiments, (b) comprises contacting in an organic solvent. In some embodiments, (d) comprises contacting in an organic solvent. In some embodiments, the organic solvent comprises an alcohol, acetonitrile, dichloromethane, dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethylacetate, hexamethylphosphoramide (HMPA), or tetrahydrofuran. In some embodiments, the organic solvent comprises acetonitrile. In some embodiments, the organic solvent comprises DMF. In some embodiments, the method further comprises heating.
  • the heating comprises heating to a temperature of at least 50°C, 60°C, 70°C, 80°C, or at least 90°C.
  • the polymerization comprises free radical polymerization, atom transfer radical polymerization (ATRP), emulsion polymerization, or precipitation polymerization.
  • the macromolecule structure comprises a composition described herein. WSGR Docket No.53344-792.601 [0014]
  • a use a macromolecule structure comprising 2-hydroxyethyl methacrylate, ethylene glycol dimethacrylate, and methacrylic acid as monomer units for binding proteins in a biological sample.
  • provided herein is a use of a particle comprising a macromolecule structure comprising the structure of Formula (A-D) and the structure of Formula (A-A) as recurring units for binding proteins in a biological sample.
  • a particle comprising a macromolecule structure comprising the structure of Formula (A-C) and the structure of Formula (A-B) as recurring units for binding proteins in a biological sample is a use of a composition provided herein for binding proteins in a biological sample.
  • compositions comprising a plurality of particles, wherein the particles comprise an outer polymer surface and a magnetic core, wherein the outer polymer surface comprises 2-hydroxyethyl methacrylate, ethylene glycol dimethacrylate, and methacrylic acid as monomer units.
  • a composition comprising a plurality of particles, wherein the particles comprise an outer polymer surface and a magnetic core, wherein the outer polymer surface comprises ethylene glycol dimethacrylate, monomer 6, and at least one of: monomer 7, monomer 8, monomer 9, and glycidyl methacrylate.
  • composition obtained by a method comprising polymerizing 2-hydroxyethyl methacrylate, ethylene glycol dimethacrylate, and methacrylic acid in the presence of vinyl-functionalized magnetic particles.
  • a method comprising: (a) contacting a plasma or serum sample comprising one or more proteins with a composition provided herein, thereby adsorbing at least a portion of the proteins to the magnetic particles; (b) separating the adsorbed proteins and the magnetic particles from the plasma or serum; WSGR Docket No.53344-792.601 (c) eluting and optionally digesting the adsorbed proteins from the magnetic particles, thereby providing one or more isolated proteins; (d) optionally purifying the isolated proteins using solid phase extraction; and (e) analyzing the isolated proteins using mass spectrometry.
  • a method of making polymer-coated magnetic particles comprising polymerizing 2-hydroxyethyl methacrylate, ethylene glycol dimethacrylate, and methacrylic acid in the presence of vinyl-functionalized magnetic particles.
  • a method of making polymer-coated magnetic particles comprising: (i) polymerizing glycidyl methacrylate, ethylene glycol dimethacrylate, and methacrylic acid in the presence of vinyl-functionalized magnetic particles to form polymer-coated particles; (ii) reacting the polymer-coated magnetic particles with an alkylene diamine to form amine-modified magnetic particles; and (iii) reacting the amine- modified magnetic particles with an optionally substituted succinic acid anhydride.
  • FIG. 1 shows an exemplary preparation scheme for particle (e.g., comprising a macromolecule structure) (A2).
  • FIG. 2 shows an exemplary preparation scheme for particle (e.g., comprising a macromolecule structure)(A3).
  • FIG.3 shows protein group (PG) counts for particles (e.g., comprising macromolecule structures) (A1), (A2), and (A1)/(A2) multiplexed in various plasmas.
  • FIG.4 shows PG counts for particles (e.g., comprising macromolecule structures) (A3), (A4), (A5), and (A6) in 1M Tris (pH 9.5) along with PG counts for multiplexed particles (e.g., comprising macromolecule structures) (A3)&(A4), (A3)&(A5), and (A3)&(A6).
  • FIG. 5A shows a scheme for loading of a 96-well plate to perform a method provided herein with 40 samples.
  • FIG.5B shows a scheme for loading a 96-well plate to perform a method provided herein with 80 samples.
  • FIG.6 shows peptide yield and protein group counts evaluated for varying multiplexed compositions of (A1) and (A2) described herein at various concentrations.
  • an alkyl comprises one to five carbon atoms (i.e., C 1 -C 5 alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (i.e., C 1 -C 4 alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (i.e., C1-C3 alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (i.e., C1-C2 alkyl). Whenever it appears herein, a numerical range such as “C1-C3 alkyl” means that the alkyl group consists of 1 carbon atom, 2 carbon atoms, or 3 carbon atoms.
  • an alkyl comprises one carbon atom (i.e., C1 alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms WSGR Docket No.53344-792.601 (i.e., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (i.e., C5- C8 alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (i.e., C2-C5 alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (i.e., C 3 -C 5 alkyl).
  • the alkyl group is selected from methyl, ethyl, 1propyl (n-propyl), 1-methylethyl (isopropyl), 1-butyl (nbutyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1dimethylethyl (tertbutyl), 1pentyl (n-pentyl).
  • examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2- methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, tert-amyl, and hexyl, and longer alkyl groups, such as heptyl,
  • the alkyl is optionally substituted with oxo, halogen, -CN, -CF 3 , OH, or -OMe. In some embodiments, the alkyl is optionally substituted with halogen such as F. In some embodiments, the alkyl is unsubstituted.
  • Alkyl groups may be a straight chain or comprise one or more branched chains. [0040] As used herein, C1-Cx (or C1-x) includes C1-C2, C1-C3... C1-Cx. By way of example only, a group designated as “C1-C4” indicates that there are one to four carbon atoms in the moiety, i.e.
  • Alkoxy refers to a radical bonded through an oxygen atom of the formula –O-alkyl, where alkyl is an alkyl chain as defined above. Unless stated otherwise specifically in the specification, an alkoxy group can be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, an alkoxy is optionally substituted with oxo, halogen, -CN, - CF3, OH, -OMe, NH2, or -NO2.
  • an alkoxy is optionally substituted with oxo, halogen, -CN, -CF 3 , OH, or -OMe. In some embodiments, the alkoxy is optionally substituted with halogen. In some embodiments, the alkoxy is unsubstituted.
  • Alkenyl refers to an optionally substituted straight or branched hydrocarbon chain radical group containing at least one carbon-carbon double bond, and preferably having from two to twelve carbon atoms (i.e., C 2 -C 12 alkenyl).
  • an alkenyl comprises two to eight carbon atoms (i.e., C 2 -C 8 alkenyl). In certain embodiments, an alkenyl comprises four to eight carbon atoms (i.e., C4-C6 alkenyl). In other embodiments, an alkenyl comprises six to eight carbon atoms (i.e., C 6 -C 8 alkenyl). In certain embodiments, an alkenyl comprises at least one double bond at the end of a carbon chain. In other embodiments, an alkenyl comprises at least one double bond in the middle of a carbon chain. The group can be in either the cis or trans configuration about the double bond(s) and should be understood to include both isomers.
  • an alkenyl group is optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • an alkenyl is optionally substituted with oxo, halogen, -CN, -CF 3 , OH, -OMe, NH 2 , or -NO 2 .
  • an alkenyl is optionally substituted with oxo, halogen, -CN, -CF3, OH, or -OMe.
  • an alkenyl is optionally substituted with oxo, halogen, -CN, -CF 3 , OH, -OMe, NH2, or -NO2. In some embodiments, an alkenyl is optionally substituted with oxo, halogen, -CN, -CF 3 , OH, or -OMe. In some embodiments, the alkenyl is optionally substituted with halogen. In some embodiments, the alkenyl is unsubstituted.
  • Alkynyl refers to an optionally substituted straight or branched hydrocarbon chain radical group containing at least one carbon-carbon triple bond, and preferably having from two to twelve carbon atoms (i.e., C 2 -C 12 alkynyl).
  • an alkynyl comprises two to eight carbon atoms (i.e., C2-C8 alkynyl).
  • an alkynyl comprises two to six carbon atoms (i.e., C2-C6 alkynyl).
  • an alkynyl comprises two to four carbon atoms (i.e., C2-C4 alkynyl).
  • an alkynyl group is optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • an alkynyl is optionally substituted with oxo, halogen, -CN, -CF3, OH, -OMe, NH2, or -NO 2 .
  • an alkynyl is optionally substituted with oxo, halogen, -CN, -CF 3 , OH, or -OMe.
  • alkynyl is optionally substituted with halogen. In some embodiments, the alkynyl is unsubstituted.
  • alkylene or "alkylene chain” refers to an optionally substituted straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group containing no unsaturation, and preferably having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, nbutylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond.
  • an alkylene comprises one to ten carbon atoms (i.e., C1-C8 alkylene). In certain embodiments, an alkylene comprises one to eight carbon atoms (i.e., C 1 -C 8 alkylene). In other embodiments, an alkylene comprises one to five carbon atoms (i.e., C1-C5 alkylene). In other embodiments, an alkylene comprises one to four carbon atoms (i.e., C 1 -C 4 alkylene).
  • an alkylene group can be optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • an alkylene is optionally substituted with oxo, halogen, -CN, -CF3, OH, -OMe, NH2, or -NO2.
  • an alkylene is optionally substituted with oxo, halogen, -CN, -CF3, OH, or - OMe.
  • the alkylene is optionally substituted with halogen.
  • the alkylene is -CH2-, -CH2CH2-, or -CH2CH2CH2-.
  • the alkylene is -CH2-.
  • the alkylene is -CH2CH2-.
  • the alkylene is -CH2CH2CH2-.
  • the alkylene is unsubstituted.
  • Aryl refers to a radical derived from a hydrocarbon ring system comprising at least one aromatic ring. In some embodiments, an aryl comprises hydrogens and 5 to 30 carbon atoms.
  • the aryl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused (when fused with a cycloalkyl or heterocycloalkyl ring, the aryl is bonded through an aromatic ring atom) or bridged ring systems.
  • the aryl is a 6- to 10- membered aryl.
  • the aryl is a 6-membered aryl.
  • an aryl can be optionally substituted, for example, with halogen, amino, alkylamino, aminoalkyl, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, -S(O)2NH-C1-C6alkyl, and the like.
  • an aryl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, OH, -OMe, NH2, -NO2, -S(O)2NH2, -S(O) 2 NHCH 3, -S(O) 2 NHCH 2 CH 3 , -S(O) 2 NHCH ( CH 3 ) 2 , -S(O) 2 N(CH 3 ) 2 , or -S(O) 2 NHC(CH 3 ) 3 .
  • an aryl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, OH, or -OMe.
  • the aryl is optionally substituted with halogen.
  • the aryl is substituted with alkyl, alkenyl, alkynyl, haloalkyl, or heteroalkyl, wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl is independently unsubstituted, or substituted with halogen, methyl, ethyl, -CN, -CF3, OH, -OMe, NH2, or -NO2.
  • the aryl is unsubstituted.
  • Alkyl refers to a radical of the formula R c aryl where R c is an alkylene chain as defined above, for example, methylene, ethylene, and the like.
  • Alkenyl refers to a radical of the formula –R d aryl where R d is an alkenylene chain as defined above.
  • Alkynyl refers to a radical of the formula R e aryl, where R e is an alkynylene chain as defined above.
  • Carbocycle refers to a saturated, unsaturated, or aromatic rings in which each atom of the ring is carbon.
  • an aromatic ring e.g., phenyl
  • a saturated or unsaturated ring e.g., cyclohexane, cyclopentane, or cyclohexene.
  • a bicyclic carbocycle includes any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits.
  • a bicyclic carbocycle includes any combination of ring sizes such as 4-5 fused ring systems, 5-5 fused ring systems, 5-6 fused ring systems, 6-6 fused WSGR Docket No.53344-792.601 ring systems, 5-7 fused ring systems, 6-5 fused ring systems, 6-7 fused ring systems, 5-8 fused ring systems, and 6-8 fused ring systems.
  • Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl, and naphthyl.
  • saturated carbocycle refers to carbocycles with at least one degree of unsaturation and excluding aromatic carbocycles.
  • unsaturated carbocycles include cyclohexadiene, cyclohexene, and cyclopentene.
  • saturated cycloalkyl refers to a saturated carbocycle.
  • Exemplary carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl, norbornyl, and naphthyl.
  • Carbocycles can be optionally substituted by one or more substituents such as those substituents described herein.
  • Cycloalkyl refers to a stable, partially or fully saturated, monocyclic or polycyclic carbocyclic ring, which can include fused (when fused with an aryl or a heteroaryl ring, the cycloalkyl is bonded through a non-aromatic ring atom), bridged, or spiro ring systems.
  • Representative cycloalkyls include, but are not limited to, cycloalkyls having from three to fifteen carbon atoms (C3-C15 cycloalkyl), from three to ten carbon atoms (C3-C10 cycloalkyl), from three to eight carbon atoms (C3-C8 cycloalkyl), from three to six carbon atoms (C3-C6 cycloalkyl), from three to five carbon atoms (C3-C5 cycloalkyl), or three to four carbon atoms (C3-C4 cycloalkyl).
  • the cycloalkyl is a 3- to 6-membered cycloalkyl.
  • the cycloalkyl is a 5- to 6-membered cycloalkyl.
  • Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic cycloalkyls or carbocycles include, for example, adamantyl, norbornyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cis-decalin, trans-decalin, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl.
  • Partially saturated cycloalkyls include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Unless stated otherwise specifically in the specification, a cycloalkyl is optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • a cycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, a cycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, - CF 3 , -OH, or -OMe. In some embodiments, the cycloalkyl is optionally substituted with halogen. In some embodiments, the cycloalkyl is unsubstituted.
  • haloalkyl or “haloalkane” refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, for example, trifluoromethyl, dichloromethyl, bromomethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.
  • the alkyl part of the fluoroalkyl radical is optionally further substituted.
  • the alkyl is substituted with one hydroxyl. In some embodiments, the alkyl is substituted with one, two, or three hydroxyls. Hydroxyalkyl include, for example, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, or hydroxypentyl. In some embodiments, the hydroxyalkyl is hydroxymethyl.
  • Amoxyalkyl refers to an alkyl radical, as defined above, that is substituted by one or more amines. In some embodiments, the alkyl is substituted with one amine. In some embodiments, the alkyl is substituted with one, two, or three amines.
  • C1-Cx alkyl substituted with disulfide as used herein may refer to a disulfide of the structure R-S-S-R’, where R and R’ may be identical or different. Each R and R’ may be independently selected from C 1 -C y alkyl, such that the length of R and R’ is the length of the C1-Cx alkyl.
