Functionalized macromolecules, and systems, synthetic methods and uses thereof

Abstract

Disclosed herein are macromolecules, compositions thereof, and methods of making the same. In one aspect, macromolecules are immobilized to a surface. In one aspect, provided herein are compositions, systems, and kits comprising macromolecules.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 378,225, filed Oct. 3, 2022, and U.S. Provisional Application No. 63 / 382,289, filed Nov. 3, 2022, the content of each of which is incorporated herein by reference in its entirety. Background of the Invention

[0003] The analysis of low-abundance biomolecules is a significant challenge in proteomics. Although some recent advancements have improved the capture of low-abundance biomolecules from samples, the compositions that facilitate the detection of low-abundance biomolecules can be improved. Accordingly, there is a need for improved structures and compositions for detecting low-abundance biomolecules from samples. The present disclosure provides structures and their compositions to address this need. Summary of the Invention

[0004] In one aspect, described herein is a macromolecule comprising repeating units of formula (I-A):

[0005]

[0006] wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl;

[0007] R1 is hydrogen, an optionally substituted succinate, a C1-C6 alkylsulfone, or a phthalate,

[0008] R2 is a C1-C6 hydroxy, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted C3-C6 heterocycloalkyl, a C1-C 12 amine, an optionally substituted C3-C6 bicycloalkylmethane, a C1-C6 alkylguanidine, a C1-C6 ether, an optionally substituted -C1-C6 disulfide, a C1-C6 thiol, an optionally substituted succinate, an optionally substituted C1-C 12 alkylamine, a C1-C6 alkylacetamide, a C5-C 11 optionally substituted cycloalkyl, or a C1-C6 aminophthalate; or

[0009] R1 and R2 together with the nitrogen to which they are attached form an optionally substituted heterocycle; or

[0010] R1 and R2 together with the nitrogen to which they are attached form an azide group; and

[0011] q is an integer between 1 and 6.

[0012] In some embodiments, Y1 is a C1-C3 alkyl group. In some embodiments, Y1 is a C1 alkyl group. In some embodiments, each of Y2 and Y3 is hydrogen. In some embodiments, q is an integer between 1 and 3. In some embodiments, q is 1.

[0013] In some embodiments, R1 is hydrogen, and R2 is selected from optionally substituted C3-C6 bicycloalkylmethanes, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted C3-C6 heterocycloalkyls, C1-C6 hydroxy groups, C1-C6 ethers, optionally substituted -C1-C6 disulfides, optionally substituted succinates, optionally substituted C1-C6 alkylamines, C1-C6 alkylacetamides or C1-C6 alkylguanidines.

[0014] In some embodiments, R2 is an optionally substituted dicyclohexylmethane. In some embodiments, R2 is aminodicyclohexylmethane.

[0015] In some embodiments, R2 is an optionally substituted aryl. In some embodiments, R2 is halotoluene. In some embodiments, R2 is 2-fluorotoluene.

[0016] In some embodiments, R2 is a C1-C6 hydroxy group. In some embodiments, R2 is a C3-C6 hydroxy group. In some embodiments, R2 is -(CH2)6OH.

[0017] In some embodiments, R2 is a C1-C6 ether. In some embodiments, R2 is -CH2OCH3, -CH2CH2OCH3, -CH2CH2OCH2CH3 or -CH2OCH2CH3. In some embodiments, R2 is -CH2CH2OCH3.

[0018] In some embodiments, R2 is a C1-C6 acetamide. In some embodiments, R2 is -(CH2)2 acetamide.

[0019] In some embodiments, R2 is an optionally substituted di-C1-C6 alkyldisulfide. In some embodiments, R2 is -CH2CH2-S-S-CH2CH2NH2.

[0020] In some embodiments, R2 is an optionally substituted succinate. In some embodiments, R2 is -(CH2) 1- 6NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2)6NH(C=O)CH2CH2COOH.

[0021] In some embodiments, R2 is an optionally substituted heteroaryl. In some embodiments, R2 is -(CH2)1-6 Imidazole. In some embodiments, R2 is -(CH2)3imidazole. In some embodiments, R2 is a disubstituted C2-C4 imidazole. In some embodiments, R2 is dipropylimidazole.

[0022] In some embodiments, R2 is an optionally substituted heterocycloalkyl. In some embodiments, R2 is -(CH2) 1-6 Pyrrolidine. In some embodiments, R2 is -(CH2)2pyrrolidine.