  • heteroalkyl refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g., oxygen, nitrogen (e.g., -NH-, -N(alkyl)- ), sulfur, or combinations thereof.
  • a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl.
  • a heteroalkyl is a C 1 -C 6 heteroalkyl wherein the heteroalkyl is comprised of 1 to 6 carbon atoms and one or more atoms other than carbon, e.g., oxygen, nitrogen (e.g.
  • heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl.
  • heteroalkyl examples include, for example, -CH2OCH3, -CH2CH2OCH3, -CH2CH2OCH2CH2OCH3, or - CH(CH3)OCH3.
  • a heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, OH, or -OMe. In some embodiments, the heteroalkyl is optionally substituted with halogen. In some embodiments, the heteroalkyl is unsubstituted.
  • “Heterocycloalkyl” refers to a stable 3 to 24membered partially or fully saturated ring radical comprising 2 to 23 carbon atoms and at least one ring heteroatoms. In some embodiments, a heterocycloalkyl contains from one to 8 heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous, and sulfur.
  • the heterocycloalkyl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused (when fused with an aryl or a heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocycloalkyl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized.
  • heterocycloalkyls include, but are not limited to, heterocycloalkyls having from two to fifteen carbon atoms (C 2 -C 15 heterocycloalkyl), from two to ten carbon atoms (C 2 -C 10 heterocycloalkyl), from two to eight carbon atoms (C 2 -C 8 heterocycloalkyl), from two to six carbon atoms (C2-C6 heterocycloalkyl), from two to five carbon atoms (C2-C5 heterocycloalkyl), or two to four carbon atoms (C2-C4 heterocycloalkyl).
  • the heterocycloalkyl is a 3- to 6-membered heterocycloalkyl.
  • heterocycloalkyl also includes all ring forms of the carbohydrates, including but not limited to, the monosaccharides, the disaccharides, and the oligosaccharides. It is understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including the heteroatoms) that make up the heterocycloalkyl (i.e. skeletal atoms of the heterocycloalkyl ring).
  • a heterocycloalkyl is optionally substituted, for example, with oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • a heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, OH, -OMe, NH 2 , or -NO 2 .
  • Heterocycles include e.g., 3- to 10-membered monocyclic rings and 6- to 12-membered bicyclic rings (such as spiro, fused, or bridged rings).
  • the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which optionally includes fused, bridged, or spirocyclic ring systems.
  • the heteroatoms in the heterocyclyl radical are optionally oxidized.
  • One or more nitrogen atoms, if present, are optionally quaternized.
  • the heterocyclyl radical can be partially or fully saturated.
  • the heterocyclyl is attached to the rest of the molecule through any atom of the ring(s).
  • heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2oxopiperazinyl, 2oxopiperidinyl, 2oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1oxothiomorph
  • heterocyclyl is meant to include heterocyclyl radicals as defined above that are optionally substituted by one or more substituents.
  • a heterocyclyl can be optionally substituted by one or more substituents selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally WSGR Docket No.53344-792.601 substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -R b -OR a , -R b -OC(O
  • Heteroaryl or “aromatic heterocycle” refers to a ring system radical comprising carbon atom(s) and one or more ring heteroatoms (e.g., selected from the group consisting of nitrogen, oxygen, phosphorous, silicon, and sulfur), and at least one aromatic ring.
  • a heteroaryl is a 5 to 14membered ring system radical comprising one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen, phosphorous, and sulfur.
  • the heteroaryl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused (when fused with a cycloalkyl or heterocycloalkyl ring, the heteroaryl is bonded through an aromatic ring atom) or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized.
  • the heteroaryl is a 5- to 10-membered heteroaryl.
  • the heteroaryl is a 5- to 6-membered heteroaryl.
  • a heteroaryl is optionally substituted, for example, with halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like.
  • a heteroaryl is optionally substituted with halogen, methyl, ethyl, -CN, -CF 3 , OH, -OMe, NH2, or -NO2.
  • a heteroaryl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, OH, or -OMe. In some embodiments, the heteroaryl is optionally substituted with halogen. In some embodiments, the heteroaryl is unsubstituted. [0063] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons or substitutable heteroatoms, e.g., NH, of the structure.
  • the permissible substituents include acyclic and cyclic, branched, and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, WSGR Docket No.53344-792.601 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9.
  • a nested sub-range of an exemplary range of 1 to 50 can comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
  • the compounds and structures provided herein may be stereoisomeric.
  • a compound or structure of the disclosure may form a stereoisomer.
  • the stereoisomer may be a diastereomer (e.g., a cis/trans isomer, E/Z isomer, conformer, or rotamer).
  • the stereoisomer may be an enantiomer (R,S enantiomers or +/- enantiomers).
  • the compound or structure of the disclosure may be enantiopure (e.g., 100% pure).
  • the compound or structure may form a racemic mixture of enantiomers (e.g., 50% pure).
  • a compound or structure of the disclosure may stabilize as a stereoisomer, where the compound or structure of the disclosure comprises at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, or more of a mixture of the compound or structure and the corresponding stereoisomer.
  • the combination of particles (e.g., comprising WSGR Docket No.53344-792.601 macromolecule structures), such as described herein, provide a synergistic relationship that enhances the macromolecule structure’s ability to isolate biomolecules. This increased ability in some instances is compared to the ability of a single macromolecule structure to isolate biomolecules on their own.
  • the methods and macromolecules provided herein aid in avoiding the need for alternative isolation/purification methods such as solid phase chromatography (e.g., reverse-phase/ion exchange) or suspension trapping which may be solvent intensive and may lead to poor recovery, reproducibility issues, or insufficient purification.
  • the methods, systems, and compositions provided herein allow for the use of a diverse range of biological sample types, including but not limited to plasma, serum, cells, and tissues, and at lower required sample volumes than comparable methods.
  • the methods provided herein allow for the preparation of biomolecule samples for analysis by mass spectrometry (MS), such as by selective removal of MS incompatible components from a biological sample.
  • MS mass spectrometry
  • two particles are used, a first particle with a neutral to negative surface charge, and a second particle, with a highly negative surface charge (e.g., more negative than the first particle).
  • two macromolecule structures are used, a first macromolecule structure with a neutral to negative surface charge, and a second macromolecule structure, with a highly negative surface charge (e.g., more negative than the first macromolecule structure).
  • the combination of the two differing particles e.g., comprising macromolecule structures
  • provides a synergistic relationship enabling the isolation, purification, quantification, or identification of a larger number of biomolecules than the two particles (e.g., comprising macromolecule structures) on their own.
  • compositions comprising two or more (e.g., structurally unique) particles (e.g., comprising macromolecule structures).
  • the compositions comprise a first particle (e.g., comprising a macromolecule structure) and a second particle (e.g., comprising a macromolecule structure).
  • the first particle e.g., comprising a macromolecule structure
  • the second particle e.g., comprising a macromolecule structure
  • the first particle e.g., comprising a macromolecule structure
  • the second particle (e.g., comprising a macromolecule structure) comprises a greater negative (e.g., more negative) surface charge than that of the first particle (e.g., comprising a macromolecule structure).
  • surface charge herein is measured by zeta potential.
  • zeta potential may be measured by electrophoretic light scattering (ELS).
  • ELS electrophoretic light scattering
  • zeta potential is measured by DLS in 1.5 mM KCl (pH 7.0).
  • zeta potential is measured by DLS in 5% PBS (pH 6.8-7.4).
  • the surface charge (e.g., zeta potential) described herein is measured by dispersing about 20 ⁇ g of particles in 5% PBS at a pH of 6.8-7.4.
  • an instrument such as a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) may be used.
  • Particles for testing in some instances are suspended at 10 mg/mL in water with about 10 min of bath sonication prior to testing. Samples then may be diluted to approximately 0.02 wt%, and DLS performed in water at about 25°C in disposable polystyrene semi-micro cuvettes with about 1 min temperature equilibration time.
  • Results from several runs may be averaged in some instances (e.g., 3 runs of about 1 min), with a 633 nm laser in 173° backscatter mode.
  • DLS results in some instances are then analyzed using the cumulants method and zeta potential measured in 5% pH 7.4 PBS in disposable folded capillary cells (Malvern Instruments, PN DTS1070) at about 25°C with an about 1 min equilibration time.
  • three measurements are performed with automatic measurement duration with a minimum of 10 runs and a maximum of 100 runs, and a 1 min hold between measurements.
  • the Smoluchowski model is used to determine the zeta potential from the electrophoretic mobility. Used to determine the zeta potential from the electrophoretic mobility.
  • a neutral surface charge is characterized by a zeta potential of from about -5 mV to about 5 mV. In some embodiments, a neutral surface charge is characterized by a zeta potential of at most 5 mV (e.g., at most 4 mV, 3 mV, 2 mV, 1 mV, 0.75 mV, 0.5 mV, 0.25 mV, or at most 0.1 mV).
  • a neutral surface charge is characterized by a zeta potential of at least -5 mV (e.g., at least -4 mV, -3 mV, -2 mV, -1.5 mV, -1 mV, -0.75 mV, -0.5 mV, -0.25 mV, or at least -0.1 mV).
  • a neutral surface charge is characterized by a zeta potential of about 0 mV.
  • a neutral surface charge is characterized by a zeta potential of about -1 mV, 0 mV, or 1 mV.
  • the first particle (e.g., comprising the first macromolecule structure) provided herein comprises a surface charge (e.g., characterized by a zeta potential) of from about 10 mV to about -15 mV.
  • the first particle (e.g., comprising a macromolecule structure) comprises a surface charge of from about 0 to about -15 mV.
  • the first particle (e.g., comprising a macromolecule structure) comprises a surface charge of from about 0 to about -10 mV.
  • the first particle (e.g., comprising a macromolecule structure) comprises a surface charge of from about -10 to about -20 mV.
  • the first particle (e.g., comprising a macromolecule structure) comprises a WSGR Docket No.53344-792.601 surface charge of at most about 0 mV. In some embodiments, the first particle (e.g., comprising a macromolecule structure) comprises a surface charge of at most about -1 mV (e.g., -2 mV, -3 mV, -5 mV, -6 mV, -8 mV, -9 mV, or at most -10 mV).
  • -1 mV e.g., -2 mV, -3 mV, -5 mV, -6 mV, -8 mV, -9 mV, or at most -10 mV.
  • the first particle (e.g., comprising a macromolecule structure) comprises a surface charge of at least -15 mV (e.g., -12 mV, -10 mV, -8 mV, -6 mV, -5 mV, -4 mV, -3 mV, -2 mV, or -1 mV).
  • -15 mV e.g., -12 mV, -10 mV, -8 mV, -6 mV, -5 mV, -4 mV, -3 mV, -2 mV, or -1 mV.
  • the first particle (e.g., comprising a macromolecule structure) comprises a zeta potential of about 0 mV, -1 mV, -2 mV, -3 mV, -4 mV, -5 mV, -6 mV, -7 mV, -8 mV, -9 mV, or -10 mV.
  • the first particle (e.g., comprising a macromolecule structure) comprises a zeta potential of about -10 mV, -11 mV, -12 mV, -13 mV, -14 mV, -15 mV, -16 mV, -17 mV, -18 mV, -19 mV, or -20 mV.
  • a negative surface charge is characterized by a zeta potential of from about 0 mV to about -80 mV.
  • a negative surface charge is characterized by a zeta potential of from about 0 mV to about -60 mV.
  • a negative surface charge is characterized by a zeta potential of from about -5 mV to about -60 mV (e.g., about -10 mV to about -60 mV, about -20 mV to about -60 mV, about -30 mV to about -60 mV, or about -40 mV to about -60 mV).
  • a negative surface charge is characterized by a zeta potential of at least -80 mV (e.g., at least -70 mV, -60 mV, -50 mV, -40 mV, -30 mV, -20 mV, -10 mV, or at least -5 mV).
  • a negative surface charge is characterized by a zeta potential of at most 0 mV (e.g., at most -5 mV, -10 mV, -20 mV, -30 mV, -40 mV, -50 mV, or -60 mV). In some embodiments, a negative surface charge is characterized by a zeta potential of about -3 mV, -8 mV, --12 mV, -20 mV, -25 mV, -30 mV, -40 mV, -45 mV, -50 mV, or -55 mV.
  • the second particle (e.g., comprising a macromolecule structure) comprises a surface charge (e.g., characterized by a zeta potential) of from about -15 mV to about -60 mV.
  • the second particle (e.g., comprising a macromolecule structure) comprises a surface charge of about 0 mV to about -60 mV.
  • the second particle (e.g., comprising a macromolecule structure) comprises a surface charge about -35 mV to about -60 mV.
  • the second particle (e.g., comprising a macromolecule structure) comprises a surface charge about -40 mV to about -50 mV.
  • the second particle (e.g., comprising a macromolecule structure) WSGR Docket No.53344-792.601 comprises a surface charge of about -36 mV (e.g., -38 mV, -40 mV, -42 mV, -44 mV, -46 mV, - 48 mV, -50 mV, -52 mV, -54 mV, -56 mV, -58 mV, or -60 mV).
  • -36 mV e.g., -38 mV, -40 mV, -42 mV, -44 mV, -46 mV, - 48 mV, -50 mV, -52 mV, -54 mV, -56 mV, -58 mV, or -60 mV.
  • the first particle has a surface charge characterized by a zeta potential of from about -10 mV to -20 mV. In some embodiments, the first particle has a surface charge characterized by a zeta potential of -13 mV.
  • the second particle e.g., comprising a macromolecule structure
  • the second particle (e.g., comprising a macromolecule structure) has a surface charge characterized by a zeta potential of from about – 40 mV to about -60 mV. In some embodiments, the second particle (e.g., comprising a macromolecule structure) has a surface charge characterized by a zeta potential of from about -40 mV to -50 mV. In some embodiments, the second particle (e.g., comprising a macromolecule structure) has a surface charge characterized by a zeta potential of about -42 mV.
  • a surface charge of the second particle is characterized by a zeta potential of at least 4-fold more negative than the surface charge of the first particle (e.g., comprising a macromolecule structure).
  • a surface charge of the second particle is characterized by a zeta potential of at least 8-fold more negative than the surface charge of the first particle (e.g., comprising a macromolecule structure).
  • a surface charge of the second particle is characterized by a zeta potential of about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13- fold, 14-fold, or 15-fold more negative than the surface charge of the first particle (e.g., comprising a macromolecule structure).
  • the compositions herein comprise the multiple different particles (e.g., comprising macromolecule structures) in varying relative amounts.
  • the compositions comprise the first particle (e.g., comprising a macromolecule structure) and the second particle (e.g., comprising a macromolecule structure) in varying relative amounts.
  • the compositions comprise the first particle (e.g., comprising a macromolecule WSGR Docket No.53344-792.601 structure) and second particle (e.g., comprising a macromolecule structure) at a ratio of about 10:1 to about 1:10.