[0023] In some embodiments, R2 is an optionally substituted C1-C6 alkylamine. In some embodiments, R2 is -(CH2) 1-3 Dimethylamine. In some embodiments, R2 is -(CH2)2dimethylamine.

[0024] In some embodiments, R2 is a C1-C6 guanidine. In some embodiments, R2 is -(CH2)2guanidine.

[0025] In some embodiments, each of R1 and R2 is nitrogen. In some embodiments, R1 and R2 together form an optionally substituted heterocycle. In some embodiments, the optionally substituted heterocycle is a triazole. In some embodiments, the optionally substituted triazole contains a benzylamide. In some embodiments, the benzylamide is halogenated.

[0026] In some embodiments, R1 is an optionally substituted succinate, and R2 is an optionally substituted C3-C6 bicycloalkylmethane, an optionally substituted aryl, a C1-C6 thiol, or an optionally substituted succinate. In some embodiments, R1 is an optionally substituted succinate (e.g., -C(=O)CH2CH2COOH).

[0027] In some embodiments, R1 is a succinate, and R2 is an optionally substituted C3-C6 bicycloalkylmethane, an optionally substituted aryl, a C1-C6 thiol, or an optionally substituted succinate. In some embodiments, R2 is dicyclohexylmethane succinate. In some embodiments, R2 is an optionally substituted aryl. In some embodiments, R2 is 2-fluorotoluene. In some embodiments, R2 is a C1-C6 thiol. In some embodiments, R2 is -(CH2)2SH. In some embodiments, R2 is an optionally substituted succinate. In some embodiments, R2 is -(CH2) 1-12 NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2) 1-3NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2)2NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2) 10-12 NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2) 12 NH(C=O)CH2CH2COOH.

[0028] In some embodiments, R1 is a C2-C 12 alkenyl succinate, and R2 is a substituted succinate. In some embodiments, R1 is a C8 alkenyl succinate, and R2 is a C8 alkenyl ethylamino succinate.

[0029] In some embodiments, R1 is a C1-C6 sulfone, and R2 is an optionally substituted C1-C6 alkylamine. In some embodiments, R1 is -(CH2)3SOOOH, and R2 is -(CH2) 1-6 N(CH3)2(CH2CH2CH2SOOOH) or -(CH2) 1-6 N(CH2CH2CH2SOOOH)2. In some embodiments, R2 is -(CH2)2N(CH3)2(CH2CH2CH2SOOOH). In some embodiments, R2 is -(CH2)2N(CH2CH2CH2SOOOH)2.

[0030] In some embodiments, R1 is a phthalate, and R2 is a C1-C6 aminophthalate. In some embodiments, R2 is a C2-C6 aminophthalate. In some embodiments, R2 is a C2 aminophthalate. In some embodiments, R2 is a C6 aminophthalate.

[0031] In one aspect, described herein is a macromolecule comprising a repeating unit of formula (II):

[0032]

[0033] wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1-C6 alkyl;

[0034] R4 is hydrogen or a C1-C6 thiol; and

[0035] R5 is a succinate, a C1-C6 thiol, an optionally substituted aryl, or an optionally substituted -C1-C6 disulfide.

[0036] In some embodiments, Y1 is a C1-C3 alkyl group. In some embodiments, Y1 is a C1 alkyl group. In some embodiments, each of Y2 and Y3 is hydrogen.

[0037] In some embodiments, R4 is hydrogen and R5 is an optionally substituted -C1-C6 disulfide. In some embodiments, R5 is an optionally substituted di-C1-C6 alkyl disulfide. In some embodiments, R5 is -CH2CH2-S-S-CH2CH2NH2. In some embodiments, R4 is a C1-C6 thiol and R5 is a succinate. In some embodiments, R4 is a C1-C3 thiol. In some embodiments, R4 is -(CH2)2SH.

[0038] In one aspect, described herein is a macromolecule comprising repeating units of formula (III-A):

[0039]

[0040] wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1-C6 alkyl group;

[0041] X is O or NH; and

[0042] each q is independently an integer between 1 and 6.

[0043] In some embodiments, Y1 is a C1-C3 alkyl group. In some embodiments, Y1 is a C1 alkyl group. In some embodiments, each of Y2 and Y3 is hydrogen. In some embodiments, q is 2 or 3. In some embodiments, each q is 2.