  • the compositions comprise the first particle (e.g., comprising a macromolecule structure) and second particle (e.g., comprising a macromolecule structure) at a ratio of about 5:1 to about 1:5.
  • the compositions comprise the first particle (e.g., comprising a macromolecule structure) and the second particle (e.g., comprising a macromolecule structure) at a ratio of about 1:1 to about 1:10. In some embodiments, the compositions comprise the first particle (e.g., comprising a macromolecule structure) and the second particle (e.g., comprising a macromolecule structure) at a ratio of 1:1 to about 1:4 (e.g., 1:2, 1:2.5, 1:3, 1:3.5, or 1:3.75).
  • the compositions comprise the first particle (e.g., comprising a macromolecule structure) and the second particle (e.g., comprising a macromolecule structure) at a ratio of about 1:2, 1:1, 1:2, 1:2.5, 1:3, 1:3.5:, 1:3.75, 1:4, 1:4.25, 1:4.5, 1:5, 1:6, 1:7, 1:8, 1:9, or about 1:10.
  • the compositions comprise the first particle (e.g., comprising a macromolecule structure) and the second particle (e.g., comprising a macromolecule structure) at a ratio of about 1:4.
  • the ratio is a molar ratio.
  • the ratio is a mass ratio.
  • the ratio is a surface area ratio.
  • the particle e.g., comprising a macromolecule structure
  • the particle provided herein comprise a surface and a polymer or co-polymer.
  • the polymer or co-polymer is covalently attached to the surface.
  • the polymer or co-polymer is non-covalently attached to the surface.
  • the co-polymer is a random co-polymer.
  • the co-polymers herein comprise 2, 3, 4, 5, 6, or more unique monomers.
  • at least a portion of the monomers within the co- polymers act as cross-linkers.
  • the surface comprises a base polymer.
  • the particles provided herein comprise macromolecule structures comprising a polymer comprising one or more units represented by Formula (A): Formula (A) WSGR Docket No.53344-792.601
  • R is hydrogen or .
  • R is hydrogen.
  • R is .
  • when R is hydroxyl the structure of Formula (A) is represented by: .
  • R 1 is hydrogen or hydroxyl.
  • R 1 is hydrogen.
  • R 1 is hydroxyl.
  • R 3 is each independently hydrogen or C 1 -C 8 alkyl optionally substituted with one or more oxo, hydroxyl, C1-C8 alkyl, C1-C8 alkenyl, and C1-C8 alkynyl.
  • R3 is hydrogen.
  • R3 is optionally substituted C 1 -C 8 alkyl.
  • R 3 is C 1 -C 8 alkyl optionally substituted with C 1 -C 8 alkenyl, oxo(s), (in some instances two oxos), and hydroxyl.
  • R2 is , wherein p is an integer from 1 to 6.
  • R 1 and R 2 are taken together to form C2-C6 heterocyloalkyl. In some embodiments, R 1 and R 2 are taken together, forming the structure: [0090] In some embodiments, R 4 is hydrogen or C 1 -C 6 alkyl. In some embodiments, R 4 is hydrogen. In other embodiments, R 4 is C1-C6 alkyl. In some embodiments, R 4 is C1 alkyl.
  • the polymer comprises two or more units represented by Formula (A), wherein the two or more units have a different structure.
  • the two or more units comprises a first unit represented by Formula (A), wherein R is and R 2 .
  • q is 1 for the first unit.
  • the two or more units comprises a second unit represented by Formula (A) having the structure of .
  • the two or more units comprises a second unit represented by Formula (A), wherein R 1 and R 2 are taken together having the structure of .
  • the two or more units comprises a second unit represented by Formula (A), wherein R is and R 2 .
  • q is 1 for the second unit.
  • the second unit is a crosslinking unit.
  • the crosslinking unit is obtained from a monomer having two or more vinyl groups.
  • the crosslinking unit may obtained from divinyl benzene, polyethylene glycol dimethacrylate, or ethylene glycol dimethacrylate.
  • compositions comprising a macromolecular structure comprising two or more units represented by Formula (A), as described hereinabove, and Formula (B): WSGR Docket No.53344-792.601 [0093]
  • R is hydrogen or .
  • R is hydrogen.
  • R is .
  • R 2 is optionally substituted C1-C8 diamine. In some embodiments, R 2 is C 1 -C 8 diamine, N-substituted with one or more R 3 . In some embodiments, R 2 is C 2 diamine, N-substituted with one or more R 3 . [0098] In some embodiments, R 3 is each independently hydrogen or C1-C8 alkyl optionally substituted with one or more oxo, hydroxyl, C1-C8 alkyl, C1-C12 alkenyl, and C1-C8 alkynyl.
  • R 3 is each independently hydrogen or C 1 -C 8 alkyl optionally substituted with one or more oxo, hydroxyl, C1-C8 alkyl, C1-C8 alkenyl, and C1-C8 alkynyl. In some embodiments, R 3 is hydrogen. In some embodiments, R 3 is optionally substituted C1-C8 alkyl. In some embodiments, R 3 is C 1 -C 8 alkyl optionally substituted with C 1 -C 8 alkenyl, (e.g., 2) oxo, and hydroxyl.
  • R 3 is C 1 -C 8 alkyl optionally substituted with C 1 -C 12 alkenyl, (e.g., 2) oxo, and hydroxyl.
  • R2 is , wherein p is an integer from 1 to 6. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. [00100] In some embodiments, R 1 and R 2 are taken together to form C2-C6 heterocyloalkyl.
  • the composition comprises one or more units (e.g., of Formula (A)) represented by Formula (C): Formula (C) [00105]
  • R 3 is each independently hydrogen or C 1 -C 8 alkyl optionally substituted with one or more oxo, hydroxyl, C1-C8 alkyl, C1-C8 alkenyl, and C1-C8 alkynyl.
  • R 3 is hydrogen.
  • R 3 is optionally substituted C1-C8 alkyl.
  • R 3 is C 1 -C 8 alkyl optionally substituted with C 1 -C 8 alkenyl, (e.g., 2) oxo, and hydroxyl. In some embodiments, one hydrogen. In some embodiments, two one of R 3 is hydrogen. [00106] In some embodiments, the composition comprises one or more units (e.g., of Formula (A)) represented by Formula (A-A): Formula (A-A) [00107] In some embodiments, R 5 is C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl. In some embodiments, R 5 is C1-C10 alkyl. In some embodiments, R 5 is C1-C10 alkenyl.
  • the composition comprises one or more units (e.g., of Formula (A)) represented by Formula (A-C): Formula (A-C) [00112]
  • R 5 is C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl. In some embodiments, R 5 is C1-C10 alkyl. In some embodiments, R 5 is C1-C10 alkenyl. In some embodiments, R 5 is C 1 -C 10 alkynyl. In some embodiments, R 5 is C 8 alkenyl. In some embodiments, R 5 is . In some embodiments, R 5 is .
  • the structure of Formula (B) is: [00116]
  • the structure of Formula (B), such as when Formula (B) acts as a crosslinking monomer is: [00117]
  • the particles comprise a macromolecule structure comprising a co-polymer of the structure (A1): WSGR Docket No.53344-792.601
  • C5H11 as used herein refers to n-C5H11.
  • C 5 H 11 refers to .
  • C 5 H 11 is .
  • the particle comprises a macromolecule structure further comprising methacrylic acid as a monomer unit.
  • the second particle comprises a macromolecule structure comprising the co-polymer of structure (A1). [00122] In some embodiments, the second particle comprises a macromolecule structure comprising a co-polymer comprising any number of units represented in Table 1. WSGR Docket No.53344-792.601 TABLE 1 WSGR Docket No.53344-792.601 [00123] In some embodiments, C 5 H 11 as used herein refers to n-C 5 H 11 . In some embodiments, C5H11 refers [00124] In some embodiments, the copolymer comprises monomer 6 and monomer 3. In some embodiments, the copolymer comprises monomer 6, monomer 3, and monomer 7.
  • the copolymer comprises monomer 6, monomer 3, and monomer 8. In some embodiments, the copolymer comprises monomer 6, monomer 3, and monomer 9. In some embodiments, the copolymer comprises monomer 6, monomer 3, and monomer 5. In some embodiments, the copolymer comprises monomer 6, monomer 7, and monomer 8. [00125] Provided herein, in some embodiments, is a particle comprising a macromolecule structure comprising a copolymer comprising two or more units represented by Formula (D) and Formula (E): WSGR Docket No.53344-792.601 [00126] In some embodiments, the particle comprises a macromolecule structure comprising a unit represented by Formula (D).
  • the particle comprises a macromolecule structure comprising a unit represented by Formula (E).
  • R 4 is each independently hydrogen or C1-C6 alkyl. In some embodiments, R 4 is hydrogen. In some embodiments, R 4 is C1-C6 alkyl. In some embodiments, R 4 is C1-C3 alkyl. In some embodiments, R 4 is C1 alkyl. [00128] In some embodiments, R 6 is C 1 -C 6 alkyl optionally substituted with R 7 . In some embodiments, R 6 is hydrogen or (CH2)pOR 7 . In some embodiments, R 6 is hydrogen. [00129] In some embodiments, R7 is hydrogen or C1-C6 alkyl.
  • p is 5. In some embodiments, p is 6. [00132] In some embodiments, is a single bond or a double bond. In some embodiments, is a single bond. In some embodiments, is a double bond. [00133] In some embodiments * is an attachment point to another unit of Formula (D) or Formula (E), such as when is a single bond. When * is an attachment point to another unit of Formula (D), the unit of Formula (B) acts as a crosslinking monomer.
  • the macromolecule structure comprises one or more units (e.g., of Formula (E)) represented by the structure of Formula (E-A): WSGR Docket No.53344-792.601 Formula (E-A) [00135]
  • R 4 is each independently hydrogen or C 1 - C6 alkyl. In some embodiments, R 4 is hydrogen. In some embodiments, R 4 is C1-C6 alkyl. In some embodiments, R 4 is C 1 -C 3 alkyl. In some embodiments, R 4 is C 1 alkyl.
  • R 7 is hydrogen or C 1 -C 6 alkyl. In some embodiments, R 7 is hydrogen.
  • R 7 is C1-C6 alkyl. In some embodiments, R 7 is C1-C3 alkyl. In some embodiments, R 7 is C1 alkyl. [00137] In some embodiments, p is an integer from 1 to 6. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5. In some embodiments, p is 6. [00138] In some embodiments, the particle comprises a macromolecule structure comprising one or more units (e.g., of Formula (E)) represented by the structure: .
  • the particle comprises a macromolecule structure comprising one or more units (e.g., of Formula (E)) represented by the structure: .
  • the particle comprises a macromolecule structure comprising one or more units (e.g., of Formula (D)) represented by the structure: WSGR Docket No.53344-792.601
  • the unit of Formula (B) acts as a crosslinking monomer.
  • the particle comprises a macromolecule structure comprising three or more units (e.g., of Formula (E)) represented by: [00144]
  • the structure of Formula (D), such as when Formula (D) acts as a crosslinking monomer is: .
  • the macromolecule structure comprises a macromolecule structure comprising a co-polymer of the structure (A2): .
  • the particle comprises a macromolecule structure comprising a co-polymer of the structure (A2’): WSGR Docket No.53344-792.601 [00147]
  • the monomers represented by n and m are randomly distributed throughout the polymer.
  • the monomers represented by n, m, and p are randomly distributed throughout the polymer.
  • the first particle comprises a macromolecule structure comprising the co-polymer of structure (A2).
  • the first particle comprises a macromolecule structure comprising the co-polymer of structure (A2’).
  • the first particle comprises a macromolecule structure further comprising methacrylic acid as a monomer unit.
  • the amount of methacrylic acid monomer units may be, in some embodiments, no more than about 10%, no more than about 5%, or no more than about 1% by weight or number of monomer units.
  • the amount of methacrylic acid monomer units may be, in some embodiments, at least 0.1%, at least 0.5%, at least 1% or at least 2% by weight or number of monomer units.
  • the copolymer may comprise monomer 14 in an amount of less than 10% (e.g., less than 8%, 6%, or 5%) by weight. In some embodiments, the copolymer comprises monomer 14 in an amount of less than 8% by weight. In some embodiments, the copolymer comprises monomer 14 in an amount of less than 6%. In some embodiments, the copolymer comprises monomer 14 in an amount of less than 5% by weight. In some embodiments, the copolymer comprises monomer 14 in an amount of less than 4% by weight. In some embodiments, the copolymer comprises monomer 14 in an amount of at least 2% by weight. In some embodiments, the copolymer comprises monomer 14 in an amount of at least 3% by weight.
  • the polymers or copolymers comprise a molecular weight of at least 0.1 kDa (e.g., WSGR Docket No.53344-792.601 at least 1 kDa, 5 kDa, 10 kDa, 20 kDa, 25 kDa, 50 kDa, 100 kDa, 250 kDa, or at least 500 kDa).
  • a molecular weight of at least 0.1 kDa e.g., WSGR Docket No.53344-792.601 at least 1 kDa, 5 kDa, 10 kDa, 20 kDa, 25 kDa, 50 kDa, 100 kDa, 250 kDa, or at least 500 kDa.
  • the polymers or copolymers comprise a molecular weight of at most 1000 kDa (e.g., at most 900 kDa, 800 kDa, 750 kDa, 600 kDa, 500 kDa, 250 kDa, 100 kDa, 75 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa, 20 kDa, 15 kDa, or at most 10 kDa).
  • kDa e.g., at most 900 kDa, 800 kDa, 750 kDa, 600 kDa, 500 kDa, 250 kDa, 100 kDa, 75 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa, 20 kDa, 15 kDa, or at most 10 kDa).
  • the polymer or copolymer comprises a molecular weight of about 0.1 kDa to about 500 kDa, about 0.5 kDa to about 250 kDa, about 0.5 kDa to about 100 kDa, about 0.5 kDa to about 70 kDa, 0.5 kDa to about 10 kDa, 0.5 kDa to about 15 kDa, or about 1 kDa to about 25 kDa.
  • “molecular weight” may refer to number average molecular weight or weight average molecular weight.
  • the polymers or copolymers provided herein may comprise any suitable number of recurring units.
  • the polymer or copolymer comprises from about 1 to about 250 (e.g., about 1 to about 500, about 1 to about 1,000, about 100 to about 1,000, about 10 to about 500, about 25 to about 750, or about 100 to about 500) recurring units.
  • the particles (e.g., comprising macromolecule structures) provided herein comprise a surface.
  • the first particle (e.g., comprising a macromolecule structure) comprises a surface.