[0044] In some embodiments, the repeating units of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’), or formula (III-A) are selected from Table 1.

[0045] In some embodiments, the repeating units of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’), or formula (III-A) are selected from

[0046]

[0047]

[0048]

[0049] In one aspect, the present disclosure describes a macromolecule comprising repeating units of a first component and a second component, wherein the first component comprises the structure of component (A), and the second component comprises the structure of component (B):

[0050]

[0051] wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl;

[0052] each of X1, X2, and X3 is independently selected from hydrogen or C1-C6 alkyl;

[0053] A is

[0054] R1 is hydrogen, an optionally substituted succinate, a C1-C6 alkyl sulfone, a phthalate,

[0055] R2 is a C1-C 12 amine, a C1-C6 hydroxy, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted C3-C6 heterocycloalkyl, two or more fused 3-6 membered rings; an optionally substituted C3-C6 bicycloalkylmethane, a C1-C6 alkylguanidine, a C1-C6 ether, an optionally substituted -C1-C6 disulfide, a C1-C6 thiol, an optionally substituted succinate, an optionally substituted C1-C6 alkylamine, a C1-C6 alkylacetamide, a C1-C6 aminophthalate, boric acid, a C1-C6 thiol, a C1-C 11 optionally substituted cycloalkyl or monosaccharide; or

[0056] R1 and R2 together with the nitrogen to which they are attached form an optionally substituted heterocycle; or

[0057] R1 and R2 together with the nitrogen to which they are attached form an azide group;

[0058] R4 is hydrogen or a C1-C6 thiol;

[0059] R5 is a succinate, an optionally substituted aryl or an optionally substituted -C1-C6 disulfide;

[0060] B is

[0061] Z is a unit of monomer (A) or monomer (B);

[0062] each q is independently an integer between 1 and 6; and

[0063] p is an integer between 1 and 20.

[0064] In some embodiments, provided herein is a macromolecule comprising repeating units of a first component and a second component, wherein the first component comprises the structure of component (A) and the second component comprises the structure of component (B’):

[0065]

[0066] wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl;

[0067] each of X1, X2, and X3 is independently selected from hydrogen or C1-C6 alkyl;

[0068] A is

[0069] R1 is hydrogen, nitrogen, an optionally substituted succinate, a C1-C6 alkyl sulfone, a phthalate,

[0070] R2 is nitrogen, a C1-C 12 amine, a C1-C6 hydroxy, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted C3-C6 heterocycloalkyl, two or more fused 3-6 membered rings; an optionally substituted C3-C6 bicycloalkylmethane, a C1-C6 alkyl guanidine, a C1-C6 ether, an optionally substituted -C1-C6 disulfide, a C1-C6 thiol, an optionally substituted succinate, an optionally substituted C1-C6 alkylamine, a C1-C6 alkylacetamide, a C1-C6 amino phthalate, boric acid or a monosaccharide;

[0071] R4 is hydrogen or a C1-C6 thiol;

[0072] R5 is a succinate or an optionally substituted -C1-C6 disulfide;

[0073] B is

[0074] q is an integer between 1 and 6; and

[0075] p is an integer between 1 and 20.

[0076] In some embodiments, B is and A is

[0077] In some embodiments, R4 is hydrogen and R5 is an optionally substituted -C1-C6 disulfide. In some embodiments, R5 is an optionally substituted di-C1-C6 alkyl disulfide. In some embodiments, R5 is -CH2CH2-S-S-CH2CH2NH2.

[0078] In some embodiments, B is and A is

[0079] In some embodiments, B is and A is In some embodiments, B is In some embodiments, B is In some embodiments, B is

[0080] In some embodiments, B is In some embodiments, B is In some embodiments, A is In some embodiments, A is In some embodiments, A is

[0081] In some embodiments, R1 is C2-C 12 alkenyl succinate, and R2 is a substituted succinate. In some embodiments, R1 is C8 alkenyl succinate, and R2 is C8 alkenyl ethylamino succinate. In some embodiments, R1 is C1-C6 alkyl sulfone, and R2 is an optionally substituted C1-C6 alkylamine. In some embodiments, R1 is -(CH2)3SOOOH, and R2 is -(CH2) 1-6 N(CH3)2(CH2CH2CH2SOOOH) or -(CH2) 1-6 N(CH2CH2CH2SOOOH)2. In some embodiments, R2 is -(CH2)2N(CH3)2(CH2CH2CH2SOOOH).

[0082] In some embodiments, R1 is a phthalate, and R2 is a C1-C6 aminophthalate. In some embodiments, R2 is a C2-C6 aminophthalate. In some embodiments, R2 is a C2 aminophthalate. In some embodiments, R2 is a C6 aminophthalate.

[0083] In some embodiments, R1 is a succinate, and R2 is a substituted succinate or an optionally substituted aryl. In some embodiments, R2 is -(CH2) 1-12 NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2) 1- 3NH(C=O)CH2CH2COOH. In some embodiments, R2 is -

[0084] (CH2)2NH(C=O)CH2CH2COOH. In some embodiments, R= is an optionally substituted aryl. In some embodiments, R2 is 2-fluorotoluene.

[0085] In some embodiments, R1 is hydrogen and R2 is C1-C 12 amine, an optionally substituted C1-C6 alkylamine, a C1-C6 acetamide, an optionally substituted heterocycloalkyl, an optionally substituted heteroaryl, a monosaccharide, two or more fused 3-6 membered rings, an optionally substituted aryl or a C1-C6 hydroxy group.

[0086] In some embodiments, R2 is C 1-12 amine. In some embodiments, R2 is a C2 amine. In some embodiments, R2 is a C6 amine. In some embodiments, R2 is an optionally substituted C1-C6 alkylamine. In some embodiments, R2 is -(CH2) 1-3 dimethylamine. In some embodiments, R2 is -(CH2)2 dimethylamine. In some embodiments, R2 is a C1-C6 acetamide. In some embodiments, R2 is -(CH2)2 acetamide. In some embodiments, R2 is an optionally substituted heterocycloalkyl. In some embodiments, R2 is -(CH2) 1-6 pyrrolidine. In some embodiments, R2 is -(CH2)2 pyrrolidine. In some embodiments, R2 is an optionally substituted heteroaryl. In some embodiments, R2 is -(CH2) 1-6 imidazole. In some embodiments, R2 is -(CH2)3 imidazole. In some embodiments, R2 is -(CH2) 1-6 pyridine. In some embodiments, R2 is -(CH2) pyridine. In some embodiments, R2 is a monosaccharide. In some embodiments, R2 is glucose. In some embodiments, R2 is D-glucose. In some embodiments, R2 is two or more fused 3-6 membered rings. In some embodiments, R2 is three 6-membered rings. In some embodiments, R2 is an optionally substituted aryl. In some embodiments, R2 is halotoluene. In some embodiments, R2 is 2-fluorotoluene. In some embodiments, R2 is a C1-C6 hydroxy group. In some embodiments, R2 is a C2-C6 hydroxy group. In some embodiments, R2 is -(CH2)2OH. In some embodiments, R2 is -(CH2)6OH.

[0087] In some embodiments, the macromolecule further comprises a second structure of component (A), wherein the first structure of component (A) is different from the second structure of component (A).

[0088] In some embodiments, in the first structure of component (A), R1 is hydrogen and R2 is a C2 alkylamine, and in the second structure of component (A), R1 is hydrogen and R2 is a C1-C6 hydroxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, two or more fused 5- to 6-membered rings, or a monosaccharide. In some embodiments, in the second structure of component (A), R2 is a C1-C6 hydroxy group. In some embodiments, R2 is a C2 alkyl hydroxy group.

[0089] In some embodiments, in the second structure of component (A), R2 is an optionally substituted aryl group. In some embodiments, R2 is 2-fluorotoluene.

[0090] In some embodiments, in the second structure of component (A), R2 is an optionally substituted heteroaryl group. In some embodiments, R2 is 1-propylimidazole.

[0091] In some embodiments, in the second structure of component (A), R2 is two or more fused 5- to 6-membered rings. In some embodiments, R2 is three fused 6-membered rings.

[0092] In some embodiments, in the second structure of component (A), R2 is a monosaccharide. In some embodiments, R2 is d-glucose.