  • the second particle e.g., comprising a macromolecule structure
  • the surface of the first particle (e.g., comprising a macromolecule structure) and the second particle (e.g., comprising a macromolecule structure) are the same. In some embodiments, the surface of the first particle (e.g., comprising a macromolecule structure) and the second particle (e.g., comprising a macromolecule structure) are different.
  • the surfaces herein comprise a particle. In some embodiments, the particles comprise a surface. In some embodiments, the particle is a microparticle. In some embodiments, the particle is a nanoparticle. [00155] In some embodiments, the particle is magnetic, for instance comprising any magnetic material suitable according to one of skill in the art.
  • the particle is paramagnetic, or comprises a paramagnetic material. In some embodiments, the particle is superparamagnetic or comprises a superparamagnetic material. WSGR Docket No.53344-792.601 [00156] In some embodiments, the particle comprises a metal material, such as but limited to a metal chalcogenide (e.g., metal oxide), metal halide.
  • a metal chalcogenide e.g., metal oxide
  • metal halide e.g., metal oxide
  • the particle comprises a core-shell structure.
  • the core of the core-shell structure comprises a paramagnetic material.
  • the particle comprises an iron oxide core.
  • the iron oxide core comprises magnetite.
  • the iron oxide core comprises maghemite.
  • the iron oxide core is functionalized with linking (e.g., tethering) moieties and/or polymers or copolymers provided elsewhere herein (e.g., bound covalently or non- covalently).
  • the particle comprises a silica shell.
  • the silica shell is functionalized with linking (e.g., tethering) moieties and/or polymers or copolymers provided elsewhere herein (e.g., bound covalently or non-covalently).
  • the particle comprises iron oxide crystals.
  • the particle comprises polystyrene.
  • the particle comprises iron oxide crystals embedded in a polystyrene core.
  • the particles (e.g., comprising macromolecule structures) (e.g., first particle (e.g., comprising a macromolecule structure)) provided herein comprise the structure: [00159]
  • the surface (e.g., of the particle) is at least partially coated with silica.
  • the particles (e.g., comprising macromolecule structures) (e.g., first particle (e.g., comprising a macromolecule structure)) provided herein comprises the structure: [00160]
  • L is a linker.
  • A is a polymer or copolymer comprising (e.g., randomly distributed) monomers of Formulas (D), (E), (E-A), or represented in Table 2.
  • A is a copolymer comprising randomly distributed monomers of Formulas (D), (E), (E-A), or represented in Table 2.
  • the particles e.g., comprising macromolecule structures
  • second particle e.g., comprising a macromolecule structure
  • the surface e.g., of the particle
  • compositions comprising a combination of: [00169] In some embodiments, L, A, B, and are described elsewhere herein.
  • the linkers provided herein comprise optionally substituted alkylene (e.g., C 1 -C 20 alkylene), optionally substituted heteroalkylene (e.g., C 1 -C 20 heteroalkylene), or optionally substituted aralkylene (e.g., C 1 -C 20 aralkylene).
  • the linker comprises optionally substituted C1-C20 alkylene (e.g., C1-C12, C1-C10, or C 1 -C 6 ).
  • the linker comprises C 1 -C 20 heteroalkylene. In some embodiments, the linker comprises C 1 -C 20 aralkylene. [00171] In some embodiments, provided herein are compositions comprising a combination of: WSGR Docket No.53344-792.601 [00172] In some embodiments, provided herein are compositions comprising a combination of: [00173] In some embodiments, provided herein are compositions comprising a combination of: [00174] In some embodiments, provided herein are compositions comprising a combination of: [00175] In some embodiments, L is a linker.
  • compositions comprising a (e.g., first) particle (e.g., comprising a macromolecule structure) comprising one or more units represented by Formulas (A)-(C), (A-A), (A-B), (A-C), (A-D), or represented in Table 1, attached (e.g., covalently) to a surface described elsewhere herein.
  • a particle e.g., comprising a macromolecule structure
  • Formulas A)-(C), (A-A), (A-B), (A-C), (A-D), or represented in Table 1, attached (e.g., covalently) to a surface described elsewhere herein.
  • compositions comprising a (e.g., second) particle (e.g., comprising a macromolecule structure) comprising one or more units represented by Formulas (D), (E), (E-A), or represented in Table 2, attached (e.g., covalently) to a surface described elsewhere herein.
  • compositions comprising a (e.g., first) particle (e.g., comprising a macromolecule structure) comprising one or more units represented by Formulas (A)-(C), (A-A), (A-B), (A-C), (A-D), or represented in Table 1, attached (e.g., covalently) to a surface described elsewhere herein, and a (e.g., second) particle (e.g., comprising a macromolecule structure) comprising one or more units represented by Formulas (D), (E), (E-A), or represented in Table 2, attached (e.g., covalently) to a surface described elsewhere herein.
  • a (e.g., first) particle e.g., comprising a macromolecule structure) comprising one or more units represented by Formulas (A)-(C), (A-A), (A-B), (A-C), (A-D), or represented in Table 1
  • a (e.g., second) particle e.g., comprising
  • the particles e.g., comprising macromolecule structures
  • the particle size can be measured by dynamic light scattering (DLS) as an indirect measure of size.
  • the DLS measurement can be an ‘intensity-weighted’ average, which means the size distribution that the mean is calculated from can be weighted by the sixth power of radius. This can be referred to herein as ‘z-average’ or ‘intensity-mean’.
  • the particles (e.g., comprising the macromolecule structures) provided herein comprise a diameter (e.g., particle size) of from about 200 nm to about 1000 nm. In some embodiments, the particles (e.g., comprising the macromolecule structures) provided herein comprise a diameter (e.g., particle size) of from about 300 nm to about 600 nm.
  • the particles comprise a diameter of from about 200 nm to about 800 nm, from about 200 nm to about 600 nm, about 300 nm to about 1000 nm, about 300 nm to about 700 nm, or about 400 nm to about 500 nm.
  • the particles (e.g., comprising the macromolecule structures) provided herein comprise a diameter of from about 400 nm to about 500 nm.
  • the particles (e.g., comprising the macromolecule structures) provided herein comprise a diameter of from about 410 nm to about 460 nm.
  • the particles provided herein have a diameter of about 350 nm to about 460 nm. In some embodiments, the particles provided herein have a diameter of about 370 nm to about 445 nm. In some embodiments, the particles (e.g., WSGR Docket No.53344-792.601 comprising the macromolecule structures) comprise a diameter of at least 100 nm (e.g., at least 200 nm, 300 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 500 nm, 600 nm, 700 nm, 750 nm, 800 nm, 900 nm, or at least 1000 nm).
  • nm e.g., at least 200 nm, 300 nm, 400 nm, 410 nm, 420 nm, 430 nm
  • the particles herein comprise a diameter of about 410 nm, 411 nm, 412 nm, 413 nm, 414 nm, 415 nm, 416 nm, 417 nm, 418 nm, 419 nm, 420 nm, 421 nm, 422 nm, 423 nm, 424 nm, 425 nm, 426 nm, 427 nm, 428 nm, 429 nm, 430 nm, 431 nm, 432 nm, 433 nm, 434 nm, 435 nm, 436 nm, 437 nm, 438 nm, 439 nm, 440 nm, 441 nm, 442 nm, 443 nm, 444 nm, 445 nm, 446 nm, 447 nm,
  • the particles described herein are freeze dried. In some instances, the particles are freeze dried before being supplied in the kits described herein.
  • the particles comprised in the kits herein may be freeze dried particles which are configured for resuspension by a user.
  • the resuspended particles, such as after freeze drying or lyophilization have a diameter that is about 20 nm greater than before freeze drying or lyophilization.
  • the resuspended particles have an average diameter of about 390 nm to about 470 nm.
  • the resuspended first particles have an average diameter of about 390 nm to about 470 nm.
  • the resuspended second particles have an average diameter of about 390 nm to about 470 nm.
  • the diameter e.g., as measured by DLS
  • the particles (e.g., as measured by DLS) is the average diameter.
  • the particles (e.g., comprising the macromolecule structures) provided herein comprise homogenous or heterogenous size distribution.
  • the particles (e.g., comprising the macromolecule structures) provided herein comprise a homogenous size distribution.
  • polydispersity index (PDI) is used as a measure WSGR Docket No.53344-792.601 for size distribution. PDI may be measured by techniques such as dynamic light scattering (DLS).
  • the particles (e.g., comprising the macromolecule structures) provided herein comprise a PDI of at least 0.01 (e.g., at least 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, at least 0.2, or at least 0.5). In some embodiments, the particles (e.g., comprising the macromolecule structures) provided herein comprise a PDI of at most 0.5 (e.g., at most 0.4, 0.35, 0.3, 0.25, 0.2, 0.175, 0.15, 0.125, or 0.1).
  • the particles (e.g., comprising the macromolecule structures) provided herein comprise a PDI of from 0.05 to 0.2. In some embodiments, the particles (e.g., comprising the macromolecule structures) provided herein comprise a PDI of from about 0.1 to about 0.2. In some embodiments, the particles (e.g., comprising the macromolecule structures) comprise a PDI of about 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.25, or 0.25. In some embodiments, the first macromolecule structure comprises a PDI of about 0.12, such as described in Example 1.
  • the second macromolecule structure comprises a PDI of about 0.14, such as described in Example 2. In some embodiments, the particles have a PDI of less than 0.2.
  • the particles (e.g., comprising the macromolecule structures) provided herein comprise varying amounts of the polymer or co-polymer, such as any amount suitable according to one of skill in the art. In some embodiments, the particles (e.g., comprising the macromolecule structures) comprise at least 1% w/w of the polymer or co-polymer.
  • the particles comprise at least 2.5% w/w (e.g., 3% w/w, 4% w/w, 5% w/w, 6% w/w, 8% w/w, 10% w/w, 12% w/w, 15% w/w, 20% w/w, or at least 25% w/w) of the polymer or copolymer.
  • the macromolecule structure comprises about 1% w/w, 2% w/w, 3% w/w, 4% w/w, 5% w/w, 6% w/w, 7% w/w, 8% w/w, 9% w/w, 10% w/w, 11% w/w, 12% w/w, 13% w/w, 14% w/w, 15% w/w, 16% w/w, 17% w/w, 18% w/w, 19% w/w, or 20% w/w.
  • the macromolecule structure comprises about 17% w/w of the polymer or copolymer.
  • the first monomer comprises the structure: WSGR Docket No.53344-792.601 [00206] In some embodiments, q is an integer from 1 to 6. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5. In some embodiments, q is 6. [00207] In some embodiments, the first monomer is glycidyl methacrylate.
  • the second monomer is ethyleneglycol dimethacrylate. In some embodiments, the second monomer comprises a crosslinking monomer. In some embodiments, the crosslinking monomer is a diene. In some embodiments, the cross-linking monomer comprises the structure: [00211] In some embodiments, R 1 is hydrogen or C1-C6 alkyl. In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is C1-C6 alkyl. In some embodiments, R 2 is hydrogen or C1- C 6 alkyl. In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is C 1 -C 6 alkyl.
  • R 3 is hydrogen or C1-C6 alkyl. In some embodiments, R 3 is hydrogen. In some embodiments, R 3 is C1-C6 alkyl. In some embodiments, R 3 is methyl. In some embodiments, R 1’ is hydrogen or C 1 -C 6 alkyl. In some embodiments, R 1’ is hydrogen. In some embodiments, R 1’ is C 1 -C 6 alkyl. In some embodiments, R 2’ is hydrogen or C 1 -C 6 alkyl. In some embodiments, R 2’ is WSGR Docket No.53344-792.601 hydrogen. In some embodiments, R 2’ is C1-C6 alkyl.
  • R 3’ is hydrogen or C1- C6 alkyl. In some embodiments, R 3’ is hydrogen. In some embodiments, R 3’ is C1-C6 alkyl. In some embodiments, R 3’ is methyl.
  • the first and/or second monomers comprise one or more impurities. In some instances, the impurities are polymerized and are comprised within the macromolecule chain. In some embodiments, impurities include impurities within the first monomer and second monomer or non-fully reacted monomers within the polymers or copolymers formed after functionalization.
  • the methods comprise contacting a surface and the mixture of monomers (e.g., comprising a first monomer and a second monomer), thereby producing a reaction mixture.
  • the surface is a surface as provided elsewhere herein.
  • the surface comprises polymerizable olefins that may polymerize with the mixture of monomers, thus covalently attaching the monomers, and the resulting polymer or copolymer, to the surface.
  • the surface is reacted with one or more monomers to create a polymer coated surface.
  • the polymer coated surface may undergo polymerization with the mixture of monomers provided herein.
  • the radical initiator comprises azobisisobutyronitrile (AIBN).
  • the method comprises contacting the macromolecule immobilized to the surface and an amine, thereby producing an aminated macromolecule.
  • the amine is an alkylamine (e.g., comprising from 1 to 6 carbons (e.g., a C1-C6 alkylamine).
  • the amine comprises the structure: [00218] In some embodiments, the amine is an alkylenediamine. In some embodiments, the amine is ethylenediamine.
  • any one of the steps in a method provided herein may be completed under inert conditions.
  • the inert conditions comprise nitrogen gas (N 2 ).
  • any one of the steps in a method provided herein may comprise heating.
  • the method comprises heating to multiple (e.g., 2 or more, or 3 or more) different temperatures during the course of the method.
  • the method comprises heating to at least 40°C (e.g., 50°C, 60°C, 70°C, 80°C, 90°C, or at least 100°C).
  • the alkylene diamine is ethylene glycol.
  • the optionally substituted succinic anhydride is an alkenylsuccinic anhydride.
  • the optionally substituted succinic anhydride is an C 4 -C 10 alkenylsuccinic anhydride.
  • the optionally substituted succinic anhydride is octenylsuccinic anhydride.
  • provided herein are compositions obtained by a method comprising polymerizing 2-hydroxyethyl methacrylate, ethylene glycol dimethacrylate, and methacrylic acid in the presence of vinyl-functionalized magnetic particles.
  • the particles e.g., comprising the macromolecule structures
  • the particles can be used in the methods disclosed in U.S. Patent Nos.10,866,242, 11,428,688, WSGR Docket No.53344-792.601 11,906,526, and PCT App. Nos. PCT/US2021/042254 and PCT/US2023/068457, each of which is incorporated by reference herein in its entirety.
  • a biological sample comprises plasma, serum, or blood. In some embodiments, a biological sample comprises blood. In some embodiments, a biological sample comprises plasma. In some embodiments, a biological sample comprises serum. In some embodiments, the biological sample is a biofluid. In some embodiments, the biological sample is a cell-free biofluid. [00234] In some embodiments, the method comprises contacting a biological sample comprising one or more biomolecules with one, two or more particles (e.g., comprising the macromolecule structures) (e.g., such as particles (e.g., comprising the macromolecule structures) provided elsewhere herein).