[0093] In some embodiments, B is and A is

[0094] In some embodiments, R1 is hydrogen and R2 is selected from C1-C 12 amine, an optionally substituted C3-C6 bicycloalkylmethane, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted C3-C6 heterocycloalkyl, a C1-C6 hydroxy group, a C1-C6 ether, an optionally substituted -C1-C6 disulfide, an optionally substituted succinate, an optionally substituted C1-C6 alkylamine, a C1-C6 alkylacetamide, boric acid, or a C1-C6 alkylguanidine.

[0095] In some embodiments, R2 is an optionally substituted dicyclohexylmethane. In some embodiments, R2 is aminodicyclohexylmethane. In some embodiments, R2 is an optionally substituted aryl. In some embodiments, R2 is a halotoluene. In some embodiments, R2 is 2-fluorotoluene. In some embodiments, R2 is boric acid. In some embodiments, R2 is phenylboronic acid. In some embodiments, R2 is a C1-C6 hydroxy group. In some embodiments, R2 is -(CH2)2OH. In some embodiments, R2 is a C1-C6 ether. In some embodiments, R2 is -CH2OCH3, -CH2CH2OCH3, -CH2CH2OCH2CH3 or -CH2OCH2CH3. In some embodiments, R2 is -CH2CH2OCH3. In some embodiments, R2 is an optionally substituted di-C1-C6 alkyl disulfide. In some embodiments, R2 is -CH2CH2-S-S-CH2CH2NH2. In some embodiments, R2 is a C1-C6 thiol. In some embodiments, R2 is -(CH2)2SH. In some embodiments, R2 is an optionally substituted succinate. In some embodiments, R2 is -(CH2) 1-6 NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2)6NH(C=O)CH2CH2COOH. In some embodiments, R2 is a C1-C 12 amine. In some embodiments, R2 is a C6-C 12 amine. In some embodiments, R2 is a C 12 amine. In some embodiments, R2 is a C1-C6 guanidine. In some embodiments, R2 is -(CH2)2guanidine. In some embodiments, R2 is an optionally substituted heteroaryl. In some embodiments, R2 is -(CH2) 1-6 imidazole. In some embodiments, R2 is -(CH2)3imidazole.

[0096] In some embodiments, R1 is a succinate and R2 is an optionally substituted C3-C6 dicycloalkylmethane, an optionally substituted aryl, a C1-C6 thiol or an optionally substituted succinate. In some embodiments, R2 is dicyclohexylmethane succinate. In some embodiments, R2 is -(CH2) 1-12 NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2) 1-3 NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2)2NH(C=O)CH2CH2COOH. In some embodiments, R2 is -(CH2) 10-12NH(C=O)CH2CH2COOH. In some embodiments, R2 is wherein R2 is -(CH2) 12 NH(C=O)CH2CH2COOH. In some embodiments, R1 is C2-C 12 alkenyl succinate, and R2 is a substituted succinate. In some embodiments, R2 is C8 alkenyl succinate, and R2 is C8 alkenyl ethylamino succinate.

[0097] In some embodiments, each of R1 and R2 is nitrogen. In some embodiments, R1 and R2 together form an optionally substituted heterocycle. In some embodiments, the optionally substituted heterocycle is a triazole. In some embodiments, the optionally substituted triazole contains a benzylamide. In some embodiments, the benzylamide is halogenated. In some embodiments, R1 is a C1-C6 alkyl sulfone, and R2 is an optionally substituted C1-C6 alkylamine.

[0098] In some embodiments, R1 is -(CH2)3SOOOH, and R2 is -(CH2) 1-6 N(CH2CH2CH2SOOOH)2. In some embodiments, R2 is -

[0099] (CH2)2N(CH2CH2CH2SOOOH)2.

[0100] In some embodiments, R1 is a phthalate, and R2 is a C1-C6 aminophthalate. In some embodiments, R2 is a C2-C6 aminophthalate. In some embodiments, R2 is a C2 aminophthalate.

[0101] In some embodiments, B is and A is

[0102] In some embodiments, R4 is a C1-C6 thiol, and R5 is a succinate. In some embodiments, R4 is a C1-C3 thiol. In some embodiments, R4 is -(CH2)2SH. In some embodiments, p is 1.

[0103] In some embodiments, B is and A is In some embodiments, p is 1. In some embodiments, q is 2 or 3. In some embodiments, q is 2.

[0104] In some embodiments, B is and A is In some embodiments, p is 1. In some embodiments, q is 2 or 3. In some embodiments, q is 2.

[0105] In some embodiments, B is and A is In some embodiments, p is 1.

[0106] In some embodiments, R1 is hydrogen and R2 is an optionally substituted C1-C6 alkylamine. In some embodiments, R2 is -(CH2) 1-3 dimethylamine. In some embodiments, R2 is -(CH2)2dimethylamine.

[0107] In some embodiments, component (A) accounts for about 10 weight percent (wt%) to about 90 wt% of the macromolecule. In some embodiments, component (A) accounts for about 20 wt% to about 80 wt% of the macromolecule. In some embodiments, component (A) accounts for about 40 wt% to about 60 wt% of the macromolecule. In some embodiments, component (A) accounts for about 50 wt% of the macromolecule. In some embodiments, component (B) or component (B’) accounts for about 10 weight percent (wt%) to about 90 wt% of the macromolecule. In some embodiments, component (B) or component (B’) accounts for about 20 wt% to about 80 wt% of the macromolecule. In some embodiments, component (B) or component (B’) accounts for about 40 wt% to about 60 wt% of the macromolecule. In some embodiments, component (B) or component (B’) accounts for about 50 wt% of the macromolecule.

[0108] In some embodiments, the structures of component (A) and component (B) or component (B’) are selected from Table 2. In some embodiments, the structures of component (A) and component (B) or component (B’) are selected from

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] In one aspect, described herein is a surface comprising a moiety of formula (IV):

[0116]

[0117] Wherein Z is a linking moiety that includes a straight chain having from 2 to 20 atoms selected from carbon, oxygen, and nitrogen, and optional substituents on any carbon and any nitrogen atoms in the straight chain;

[0118] R1 is hydrogen, an optionally substituted succinate, an optionally substituted glutarate, an optionally substituted adipate, an optionally substituted pimelate, an optionally substituted suberate, an optionally substituted azelate, or an optionally substituted sebacate; and

[0119] R2 is an optionally substituted -C1-C6 disulfide or an optionally substituted C1-C6 thiol.

[0120] In some embodiments, Z is a C1-C6 alkyl. In some embodiments, Z is a C3 alkyl. In some embodiments, Z is

[0121] In some embodiments, Z is an optionally substituted C1-C8 alkoxy. In some embodiments, Z is an optionally substituted C1-C8 ether. In some embodiments, Z is a C1-C8 alkoxy substituted with a hydroxyl group. In some embodiments, Z is a C1-C8 ether substituted with a hydroxyl group. In some embodiments, Z is:

[0122]

[0123] In some embodiments, R1 is hydrogen and R2 is a substituted -C1-C6 alkyl disulfide. In some embodiments, R2 is a substituted di-C1-C6 alkyl disulfide. In some embodiments, R2 is -CH2CH2-S-S-CH2CH2NH2. In some embodiments, R1 is a succinate and R2 is a C1-C6 thiol. In some embodiments, R2 is -(CH2)2SH.

[0124] In some embodiments, described herein are macromolecules that include repeating units of a first component and crosslinked repeating units, wherein the first component includes the structure of component (A’): In some embodiments, each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1-C6 alkyl. In some embodiments, A is In some embodiments, G’ or W’ includes Q’. In some embodiments, Q’ is a peptide. In some embodiments, W’ is In some embodiments, G’ is

[0125] In some embodiments, a macromolecule is described herein that comprises repeating units of a first component and a second component, wherein the first component comprises the structure of component (A’) and the second component comprises the structure of (B). In some embodiments, component (A’) is In some embodiments, component (B) is In some embodiments, each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1-C6 alkyl group. In some embodiments, each of X1, X2, and X3 is independently selected from hydrogen or a C1-C6 alkyl group. In some embodiments, A’ is In some embodiments, G’ or W’ comprises Q’. In some embodiments, Q’ is a peptide. In some embodiments, B is In some embodiments, Z is a unit of monomer (A’) or (B). In some embodiments, q is an integer between 1 and 6. In some embodiments, p is an integer between 1 and 20. In some embodiments, W’ is In some embodiments, G’ is

[0126] In some embodiments, a macromolecule is provided herein that comprises repeating units of a first component and a second component, wherein the first component comprises the structure of component (A’) and the second component comprises the structure of (B’):

[0127]

[0128] wherein

[0129] each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1-C6 alkyl group;

[0130] each of X1, X2, and X3 is independently selected from hydrogen or a C1-C6 alkyl group;

[0131] A is

[0132] G’ or W’ comprises Q’;

[0133] Q’ is a peptide;

[0134] B is

[0135] q is an integer between 1 and 6; and

[0136] p is an integer between 1 and 20.