  • particles e.g., comprising the macromolecule structures
  • the first and second particles are contacted with the biological sample at a total concentration (e.g., of the first and second particle) of about 0.5 mg/mL, about 0.55 mg/mL, about 0.6 mg/mL, about 0.65 mg/mL, about 0.7 mg/mL, about 0.75 mg/mL, about 0.8 mg/mL, about 0.85 mg/mL, about 0.9 mg/mL, or about 0.95 mg/mL.
  • the first and second particles are contacted with the biological sample at a total concentration (e.g., of the first and second particle) of about 0.7 mg/mL.
  • the method comprises contacting the biological sample with the second particle, wherein the second particles are at a concentration of about 0.5 mg/mL, 0.51 mg/mL, 0.52 mg/mL, 0.53 mg/mL 0.54 mg/mL, 0.55 mg/mL, 0.56 mg/mL, 0.57 mg/mL, 0.58 mg/mL, 0.59 mg/mL, or 0.6 mg/mL.
  • the method comprises contacting the biological sample with the second particle, wherein the second particles are at a concentration of about 0.56 mg/mL.
  • the method comprises contacting the biological sample with the second particle, wherein the second particles are at a concentration of about 0.36 mg/mL.
  • the method comprises contacting a biological sample (e.g., plasma or serum) comprising one or more biomolecules (e.g., proteins) with the magnetic particles in any one of the compositions provided herein.
  • a biological sample e.g., plasma or serum
  • biomolecules e.g., proteins
  • a “biomolecule corona” refers to one or more biomolecules adsorbed to a surface of a macromolecule structure described herein.
  • multiple (e.g., a plurality) of biomolecules may be adsorbed to particles (e.g., comprising the macromolecule structures) provided herein.
  • a plurality of unique biomolecules may be adsorbed to a macromolecule structure.
  • digestion comprises the use of trypsin, lysin, serine protease, chymotrypsin, pepsin, thermolysin, proteinase K, or a combination thereof.
  • the digestion comprises trypsin.
  • the digestion comprises lysin.
  • the digestion comprises serine protease.
  • the digestion comprises chymotrypsin.
  • the digestion comprises pepsin.
  • the digestion comprises thermolysin.
  • the digestion comprises proteinase K.
  • the particle (e.g., third particle) is contacted with the digested biomolecules at a concentration of about 40 mg/mL to about 45 mg/mL. In some embodiments, the particle (e.g., third particle) is contacted with the digested biomolecules at a concentration of about 40 mg/mL, about 41 mg/mL, about 42 mg/mL, about 43 mg/mL, about 44 mg/mL, about 45 mg/mL, about 46 mg/mL. about 47 mg/mL, about 48 mg/mL, about 49 mg/mL, or about 50 mg/mL.
  • a buffer as provided herein comprises a pH of about 6.7. In some embodiments, a buffer as provided herein comprises a pH of about 6.6. In some embodiments, a buffer as provided herein comprises a pH of about 6.5. In some embodiments, a buffer may be any suitable buffer according to one of skill in the art. In some embodiments, the buffer comprises phosphate buffer, Tris, HEPES, MES, MOPS, TES, CAPS, Bicine, or Bis-Tris. In some embodiments, the buffering agent is CAPS. In some embodiments, the buffering agent is HEPES. In some embodiments, the buffering agent does not comprise a primary amine.
  • the organic solvent is N,N-dimethylacetamide.
  • the organic solvents provided herein comprise a combination of two or more organic solvents.
  • the organic solvent comprises an alcohol, acetonitrile, dichloromethane, dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethyl acetate, hexamethylphosphoramide (HMPA), or tetrahydrofuran.
  • the organic solvent comprises DMF.
  • the organic solvent comprises acetonitrile.
  • eluting comprises the use of an elution buffer, such as a buffer described elsewhere herein.
  • the methods provided herein are capable of isolating at least 100 (e.g., 250, 500, 750, 1,000, 1,500, 2,000, 2,500, or at least 3000 biomolecules). In some embodiments, the methods provided herein are capable of isolating at least about 1,000 biomolecules. In some embodiments, the methods provided herein are capable of isolating at most 20,000 biomolecules (e.g., at most 15,000, 12,500, 10,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, or at most 2,500) biomolecules.
  • the combination of a first macromolecule structure and a second macromolecule structure provides a synergistic relationship allowing for identification, isolation, purification, or quantification of a larger number of biomolecules.
  • the combination of the two particles e.g., comprising the macromolecule structures
  • the wash solution comprises an aqueous solution.
  • the wash solution comprises a buffer.
  • the washing may be performed after adsorbing the one or more biomolecules to the particles (e.g., comprising the macromolecule structures), and before eluting the one or more biomolecules.
  • the methods provided herein comprise purifying the isolated biomolecules (e.g., proteins), such as by solid phase extraction.
  • the solid phase extraction comprises use of a third particle, such as a third particle described elsewhere herein.
  • the isolating comprises contacting with the third particle described elsewhere herein.
  • the solid phase comprises a polyalkylene glycol (PAG) methacrylate, polyalkylene glycol (PAG) acrylate, polyalkylene glycol (PAG) methacrylamide, polyalkylene glycol (PAG) acrylamide, polyalkylene glycol (PAG) vinyl ether, and combinations thereof.
  • the solid phase comprises polyethylene glycol (PEG) methacrylate.
  • the methods provided herein comprise removing a surfactant from the biological sample.
  • the amount of surfactants in a composition comprising the population of biomolecules is greater than the amount of surfactants in a composition comprising the one or more isolated biomolecules by about 10 wt% to about 100 wt%, 10 wt% to about 90 wt%, 20 wt% to about 100 wt%, 30 wt% to about 100 wt%, 50 wt% to about 100 wt%, 50 wt% to about 80 wt%, or about 80 wt% to about 100 wt%.
  • Non-limiting examples of surfactants that may be greater include sodium lauryl sulfate, Triton X-100, TWEEN, NP-40, CHAPS, Octyl glucoside, and decyl maltoside.
  • the surfactant is sodium lauryl sulfate.
  • the surfactant is CHAPS.
  • the surfactant is a synthetic molecule. [00274]
  • the methods provided herein comprise removing a buffering agent from the biological sample.
  • the methods provided herein comprise removing at least 90% (e.g., at least 95%, at least 97.5%, at least 98%, at least 99%, at least 99.5%) of a buffering agent. In some embodiments, the methods provided herein comprise removing about 90% to about 99.9%, about 90% to about 99%, about 90% to about 98%, about 92% to about 97.5%, or about 93% to about 99% of a buffering agent. In some embodiments, the amount of buffering agent in the composition comprising the population of biomolecules is greater than the amount of buffering agent in a composition comprising the one or more isolated biomolecules.
  • the amount of buffering agent in a composition comprising the population of biomolecules is greater than the amount of buffering agent in a composition comprising the one or more isolated biomolecules by at most 100 wt% (e.g., 99 wt%, 95 wt%, 90 wt%, 80 wt%, 70 wt%, 60 wt%).
  • the buffering agent is CAPS. In some embodiments, the buffering agent is HEPES. WSGR Docket No.53344-792.601 [00275]
  • the methods provided herein comprise removing a chaotrope from the biological sample. In some embodiments, the methods provided herein comprise removing at least 90% (e.g., at least 95%, at least 97.5%, at least 98%, at least 99%, at least 99.5%) of a chaotrope. In some embodiments, the methods provided herein comprise removing about 90% to about 99.9%, about 90% to about 99%, about 90% to about 98%, about 92% to about 97.5%, or about 93% to about 99% of a chaotrope.
  • the methods provided herein comprise removing at least 90% (e.g., at least 95%, at least 97.5%, at least 98%, at least 99%, at least 99.5%) of a base. In some embodiments, the methods provided herein comprise removing about 90% to about 99.9%, about 90% to about 99%, about 90% to about 98%, about 92% to about 97.5%, or about 93% to about 99% of a base. In some embodiments, the amount of base in the composition comprising the population of biomolecules is greater than the amount of base in a composition comprising the one or more isolated biomolecules.
  • the amount of base in a composition comprising the population of biomolecules is greater than the amount of base in a composition comprising the one or more isolated biomolecules by at least 10 wt% (e.g., at least 20 wt%, 30 wt%, 50 wt%, 80 wt%, 90 wt%, or at least 100 wt%). In some embodiments, the amount of base in a composition comprising the population of biomolecules is greater than the amount of base in a composition comprising the one or more isolated biomolecules by at least 70 wt%.
  • the amount of base in a composition comprising the population of biomolecules is greater than the amount of base in a composition comprising the one or more isolated biomolecules by at most 100 wt% (e.g., 99 wt%, 95 wt%, 90 wt%, 80 wt%, 70 wt%, or at most 60 wt%).
  • the methods provided herein comprise providing one or more isolated biomolecules that are at least 75% (e.g., at least 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99%, 99.5%, or at least 99.9%) pure. In some embodiments, the methods provided herein comprise providing one or more isolated biomolecules that are at most 99.9% (e.g., at most 99%, 98%, 97%, WSGR Docket No.53344-792.601 96%, 95%, 94%, or at most 92%) pure.
  • the methods comprise assaying the purified isolated proteins (e.g., such as purified by solid phase extraction). In some embodiments, the methods comprise analyzing the isolated proteins using mass spectrometry. In some embodiments, such as when the method comprises digestion, the isolated proteins are or comprise peptides. [00280] In some embodiments, the method comprises contacting the biological sample with a composition provided herein to form at least two biomolecule corona. In some embodiments, the method comprises assaying the at least two biomolecule corona to detect one or more biomolecules in the biological sample.
  • the assaying detects at least 2% more unique biomolecules than a method comprising contacting the biological sample with a composition comprising the second particle in absence of the first particle. In some embodiments, the assaying detects at least 5% more unique biomolecules than a method comprising contacting the biological sample with a composition comprising the second particle in absence of the first particle. In some embodiments, the assaying detects at least 7% more unique biomolecules than a method comprising contacting the biological sample with a composition comprising the second particle in absence of the first particle. In some embodiments, the assaying detects at least 10% more unique biomolecules than a method comprising contacting the biological sample with a composition comprising the second particle in absence of the first particle.
  • the assaying detects at least 60% more unique biomolecules than a method comprising contacting the biological sample with a composition comprising the first particle in absence of the second particle. In some embodiments, the assaying detects at least 70% more unique biomolecules than a method comprising contacting the biological sample with a composition comprising the first particle in absence of the second particle. [00283] In some embodiments, the method does not comprise pre-selection of the detected biomolecules before assaying. [00284] In some embodiments, the methods herein comprise detecting or identifying the one or more (e.g., isolated biomolecules).
  • the (e.g., isolated biomolecules) biomolecules can be identified, measured, and quantified using mass spectrometry, high performance liquid chromatography, LC-MS, LC- MS/MS, Edman degradation, immunoaffinity techniques, and methods disclosed in EP3548652, WO2019083856, WO2019133892, each of which is incorporated herein by reference in its entirety.
  • the methods provided herein are capable of identifying at least 50 biomolecules. In some embodiments, the methods provided herein are capable of identifying at least 100 (e.g., 250, 500, 750, 1,000, 1,500, 2,000, 2,500, or at least 3000 biomolecules).
  • the methods provided herein are capable of identifying (e.g., isolated) biomolecules over a dynamic range of at least 2 (e.g., at least 3, 4, 5, 6, 7, 8, 9, or at least 10). In some embodiments, the methods herein are capable of identifying (e.g., isolated) biomolecules over a dynamic range of at least 7, at least 8, at least 9, or at least 10.
  • the uses and methods provided herein comprise using the structures of Formula (A-D) and Formula (A-A) as recurring units for binding proteins in a biological sample.
  • the uses and methods provided herein comprise using the structures of Formula (A-C) and Formula (A-B) as recurring units for binding proteins in a biological sample.
  • the uses and methods provided herein comprise the use of any of the compositions provided herein for binding proteins in a biological sample.
  • the system comprises a plurality of particles (e.g., comprising the macromolecule structures).
  • the system comprises at least two particles (e.g., comprising the macromolecule structures).
  • the at least two particles e.g., comprising the macromolecule structures
  • the at least two particles comprise a first macromolecule structure with a neutral to negative surface charge.
  • the at least two particles e.g., comprising the macromolecule structures
  • a first macromolecule structure e.g., such as WSGR Docket No.53344-792.601 a macromolecule structure provided elsewhere herein).
  • the at least two particles comprise a second macromolecule structure (e.g., such as a macromolecule structure provided elsewhere herein).
  • the system comprise any composition provided elsewhere herein.
  • the system is configured to perform any one of the methods provided elsewhere herein.
  • the system comprises a suspension solution.
  • the suspension solution is configured to suspend the at least two particles (e.g., comprising the macromolecule structures).
  • the suspension solution comprises a buffer.
  • the suspension solution comprises Tris, EDTA, CHAPS, or HEPES buffer.
  • the suspension solution comprises HEPES buffer.
  • the suspension solution comprises a buffer at a concentration of about 10 mM, 25 mM, 30 mM, 50 mM, 75 mM, 100 mM, 125 mM, 200 mM, 250 mM, or about 300 mM. In some embodiments, the suspension solution comprises the buffer at an amount of about 300 mM. As an example, a suspension solution may comprise 300 mM HEPES buffer. In another example, the suspension solution may be 10mM Tris HCl pH 7.4, 1 mM EDTA. [00302] In some embodiments, the system comprises a biological sample, such as a biological sample provided elsewhere herein. In some embodiments, the biological sample comprises a plurality of biomolecules.
  • the system comprises an automated system comprising a network of units with differentiated functions configured to isolate one or more biomolecules from the biological sample using the at least two particles (e.g., comprising the macromolecule structures).
  • the present disclosure provides an automated system comprising a network of units as described in U.S. Patent No. 11,630,112, which is incorporated herein by reference in its entirety.
  • the network of units may comprise differentiated functions in distinguishing states of a complex biological sample using two or more particles (e.g., comprising the macromolecule structures) having surfaces with different physicochemical properties.
  • the system comprises a first unit comprising a multichannel fluid transfer instrument for transferring fluids between units within the system.
  • the first unit comprises a degree of mobility that enables access to all other units within the system.
  • the first unit comprises a capacity to perform pipetting functions.
  • the system comprises a second unit comprising a support for storing a plurality of biological samples.
  • the second unit can facilitate a transfer of the sample for mass spectrometry to a mass spectrometry unit.
  • WSGR Docket No.53344-792.601 the second unit can facilitate a transfer of the sample for analysis, such as by any analytical technique described herein.
  • the system comprises a third unit comprising a support for an array plate possessing partitions that comprise the one or more macromolecule structure for binding of the one or more biomolecules with the two or more particles (e.g., comprising the macromolecule structures).