[0137] In some embodiments, described herein is a surface that includes a moiety of formula (IV’):

[0138]

[0139] In some embodiments, Z is a linking moiety that includes a straight chain having from 2 to 20 atoms selected from carbon, oxygen, and nitrogen, and optional substituents on any of the carbon and any nitrogen atoms in the straight chain. In some embodiments, R1’ is hydrogen or a succinate ester. In some embodiments, R2’ is a C1-C6 alkyl-G’. In some embodiments, G’ includes Q’. In some embodiments, Q’ is a peptide. In a specific embodiment, the peptide does not include cysteine. In some embodiments, G’ is

[0140] In some embodiments, the peptide includes up to about 40 amino acids. In some embodiments, the peptide includes at least about 20 amino acids. In some embodiments, the peptide includes a synthetic sequence. In some embodiments, the peptide includes non-natural amino acids.

[0141] In some embodiments, the macromolecule described herein may further include a peptide. In some embodiments, the peptide binds to the macromolecule by non-specific adsorption.

[0142] In one aspect, described herein is a system that includes a surface, a macromolecule described herein that is coupled to the surface and includes repeating units of (A’) and (B), wherein the peptide includes a binding site, and a protein that interacts with the peptide at the binding site.

[0143] In one aspect, described herein is a method that includes contacting a biological sample with a surface described herein; wherein the surface includes a peptide and the peptide is configured to bind to a protein, a plurality of biomolecules, or a portion thereof from the surface, and to identify at least a plurality of biomolecules or a portion thereof from the surface, wherein the plurality of biomolecules or a portion thereof includes one or more biomolecules from at least three different biomolecules in the biological sample. In some embodiments, the biomolecule is a protein. In some embodiments, the protein includes a target protein. In some embodiments, the protein includes a vacuolar lumen, a lysosomal lumen, a spliceosome tri-snRNP complex, a U4 / U6 xU5 tri-snRNP complex, a secretory granule lumen, an intracellular organelle lumen, a membrane raft, a spliceosome snRNP complex, a spermatoproteasome complex, or a Golgi lumen protein.

[0144] In one aspect, described herein is a surface that includes a macromolecule of any of the repeating units of the present disclosure, wherein the macromolecule is immobilized on a modified surface.

[0145] In some embodiments, the macromolecule is covalently conjugated to the surface. In some embodiments, the macromolecule is electrostatically coupled to the surface. In some embodiments, the macromolecule is coupled to the surface via a polymerization event. In some embodiments, the polymerization event includes reacting with vinyl groups on the surface.

[0146] In some embodiments, the surface is a bead or a particle. In some embodiments, the surface is a particle. In some embodiments, the particle is a nanoparticle or a microparticle. In some embodiments, the particle has a diameter of about 200 nanometers (nm) to about 400 nm. In some embodiments, the particle is a superparamagnetic iron oxide particle. In some embodiments, the particle contains an iron oxide material. In some embodiments, the particle has an iron oxide core. In some embodiments, the particle has an iron oxide crystal embedded in a polystyrene core. In some embodiments, the particle contains an iron oxide core with a silica shell coating.

[0147] In some embodiments, the surface is selected from Table 3 or Table 4.

[0148] In some embodiments, the surface comprises a structure In some embodiments, the surface comprises a structure In some embodiments, the surface comprises a structure wherein represents the point of attachment of the units of component (A) or component (B) when the monomer is crosslinked. For example, the structure of can be represented by wherein represents the point of attachment of the units of component (A) or component (B). In some embodiments, the surface comprises a structure wherein represents the point of attachment of the units of component (A) or component (B) when the divinylbenzene (DVB) monomer is crosslinked. For example, the structure of is represented by wherein represents the point of attachment of the units of component (A) or component (B). In some embodiments, the surface comprises a structure wherein represents the point of attachment of the units of component (A) or component (B). In some embodiments, the surface comprises a structure wherein represents the point of attachment of the units of component (A) or component (B). In some embodiments, the surface comprises a structure wherein Represents the connection point of the unit of component (A) or component (B). In some embodiments, the surface comprises a structure wherein Represents the connection point of the unit of component (A) or component (B) when the monomer Is crosslinked. For example, The structure of can be represented by In some embodiments, the surface comprises a structure wherein Represents the connection point of the unit of component (A) or component (B). In some embodiments, the surface comprises a structure wherein Represents the connection point of the unit of component (A) or component (B).