  • the support of the second and/or third unit comprises support for a single plate, a 6 well plate, a 12 well plate, a 96 well plate, or a rack of microtubes.
  • the second and/or unit comprises a thermal unit capable of modulating the temperature of said support and a sample.
  • the second and/or third unit comprises a rotational unit capable of physically agitating and/or mixing a sample.
  • the two or more particles having surfaces with different physicochemical properties for binding a population of analytes (e.g., biomolecules) within the biological sample are immobilized to a macromolecule WSGR Docket No.53344-792.601 structure within a partition of the sensory array.
  • the two or more particles comprise different physicochemical properties for binding a population of analytes (e.g., biomolecules) within the biological sample.
  • the system comprises a step wherein the sensor array plate is transferred to an additional seventh unit that comprises a magnetized support and a thermal unit capable of modulating the temperature of said support and a sample and incubated for an additional amount of time.
  • incubating the biological sample with at least two particles (e.g., comprising the macromolecule structures) contained within the partition of the sensor array plate comprises an incubation time of at least about 10 seconds (e.g., at least about 15 seconds, 20 seconds, 25 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 90 seconds, 2 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 10 minutes, 15 minutes 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, or at least about 24 hours).
  • incubating the biological sample with the at least two particles (e.g., comprising the macromolecule structures) contained within the partition of the substrate comprises an incubation temperature of at least 4°C (e.g., at least 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or at least 40°C). In some embodiments, the incubation temperature is at most 50°C (e.g., at most 45°C, 40°C, 38°C, 35°C, 30°C, 25°C, or 20°C). In some embodiments, the incubation temperature is from about 4°C to about 40°C.
  • the automated apparatus comprises a sample preparation unit; a substrate comprising a plurality of channels; a plurality of pipettes; a plurality of solutions; a plurality of particles (e.g., comprising the macromolecule structures) as provided WSGR Docket No.53344-792.601 elsewhere herein.
  • the automated apparatus comprises a sample preparation unit.
  • the automated apparatus comprises a substrate comprising a plurality of channels.
  • the automated apparatus comprises a plurality of pipettes.
  • the automated apparatus comprises a plurality of solutions.
  • the automated apparatus comprises a plurality of particles (e.g., comprising the macromolecule structures), such as described elsewhere herein.
  • the automated apparatus is configured to form a biomolecule corona and digest the biomolecule corona (or two or more biomolecule coronas).
  • the automated apparatus is configured for digestion (e.g., by enzymolysis), such as using any digestion agent provided herein (e.g., trypsin) of the biomolecule corona.
  • the automated apparatus is configured for BCA, gel, or trypsin digestion of the biomolecule corona.
  • the automated apparatus is enclosed.
  • the automated apparatus is sterilized before use.
  • the automated apparatus is configured to a mass spectrometer.
  • the automated apparatus is temperature controlled.
  • the automated apparatus comprises a substrate comprising a plurality of partitions, a first unit comprising the biological sample, and a loading unit that is movable across the substrate and is capable of transferring a volume (e.g., a volume of buffer) between different units of the apparatus.
  • the substrate is a multi-well plate.
  • the plurality of partitions comprises a plurality of sensor elements.
  • the plurality of sensor elements may comprise particles (e.g., comprising the macromolecule structures) as provided elsewhere herein.
  • a partition from among the plurality of partitions may comprise 4 to 10 types of sensor elements.
  • a partition from WSGR Docket No.53344-792.601 among the plurality of partitions may comprise 5 to 12 types of sensor elements.
  • a partition from among the plurality of partitions may comprise 6 to 15 types of sensor elements.
  • a partition from among the plurality of partitions may comprise 8 to 20 types of sensor elements.
  • a partition from among the plurality of partitions may comprise 2 types of sensor elements.
  • a partition from among the plurality of partitions may comprise at least 2 types of sensor elements.
  • a partition from among the plurality of partitions may comprise 3 types of sensor elements. [00322] Two or more partitions from among the plurality of partitions may comprise different quantities of sensor elements.
  • a substrate partition may comprise a solution comprising a high concentration of particles (e.g., comprising the macromolecule structures).
  • Partitions from among the plurality of partitions comprise different concentrations or amounts (e.g., by mass/molar amount per unit volume of sample) of sensor elements.
  • a partition from among the plurality of partitions may comprise from 1 pM to 100 nM of sensor elements.
  • a partition from among the plurality of partitions may comprise from 10 pM to 1 nM of sensor elements.
  • a partition from among the plurality of partitions may comprise from 100 pM to 10 nM of sensor elements.
  • the loading unit may be configured to move precise volumes (e.g., within 0.1%, 0.01%, 0.001% of the specified volume).
  • the loading unit may be configured to collect a volume from the substrate or a compartment or partition within the substrate and dispense the volume back into the substrate or compartment or partition within the substrate, or to dispense the volume or a portion of the volume into a different unit, compartment, or partition.
  • the loading unit is configured to move multiple volumes simultaneously, such as 2 to 400 separate volumes.
  • the loading unit may comprise a plurality of pipette tips. [00326]
  • the loading unit may be configured to move a volume of a liquid.
  • the volume may be at least 0.1 ⁇ L (e.g., at least 0.4 ⁇ L, 0.6 ⁇ L 0.8 ⁇ L, 1 ⁇ L, 5 ⁇ L, 10 ⁇ L, 25 ⁇ L, 100 ⁇ L, or at least 250 ⁇ L). In some embodiments, the volume may be at most 5 mL (e.g., at most 2 mL, 1 mL, 750 ⁇ L, 500 ⁇ L, 250 ⁇ L, 200 ⁇ L, 100 ⁇ L, or at most 50 ⁇ L).
  • the volume may be about 0.1 ⁇ l, 0.2 ⁇ l, 0.3 ⁇ l, 0.4 ⁇ l, 0.5 ⁇ l, 0.6 ⁇ l, 0.7 ⁇ l, 0.8 ⁇ l, 0.9 ⁇ l, 1 ⁇ l, 2 ⁇ l, 3 ⁇ l, 4 ⁇ l, 5 ⁇ l, 6 ⁇ l, 7 ⁇ l, 8 ⁇ l, 9 ⁇ l, 10 ⁇ l, 12 ⁇ l, 15 ⁇ l, 20 ⁇ l, 25 ⁇ l, 30 ⁇ l, 40 ⁇ l, 50 ⁇ l, 60 ⁇ l, 70 ⁇ l, 80 ⁇ l, 90 ⁇ l, 100 ⁇ l, 120 ⁇ l, 150 ⁇ l, 180 ⁇ l, 200 ⁇ l, 250 ⁇ l, 300 ⁇ l, 400 ⁇ l, 500 ⁇ l, 600 ⁇ l, 800 ⁇ l, 1 ml, or more than 1 ml.
  • the solution comprises a wash solution, a resuspension solution, a denaturing solution, a buffer, a reagent (e.g., a reducing reagent), or any combination thereof.
  • the solution comprises a biological sample.
  • the loading unit can be capable of partitioning a sample. In some embodiments, this comprises dividing a sample into a number of partitions. A sample can be divided into at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 500, or more partitions.
  • the volume of biological sample loaded into a partition may be about 0.1 ⁇ l, 0.2 ⁇ l, 0.3 ⁇ l, 0.4 ⁇ l, 0.5 ⁇ l, 0.6 ⁇ l, 0.7 ⁇ l, 0.8 ⁇ l, 0.9 ⁇ l, 1 ⁇ l, 2 ⁇ l, 3 ⁇ l, 4 ⁇ l, 5 ⁇ l, 6 ⁇ l, 7 ⁇ l, 8 ⁇ l, 9 ⁇ l, 10 ⁇ l, 12 ⁇ l, 15 ⁇ l, 20 ⁇ l, 25 ⁇ l, 30 ⁇ l, 40 ⁇ l, 50 ⁇ l, 60 ⁇ l, 70 ⁇ l, 80 ⁇ l, 90 ⁇ l, 100 ⁇ l, 120 ⁇ l, 150 ⁇ l, 180 ⁇ l, 200 ⁇ l, 250 ⁇ l, 300 ⁇ l, 400 ⁇ l, 500 ⁇ l, 600 ⁇ l, 800 ⁇ l, 1 ml, or more than 1 ml.
  • the automated apparatus may dilute a sample or sample partition by 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold or greater.
  • the automated apparatus may dilute a sample or sample partition by about 2-fold to about 5-fold.
  • the automated apparatus may perform different dilutions on two samples or sample partitions.
  • the system may perform different dilutions on each partition from among a plurality of partitions. For example, the system may perform different dilutions on each of the 96 sample partitions in a 96 well plate.
  • the different dilutions comprise different degrees of dilution (e.g., 2- fold vs. 4-fold). In some cases, the different dilutions comprise dilution with different solutions (e.g., different buffers). In some cases, two sample partitions may be made to differ in one or more chemical properties, such as pH, salinity, or viscosity.
  • the system may modify the chemical composition of a sample or sample partition. The system may modify or adjust the pH, salinity, osmolarity, dielectric constant, viscosity, buffer types, salt types, sugar types, detergent types, or any combination thereof for a sample or sample partition.
  • the automated apparatus may comprise a unit comprising a resuspension solution.
  • the loading unit may be capable of transferring a volume of the resuspension solution to a partition from among the plurality of partitions of the substrate. In some cases, this results in the dilution of a sample present within the partition and can further result in the desorption of a plurality of biomolecules from a biomolecule corona disposed on a sensor element within the partition.
  • the transfer of a volume of the resuspension solution into a partition may result in the desorption of 10% to 20% of the biomolecules from a biomolecule corona.
  • the transfer of a volume of the resuspension solution into a partition may result in the desorption of 20% to 30% of the biomolecules from a biomolecule corona.
  • the transfer of a volume of the resuspension solution into a partition may result in the desorption of 30% to 40% of the biomolecules from a biomolecule corona.
  • the transfer of a volume of the resuspension solution into a partition may result in the desorption of 40% to 50% of the biomolecules from a biomolecule corona.
  • the transfer of a volume of the resuspension solution into a partition may result in the desorption of 50% to 60% of the biomolecules from a biomolecule corona.
  • the transfer of a volume of the resuspension solution into a partition may result in the desorption of 60% to 70% of the biomolecules from a biomolecule corona.
  • the transfer of a volume of the resuspension solution into a partition may result in the desorption of 70% to 80% of the biomolecules from a biomolecule corona.
  • the transfer of a volume of the resuspension solution into a partition may result in the desorption of 80% to 90% of the biomolecules from a biomolecule corona.
  • the transfer of a volume of the resuspension solution into a partition may result in the desorption of more than 90% of the biomolecules from a biomolecule corona.
  • multiple rounds of desorption are performed. In each round, the supernatant comprising the desorbed biomolecules may be collected, analyzed, or discarded. The types and abundances of biomolecules in the supernatant may differ between desorption rounds.
  • the automated apparatus may perform one or more desorption and discard cycles (i.e., washes), followed by one or more desorption cycles comprising sample collection and/or analysis.
  • the resuspension solution may be tailored to optimize enrichment of particular biomarkers.
  • the resuspension solution may comprise a buffer, such as Tris-EDTA (TE), CHAPS, PBS, citrate, HEPES, MES, CHES, or another bio buffer.
  • the resuspension solution may comprise Tris EDTA (TE) 150mM KCl 0.05% CHAPS buffer.
  • the resuspension solution may comprise 10 mM TrisHCl pH 7.4, 1 mM EDTA.
  • the resuspension solution may comprise 300 mM HEPES.
  • the resuspension solution may also contain or be highly purified water (e.g., distilled or deionized water). Biomolecule desorption may be augmented by heating or agitation by an incubation element.
  • the denaturing solution may comprise a chemical denaturant such as guanidine, urea, sodium deoxycholate, acetonitrile, trichloroacetic acid, acetic acid, sulfosalicylic acid, sodium bicarbonate, ethanol, perchlorate, dodecyl sulfate, or any combination thereof.
  • the denaturing solution may comprise a reductant, such as 2-mercaptoethanol, dithiothreitol, or tris(2-carboxyethyl)phosphine.
  • the protease may be trypsin.
  • the denaturing solution may be added to a partition following desorption.
  • the denaturing solution may be added to a partition comprising biomolecule coronas.
  • the automated apparatus may comprise a magnet or array of magnets.
  • the automated apparatus may be capable of moving the substrate onto and off of the magnet or array of magnets.
  • the array of magnets may be structured so that a plurality of magnets from the array of magnets can rest directly underneath a plurality of partitions from the substrate.
  • the magnet may be capable of immobilizing magnetic sensor elements (e.g., magnetic particles (e.g., comprising the macromolecule structures) provided herein) within a partition on the substrate.
  • the magnet may prevent magnetic particles (e.g., comprising the macromolecule structures) from being removed from a partition during a wash step.
  • the magnet may also create a pellet from a collection of magnetic particles (e.g., comprising the macromolecule structures).
  • the magnet may create a macromolecule structure pellet in less than 10 minutes.
  • the magnet may create a particle pellet in less than 5 minutes.
  • the macromolecule structure pellet may comprise a particle with a biomolecule corona.
  • the automated apparatus may comprise a purification unit.
  • the purification unit may comprise a plurality of partitions comprising an adsorbent or resin.
  • the purification unit may comprise a solid-phase extraction array or plate.
  • the solid-phase extraction array or plate may comprise a polar stationary phase material.
  • the solid-phase extraction array or plate may comprise a non-polar stationary phase material.
  • the solid-phase extraction array or plate may comprise a C18 stationary phase material (e.g., octadecyl group silica gel).
  • the automated apparatus comprises a unit with a conditioning solution for the purification unit (e.g., a conditioning solution for a solid-phase extraction material).
  • the automated apparatus may comprise a unit with an elution solution for removing biomolecules from the purification unit.
  • the purification unit may comprise a solution comprising the third particles described herein.
  • a supernatant is removed from the sensor array plate.
  • the automated apparatus may perform a series of wash steps.
  • a wash step may remove biomolecules that are not bound to the sensor elements within the partition.
  • a wash step may desorb a subset of biomolecules bound to sensor elements within a partition.
  • a wash step may result in the desorption and removal of a subset of soft corona analytes, while leaving the majority of hard corona analytes bound to the sensor element.
  • WSGR Docket No.53344-792.601 the present disclosure provides an automated apparatus to identify proteins in a biological sample, the automated apparatus comprising: a sample preparation unit; a substrate comprising a plurality of channels; a plurality of pipettes; a plurality of solutions, a plurality of particles (e.g., comprising the macromolecule structures), such as particles (e.g., comprising the macromolecule structures) provided elsewhere herein, and wherein the automated apparatus is configured to form a biomolecule corona and digest the biomolecule corona.
  • the automated apparatus comprises a sample preparation unit. In some embodiments, the automated apparatus comprises a substrate comprising a plurality of channels. In some embodiments, the automated apparatus comprises a plurality of pipettes. In some embodiments, the automated apparatus comprises a plurality of solutions. In some embodiments, the automated apparatus comprises a plurality of particles (e.g., comprising the macromolecule structures). In some embodiments, the automated apparatus is configured to form a biomolecule corona and digest the biomolecule corona. [00341] In some embodiments, the automated apparatus or system is enclosed. Kits [00342] Provided herein, in some embodiments, are kits for isolating one or more biomolecules from a biological sample.
  • the plurality (e.g., at least two) of particles (e.g., comprising the macromolecule structures) can be packaged together to comprise a combination of particles (e.g., comprising the macromolecule structures) in a single package.
  • the particles (e.g., comprising the macromolecule structures) of the kits provided herein may be freeze dried and stored in sealed containers.
  • the kits comprise particles (e.g., comprising the macromolecule structures), such as particles (e.g., comprising the macromolecule structures) provided elsewhere herein.
  • the kits comprise at least two particles (e.g., comprising the macromolecule structures).
  • the particles comprise a first macromolecule structure.
  • the first macromolecule structure comprises a neutral to negative surface charge.
  • the particles (e.g., comprising the macromolecule structures) comprise a second macromolecule structure.
  • the second macromolecule structure comprises a negative surface charge.
  • the second macromolecule structure comprises a greater negative surface charge than that of the first macromolecule structure.
  • the kits provided herein comprise a buffer.
  • Non-limiting examples of buffers that may be greater include Tris, phosphate buffer, HEPES, MES, MOPS, TES, TE, CAPS, Bicine, and Bis-Tris.
  • the buffer is CAPS.
  • the buffer is HEPES.
  • the buffer comprises 200-750, 100- 750, 250-750, 300-7250, 400-750, 400-1000, 500-1000, or 500-750 mM HEPES.
  • the buffer is 300 mM HEPES.
  • buffers include but are not limited to a digestion buffer, resuspension buffer, dilution buffer, denaturation buffer, or a lysis buffer.
  • kits provided herein comprise a washing agent (e.g., a washing solution). In some embodiments, a washing agent comprise a wash solution as described elsewhere herein. [00347] In some embodiments, the kits provided herein comprise an elution agent. In some embodiments, the elution agent is an elution buffer as described elsewhere herein. [00348] In some embodiments, the kits comprise a digestion solution or digestion agent, such as a digestion solution or digestion agent provided elsewhere herein. In some embodiments, the digestion solution comprises Trypsin. In some embodiments, the digestion solution comprises Trypsin/LysC protease.
  • kits comprise a denaturing solution or denaturing agent.
  • the denaturing agent comprises at least one of: sodium dodecyl sulfate, acetic acid, trichloroacetic acid, sulfosalicylic acid, sodium bicarbonate, ethanol, formaldehyde, glutaraldehyde, urea, guanidium chloride, lithium perchlorate, 2-mercaptoethanol, dithiothreitol, tris(2-carboxyethyl)phosphine (TCEP), or any combination thereof.
  • the kit comprises a reducing agent.
  • the reducing agent comprises TCEP, dithiothreitol, beta-mercaptoethanol, glutathione, cysteine, or any combination thereof.
  • the kit comprises an alkylating agent.
  • the alkylating agent comprises iodoacetamide, iodoacetic acid, acrylamide, chloroacetamide, or any combination thereof.
  • the kit comprises a solid support for solid phase extraction.
  • the kit comprises a polar stationary phase material.
  • the kit comprises a non-polar stationary phase material.
  • the kit comprises WSGR Docket No.53344-792.601 a C18 stationary phase material (e.g., octadecyl group silica gel).
  • the kit comprises conditioning solution for the solid phase extraction material.
  • the solid support comprises magnetic particles.
  • the kit comprises an organic solvent (e.g., acetonitrile) to precipitate biomolecules onto the magnetic particles.
  • the kit comprises cleanup particles.
  • the kit comprises a wash solution.
  • the kit comprises a multi-well plate. In some embodiments, the multi-well plate is a 4 well plate.
  • the multi-well plate is a In some embodiments, the multi-well plate is a 12 well plate. In some embodiments, the multi-well plate is a 24 well plate. In some embodiments, the multi-well plate is a 48 well plate. In some embodiments, the multi-well plate is a 96 well plate. In some embodiments, the multi-well plate is a 384 well plate. In some embodiments, the multi-well plate is a 1536 well plate. [00356] In some embodiments, the kit comprises a diluent. In some embodiments, the diluent is an organic solvent. In some embodiments, the diluent is water. In some embodiments, the diluent is a buffer.
  • the diluent is an organic solvent, water, a buffer, or any combination thereof.
  • the kit comprises a reconstitution solution.
  • the reconstitution solution may be suitable to reconstitute lyophilized particles described herein.
  • the kit further comprises an organic solvent, such as an organic solvent described elsewhere herein.
  • the kit further comprises a cysteine blocking reagent.
  • the cysteine blocking reagent comprises methyl methanethiosulfonate, iodoacetamide, N-ethylmaleimide, methylsulfonyl benzothiazole, or any combination thereof.
  • Kits may also, in some embodiments, comprise one or more of outer packaging, lot numbers, and instructions for use of the kit with the methods provided herein.
  • Kits may also, in some embodiments, comprise one or more of outer packaging, lot numbers, and instructions for use of the kit with the methods provided herein.
  • Example 1 Preparation of a First Particle
  • a particle comprising randomly distributed ethyleneglycol dimethacrylate and hydroxyethylmethacrylate were prepared according to FIG. 1.
  • Olefin functionalized superparamagnetic iron oxide@silica nanoparticles (12 g) were provided and optionally dispersed in acetonitrile.
  • HEMA hydroxyethyl)methacrylate
  • EGDMA ethyleneglycol dimethacrylate
  • AIBN 1.4 g, 8.8 mmol
  • EGDMA 23g, 116 mmol
  • GMA glycidylmethacrylate
  • the resulting macromolecule structure was washed with ethanol once and at least three times with water. were added and allowed to stir at 60°C overnight (16 hrs). The resulting macromolecule structure was washed with ethanol once and at least three times with water.
  • the particle size, zeta potential, and % polymer were dependent on the length of time before quenching with benzoquinone in the first reaction (Table 3 and Table 4). TABLE 3 TABLE 4 Example 3: Performance of Multiplexed Particles [00366]
  • the particles (e.g., comprising the macromolecule structures) provided herein were assessed for their performance in isolating protein groups both individually and while multiplexed.
  • the particles were exposed to three different plasma samples, both on their own, and as a combination of (A2) and (A1).
  • the ratio of (A2):(A1) was about 4:1.
  • the total concentration of particles (e.g., comprising the macromolecule structures) in each of the experiments was about 0.31 mg/mL with a particle:plasma volume ratio of about 100 ⁇ L:100 ⁇ L. The results are depicted in FIG.
  • Particles (A1) and (A2) were evaluated at varying concentrations in equal volumes of particles and plasma (100 ⁇ L:100 ⁇ L) as shown in FIG. 6. Concentrations of total particles evaluated were 0.45 mg/mL, 0.45 mg/mL, and 0.6 mg/mL.
  • Peptide mass ( ⁇ g) and protein group counts were evaluated for samples with a multiplexed composition of (A1) and (A2) in a ratio of 4:1, 1:4, and (A2) alone.
  • the ratios are weight ratios, for example, in the 4:1 (A1):(A2) multiplexed samples, 0.36 mg/mL of (A1) and 0.09 mg/mL of (A2) may be combined to arrive at the composition used.
  • the results in FIG.6 may indicate that multiplexing of (A1) and (A2) can result in higher peptide mass yields at all concentrations of nanoparticle used.
  • FIG. 6 also may indicate that increasing amounts of (A2) relative to (A1) e.g., 1:4 (A1):(A2) may result in increased protein group identification.
  • particles e.g., comprising the macromolecule structures
  • A3, A4), (A5), and (A6) were similarly assessed for a synergistic relationship when multiplexed.
  • (A3), (A4), and (A5) had highly negative surface charges, whereas (A6) had an almost neutral surface charge.
  • WSGR Docket No.53344-792.601 [00369] In this instance, the protein group count improvement from multiplexing was assessed, complexing (A3) with other negative particles (e.g., comprising the macromolecule structures) or almost neutral particles (e.g., comprising the macromolecule structures) (A6).
  • Biomolecule Assay Protocol Methods of assaying biomolecules may be completed using the following protocol.
  • the assay protocol and methods herein may provide high-throughput, unbiased, deep, rapid proteomics analysis of biological samples, such as plasma, serum, and tissue or cell lysates (or other biological samples provided herein).
  • the protocol may comprise an assay step, a peptide quantification step, and a peptide reconstitution step, which may be run separately.
  • Materials may be used to assay 40 or 80 biomolecules (peptide) samples on a single 96 well plate.
  • the estimated time for each step of an exemplary method provided herein is shown in Table 6, such as for an 80 or 40 sample analysis.
  • Table 6 WSGR Docket No.53344-792.601
  • the assay may require use of an assay kit, such as a kit provided herein (i.e., assay kit). In cases where peptide quantification and peptide reconstitution are desired, an additional kit may be required (i.e., a PQR kit).
  • the assay kit for analysis of 40 or 80 samples may contain the following reagents in Table 6.
  • the kit may also include a labware box including a binding reservoir, black lid, 2 cleanup plates, clear lid, collection plate, nanoparticle plate, preparation plate, 2 reagents reservoirs, and a sample transfer plate.
  • the labware box may not be refrigerated whereas the reagents kit (i.e., Table 7) may be refrigerated.
  • the method may also make use of a peptide calibration kit.
  • biomolecule (peptide) preparation, quantification, and reconstitution may require the following additional equipment: (1) a centrifuge with plate adapter that can achieve a relative centrifugal force of 500 x g and accommodate a standard 96 well microplate; (2) a fluorometric microplate reader that can measure peptide concentration via fluorescence (Ex. 390, Em.475 nm) in a 96-well plate format; (3) a refrigerated microcentrifuge that can achieve a relative centrifugal force of 5000 x g, maintain a temperature of 4°C, and accommodate sample tubes; and (4) a vacuum concentrator capable of maintaining refrigerated temperature at vacuum and accommodate a standard 96 well microplate.
  • process control a pooled plasmas sample that is processed through the entirety of the workflow, including corona formation, trypsin digestion, and peptide cleanup
  • digestion control a pooled plasma sample that is processed through workflow steps after the corona formation, including trypsin digestion and peptide cleanup, used for diagnostic and troubleshooting efforts
  • user control an available well for the end user to supply their own on plate control sample of interest
  • cleanup control a digested peptide sample generated from pooled plasma sample that is only processed through the peptide cleanup steps of the assay, also used for diagnostic and troubleshooting efforts.
  • the centrifuge is first set to 4°C. If the biological (e.g., plasma) samples are frozen, they are removed from the freezer and thawed in an ice-water bath. The plasma samples should not be allowed to warm to room temperature and should be kept on ice or at 4°C until they are loaded into the sample prep plate and into the instrument. The labware is removed from the above-mentioned labware box and the Trypsin/LysC tube is placed on ice.
  • biological e.g., plasma
  • Required materials for the peptide quantification may include the PQR kit, described above, as well as an acid-resistant CentriVap centrifugal vacuum concentrator, a fluorescence intensity microplate reader, 50 ⁇ L nested conductive tips (NCTs), 300 ⁇ L NCTs, aluminum sealing foil 5 x 3 in, a Pierce quantitative fluorometric peptide assay, and pipettes with tips varying from 1-10 mL to 10-1000 ⁇ L.
  • the PQR kit may include a black lid, quantification plate, quantification reservoir, reconstitution reservoir, recovery solution, and standards prep plate.
  • Peptide reconstitution may be completed, which can reconstitute the dried peptides to the concentration and volume needed for mass spectrometry.
  • the peptide reconstitution method may require use of the PQR kit described above as well as 300 ⁇ L NCTs, aluminum sealing foil 5 x 3 in, Axygen AxyMats 96 round well sealing mat for PCR microplates, peptide reconstitution buffer, and pipettes with tips varying from 1-10 mL to 10-1000 ⁇ L.
  • an appropriate sized bottle large enough to hold a sufficient volume to make a single batch for the anticipated study size is obtained, assuming 4 mL per plate.
  • Embodiment 1 A composition comprising two or more macromolecule structures comprising: (a) a first macromolecule structure comprising a neutral to negative surface charge; and (b) a second macromolecule structure comprising a greater negative surface charge than the first macromolecule structure.
  • Embodiment 2. The composition of embodiment 1, wherein the first macromolecule structure comprises a surface charge of about 0 mV to about -15 mV.
  • Embodiment 3. The composition of any one of the preceding embodiments, wherein the second macromolecule structure comprises a surface charge of about -35 mV to about -60 mV.
  • composition of any one of embodiments 1-7, wherein the second macromolecule structure comprises a polymer comprising one or more units represented by Formula (A): WSGR Docket No.53344-792.601 Formula (A) wherein, R is hydrogen R 1 is hydrogen or hydroxyl; q is an integer from 1 to 6; R 2 is C1-C8 diamine, N-substituted with one or more R 3 ; R 3 is each independently hydrogen or C 1 -C 8 alkyl optionally substituted with one or more oxo, hydroxyl, C 1 -C 8 alkyl, C 1 -C 8 alkenyl, and C 1 -C 8 alkynyl; or R 1 and R 2 are taken together to form C2-C6 heterocycloalkyl; and R 4 is hydrogen or C1-C6 alkyl.
  • R is hydrogen
  • R 1 is hydrogen or hydroxyl
  • q is an integer from 1 to 6
  • R 2 is C1-C8 diamine, N-substitute
  • Embodiment 10 The composition of embodiment 5 or 9, wherein R is .
  • Embodiment 11 The composition of any one of embodiments 5-10, wherein R 1 is hydroxyl.
  • Embodiment 12. The composition of any one of embodiments 5-11, wherein R 2 is a C2 diamine, N-substituted with one or more R 3 .
  • Embodiment 13 The composition of any one of embodiments 5-11, wherein R 2 is , wherein p is an integer from 1 to 6.
  • Embodiment 17 The composition of any one of embodiments 8-16, wherein at least one R 3 is C1-C8 alkyl substituted with at least one of oxo, hydroxyl, and C1-C8 alkenyl.
  • composition of any one of embodiments 8-17, wherein the structure of Formula (A) is represented by Formula (A-A): WSGR Docket No.53344-792.601 Formula (A-A) wherein, R 5 is C 1 -C 10 alkyl, C 1 -C 10 alkenyl, or C 1 -C 10 alkynyl.
  • Embodiment 19 The composition of any one of embodiments 8-17, wherein the structure of Formula (A) is represented by Formula (A-B): Formula (A-B) wherein, R 5 is C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl.
  • Embodiment 20 Embodiment 20.
  • composition of any one of embodiments 8-17, wherein the structure of Formula (A) is Formula Formula (A-C) wherein, R 5 is each independently C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl.
  • R 5 is each independently C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl.
  • the composition of embodiment 17, wherein the structure of Formula (A- C) is: .
  • Embodiment 22 The composition of any one of embodiments 8-13, wherein the structure of Formula (A) is represented by Formula (A-D): Formula (A-D) Embodiment 23.
  • Embodiment 25 The composition of any one of embodiments 18-20, wherein R 5 is C 1 -C 10 alkenyl.
  • Embodiment 26 The composition of embodiment 8 or 9, wherein R is hydrogen.
  • Embodiment 27 The composition of embodiment 26, wherein the structure of Formula (A) .
  • Embodiment 28 The composition of any one of embodiments 8-10, wherein R 1 and R 2 are taken together to form C 2 heterocycloalkyl.
  • Embodiment 29 The composition of any one of embodiments 9-28, wherein m is an integer of 1.
  • Embodiment 30 The composition of any one of embodiments 9-28, wherein m is an integer of 1.
  • Embodiment 34 The composition of embodiment 33, wherein R 6 is (CH2)pOR 7 .
  • Embodiment 35 The composition of embodiment 33, wherein R 6 is (CH2)pOR 7 .
  • composition of any one of embodiments 33-40, wherein the structure of Formula (D) is: WSGR Docket No.53344-792.601 .
  • Embodiment 42. The composition of any one of embodiments 33-41, wherein the copolymer comprises the structure: .
  • Embodiment 43. The composition of any one of the preceding embodiments, wherein the first macromolecule structure comprises a surface.
  • Embodiment 44. The composition of any one of the preceding embodiments, wherein the second macromolecule structure comprises a surface.
  • the composition of embodiment 43 or 44, wherein the surface comprises a particle.
  • Embodiment 46. The composition of embodiment 45, wherein the particle is a nanoparticle.
  • composition of embodiment 45 wherein the particle is a microparticle.
  • Embodiment 48 The composition of any one of embodiments 45-47, wherein the particle comprises a diameter of from about 100 nm to about 750 nm.
  • Embodiment 49 The composition of any one of embodiments 45-48, wherein the particle comprises a diameter of from about 100 nm to about 500 nm.
  • Embodiment 50 The composition of any one of embodiments 45-49, wherein the particle comprises a polydispersity index (PDI) of from about 0.01 to about 0.2.
  • Embodiment 51 The composition of any one of embodiments 45-50, wherein the particle comprises a PDI of from about 0.1 to about 0.2.
  • Embodiment 52 The composition of any one of embodiments 45-50, wherein the particle comprises a PDI of from about 0.1 to about 0.2.
  • the composition of any one of embodiments 45-52, wherein the particle comprises is a superparamagnetic iron oxide nanoparticle.
  • Embodiment 58. The composition of any one of embodiments 45-57, wherein the polymer is non-covalently coupled to the surface.
  • Embodiment 59. The composition of any one of embodiments 45-58, wherein the polymer or copolymer is covalently coupled to the surface via a linker.
  • the composition of any one of embodiments 45-59, wherein the first macromolecule structure comprises the structure: wherein, the surface; L is a linker; and A is the copolymer of any one of embodiments 33-42.
  • composition of embodiment 64, wherein the stabilizing agent comprises a metal salt.
  • Embodiment 66 The composition of embodiment 65, wherein the metal salt comprises aluminum chloride.
  • Embodiment 67 The composition of any one of embodiments 9-66, wherein the copolymer is a random copolymer.
  • Embodiment 68 The composition of any one of embodiments 9-66, wherein the copolymer is a block copolymer.
  • Embodiment 69. The composition of any one of embodiments 8-68, wherein the composition comprises at least 1 wt% of the polymer or copolymer.
  • Embodiment 70 The composition of embodiment 64, wherein the stabilizing agent comprises a metal salt.
  • Embodiment 66 The composition of embodiment 65, wherein the metal salt comprises aluminum chloride.
  • Embodiment 67 The composition of any one of embodiments 9-66, wherein the copolymer is a random copolymer.
  • Embodiment 68 The composition of any one
  • Embodiment 71. The composition of any one of embodiments 8-70, wherein the polymer or copolymer comprises a molecular weight of from about 0.5 kDa to about 25 kDa.
  • Embodiment 72. The composition of any one of embodiments 8-71, wherein the polymer or copolymer comprises a molecular weight of from about 0.5 kDa to about 10 kDa.
  • Embodiment 73 Embodiment 73.
  • a method of isolating one or more biomolecules from a biological sample comprising: (a) contacting the biological sample comprising one or more biomolecules with a composition of any one of embodiments 1-72 to bind the one or more biomolecules to the at least two macromolecule structures, thereby forming at least two biomolecule corona; and (b) eluting the one or more biomolecules from the at least two macromolecule structures, thereby providing one or more isolated biomolecules, wherein, the at least two macromolecule structures comprise: (i) a first macromolecule structure comprising a neutral to negative surface charge; and WSGR Docket No.53344-792.601 (ii) a second macromolecule structure comprising a greater negative surface charge than the first macromolecule structure.
  • Embodiment 74 The method of embodiment 73, wherein the method further comprises separating the one or more biomolecules and the at least two macromolecule structures from the biological sample.
  • Embodiment 75 The method of embodiment 73 or 74, wherein the method further comprises optionally digesting the one or more biomolecules.
  • Embodiment 76 The method of embodiment 73, wherein the method further comprises optionally digesting the one or more biomolecules.
  • a method of isolating one or more biomolecules from a biological sample comprising: (a) contacting a population of biomolecules comprising one or more biomolecules with a composition of any one of embodiments 1-72, thereby forming at least two biomolecule corona; (b) separating and optionally digesting the one or more biomolecules and the at least two macromolecule structures from the biological sample; and (c) eluting and optionally digesting the one or more biomolecules from the at least two macromolecule structures, thereby providing one or more isolated biomolecules, wherein, the at least two macromolecule structures comprise: (i) a first macromolecule structure comprising a neutral to negative surface charge; and (ii) a second macromolecule structure comprising a greater negative surface charge than the first macromolecule structure.
  • Embodiment 77 The method of any one of embodiments 73-76, wherein the first macromolecule structure comprises a surface charge of from about 0 mV to about -15 mV.
  • Embodiment 78 The method of any one of embodiments 73-77, wherein the second macromolecule structure comprises a surface charge of from about -35 mV to about -60 mV.
  • Embodiment 79 The method of any one of embodiments 73-78, wherein the surface charge is characterized by a zeta potential.
  • Embodiment 80 The method of any one of embodiments 73-79, wherein the first macromolecule structure comprises the composition of any one of embodiments 33-72.
  • the method of any one of embodiments 73-80, wherein the second macromolecule structure comprises the composition of any one of embodiments 5-32 or 43- 72.
  • Embodiment 82. The method of any one of embodiments 73-81, wherein the one or more biomolecules comprises proteins, peptides, or a combination thereof.
  • Embodiment 83. The method of any one of embodiments 73-82, wherein the biological sample comprises plasma, serum, or blood.
  • the method of any one of embodiments 73-82, wherein the biological sample comprises biofluid.
  • Embodiment 85. The method of embodiment 84, wherein the biofluid is a cell-free biofluid.
  • kits for isolating one or more biomolecules from a biological sample comprising: at least two macromolecule structures; (i) a first macromolecule structure with a neutral to negative surface charge; and (ii) a second macromolecule structure with a greater negative surface charge than the first macromolecule structure; Embodiment 128.
  • the kit further comprises a washing agent configured to wash the one or more biomolecules bound to the at least two macromolecule structures.
  • the kit further comprises an elution agent configured to elute the one or more biomolecules from the at least two macromolecule structures.
  • Embodiment 130 The kit of embodiment 127, wherein the kit further comprises a denaturing agent.
  • a method of preparing a mixture of at least two macromolecule structures, the at least two macromolecule structures comprising recurring units of a first monomer and a second monomer or a first monomer and a third monomer comprising: (a) obtaining a first macromolecule structure comprising a neutral to negative surface charge; (b) obtaining a second macromolecule structure comprising a greater negative surface charge than the first macromolecule structure; and (c) forming a mixture comprising the first and second macromolecule structures.
  • Embodiment 139 The method of embodiment 138, wherein the first macromolecule structure comprises a surface charge of about 0 mV to about -15 mV.
  • Embodiment 140 Embodiment 140.
  • the method of embodiment 138 or 139, wherein the second macromolecule structure comprises a surface charge of about -35 mV to about -60 mV.
  • Embodiment 141. The method of any one of embodiments 138-140, wherein the surface charge is characterized by a zeta potential.
  • Embodiment 142. The method of any one of embodiments 138-141, wherein the first macromolecule structure comprises the composition of any one of embodiments 33-72.
  • the method of any one of embodiments 138-142, wherein the second macromolecule structure comprises the composition of any one of embodiments 8-32 or 43- 72.
  • a method of preparing a macromolecule structure comprising recurring units of a first component and a second component, the method comprising: (a) providing a mixture of monomers in a solvent comprising a first monomer and a second monomer, wherein the first monomer comprises: wherein q is an integer from 1 to 6; and the second monomer comprises: wherein m is an integer from 1 to 6; (b) contacting a surface and the mixture of monomers, thereby producing a reaction mixture; (c) polymerizing the mixture of monomers to produce a macromolecule immobilized to the surface; (d) contacting the macromolecule immobilized to the surface and an amine, thereby producing an aminated macromolecule; and (e) optionally, contacting the aminated macromolecule with an anhydride optionally substituted with R 2 , wherein R 2 is C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl.
  • Embodiment 145 The method of embodiment 144, wherein the surface comprises a particle.
  • Embodiment 146. The method of embodiment 145, wherein the particle is a nanoparticle.
  • Embodiment 147. The method of embodiment 145, wherein the particle is a microparticle.
  • Embodiment 148. The method of any one of embodiments 145-147, wherein the particle comprises a diameter of from about 100 nm to about 750 nm.
  • Embodiment 149 The method of any one of embodiments 145-148, wherein the particle comprises a diameter of from about 100 nm to about 500 nm.
  • PDI polydispersity index
  • WSGR Docket No.53344-792.601 Embodiment 151.
  • the method of any one of embodiments 145-151, wherein the particle comprises iron oxide.
  • the method of any one of embodiments 145-152, wherein the particle comprises is a superparamagnetic iron oxide nanoparticle.
  • Embodiment 155 The method of any one of embodiments 145-154, wherein the particle comprises an iron oxide core and a silica shell.
  • Embodiment 156 The method of any one of embodiments 145-154, wherein the particle comprises iron oxide crystals embedded in a polystyrene core.
  • Embodiment 157 The method of any one of embodiments 144-155, wherein (b) comprises contacting in an organic solvent.
  • Embodiment 158 The method of any one of embodiments 144-156, wherein (d) comprises contacting in an organic solvent.
  • Embodiment 159 The method of any one of embodiments 144-156, wherein (d) comprises contacting in an organic solvent.
  • Embodiment 162 wherein the heating comprises heating to a temperature of at least 50°C, 60°C, 70°C, 80°C, or at least 90°C.
  • Embodiment 164 The method of any one of embodiments 144-163, wherein the polymerization comprises free radical polymerization, atom transfer radical polymerization (ATRP), emulsion polymerization, or precipitation polymerization.
  • Embodiment 165 The method of any one of embodiments 144-164, wherein the macromolecule structure comprises a composition of any one of embodiments 1-42. WSGR Docket No.53344-792.601 Embodiment 166.
  • Embodiment 167 Use of a macromolecule structure comprising 2-hydroxyethyl methacrylate, ethylene glycol dimethacrylate, and methacrylic acid as monomer units for binding proteins in a biological sample.
  • Embodiment 167 Use of a macromolecule structure comprising the structure of Formula (A-D) and the structure of Formula (A-A) as recurring units for binding proteins in a biological sample.
  • Embodiment 168. Use of a macromolecule structure comprising the structure of Formula (A-C) and the structure of Formula (A-B) as recurring units for binding proteins in a biological sample.
  • Embodiment 169 Use of the composition of any one of embodiment 1-72 for binding proteins in a biological sample.
  • Embodiment 170 Use of the composition of any one of embodiment 1-72 for binding proteins in a biological sample.
  • a composition comprising a plurality of particles, wherein the particles comprise an outer polymer surface and a magnetic core, wherein the outer polymer surface comprises 2-hydroxyethyl methacrylate, ethylene glycol dimethacrylate, and methacrylic acid as monomer units.
  • Embodiment 171. A composition comprising a plurality of particles, wherein the particles comprise an outer polymer surface and a magnetic core, wherein the outer polymer surface comprises ethylene glycol dimethacrylate, monomer 6, and at least one of: monomer 7, monomer 8, monomer 9, and glycidyl methacrylate.
  • Embodiment 172 Embodiment 172.

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Abstract

L'invention concerne des compositions, des procédés, des systèmes et des kits destinés à isoler une ou plusieurs biomolécules à partir d'une solution biologique. Dans certains cas, les compositions, les procédés, les systèmes et les kits selon l'invention peuvent comprendre une, deux particules (par exemple, comprenant des structures macromoléculaires) ou plus, de charge de surface différente. Dans certains modes de réalisation, les particules (par exemple, comprenant des structures macromoléculaires) comprennent une première particule (par exemple, comprenant une structure macromoléculaire) présentant une charge de surface neutre à négative et une seconde particule (par exemple, comprenant une structure macromoléculaire) présentant une charge de surface négative supérieure à celle de la première particule (par exemple, comprenant une structure macromoléculaire). Dans certains cas, les combinaisons de particules selon l'invention fournissent une relation synergique, améliorant le nombre de biomolécules qui peuvent être isolées, purifiées, identifiées ou similaires à partir d'une solution biologique.
PCT/US2025/022713 2024-04-03 2025-04-02 Procédés, kits et systèmes de préparation de biomolécules Pending WO2025212750A1 (fr)

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WO2023245075A2 (fr) * 2022-06-15 2023-12-21 Seer, Inc. Systèmes et méthodes pour dosages de biomolécules

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WO1993012184A1 (fr) * 1991-12-03 1993-06-24 Rohm And Haas Company Procede d'absorption de particules
US20050009002A1 (en) * 2001-03-20 2005-01-13 Depu Chen Processes for producing coated magnetic microparticles and uses thereof
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WO2014089160A1 (fr) * 2012-12-04 2014-06-12 Phosphorex, Inc. Microparticules et nanoparticules ayant des charges de surface négatives
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