[0149] In one aspect, a method is described herein that includes: (a) contacting a biological sample with a surface provided herein; (b) releasing a plurality of biomolecules or portions thereof from the surface; and (c) identifying at least a plurality of biomolecules or portions thereof from the surface. In some embodiments, the surface includes at least two unique surfaces.

[0150] In some embodiments, the at least two unique surfaces comprise a structure wherein Represents the connection point of the unit of component (A) or component (B). In some embodiments, the at least two unique surfaces comprise a structure wherein Represents the connection point of the unit of component (A) or component (B). In some embodiments, the at least two unique surfaces comprise a structure wherein Represents the connection point of the unit of component (A) or component (B). In some embodiments, the at least two unique surfaces comprise wherein Represents the connection point of the unit of component (A) or component (B). In some embodiments, the at least two unique surfaces comprise a structure wherein Represents the connection point of the unit of component (A) or component (B). In some embodiments, the at least two unique surfaces comprise a structure wherein Independently represents the connection point of the unit of component (A) or component (B).

[0151] In some embodiments, the biomolecule comprises a protein. In some embodiments, the protein comprises a targeting protein. In some embodiments, the protein comprises a vacuolar lumen, lysosomal lumen, spliceosome tri-snRNP complex, U4 / U6 xU5 tri-snRNP complex, secretory granule lumen, intracellular organelle lumen, membrane raft, spliceosome snRNP complex, sperm proteasome complex, or Golgi lumen protein.

[0152] In one aspect, the present disclosure describes a method of preparing a surface having a repeating unit comprising a first component and a second component, the method comprising: (a) providing a monomer mixture comprising a first monomer and a second monomer in a solvent, wherein the first monomer comprises a vinyl group and wherein the second monomer comprises an epoxy group; (b) contacting the surface with the monomer mixture to produce a reaction mixture; (c) initiating radical polymerization to produce a macromolecule immobilized on the surface; (d) contacting the macromolecule immobilized on the surface with an amine to produce an aminated macromolecule; and (e) optionally, contacting the aminated macromolecule with a compound comprising a succinate, phthalate, or propanesulfone.

[0153] In some embodiments, the surface is a bead or a particle. In some embodiments, the surface is a particle. In some embodiments, the particle is a nanoparticle or a microparticle. In some embodiments, the particle has a diameter of from about 200 nanometers (nm) to about 400 nm. In some embodiments, the particle is a superparamagnetic iron oxide particle. In some embodiments, the particle comprises an iron oxide material. In some embodiments, the particle has an iron oxide core. In some embodiments, the particle has an iron oxide crystal embedded in a polystyrene core. In some embodiments, the particle comprises an iron oxide core having a silica shell coating.

[0154] In some embodiments, the solvent is a polar solvent. In some embodiments, the solvent is acetonitrile, THF, or DMF.

[0155] In some embodiments, the radical polymerization is initiated with a radical initiator. In some embodiments, the radical initiator is AIBN.

[0156] In some embodiments, the method comprises contacting the macromolecule immobilized on the surface with a quencher after (c) and before (d). In some embodiments, the quencher is introduced into the reaction mixture when the macromolecule immobilized on the surface has a diameter of from about 300 nm to about 500 nm. In some embodiments, the diameter is from about 325 nm to about 375 nm. In some embodiments, the quencher comprises benzoquinone.

[0157] In some embodiments, the method further includes purifying the macromolecule immobilized on the surface. In some embodiments, purification includes washing the macromolecule immobilized on the surface with THF or ethanol.

[0158] In some embodiments, the first monomer is divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), or N,N'-methylenebisacrylamide (MBA). In some embodiments, the second monomer comprises glycidyl methacrylate. In some embodiments, the amine is a C1-C 12 alkylamine, a C1-C6 hydroxyamine, or a C1-C6 alkoxyethylamine. In some embodiments, the C1-C 12 alkylamine is diethylamine. In some embodiments, the C1-C6 hydroxyamine is ethanolamine or hexanolamine. In some embodiments, the C1-C6 alkoxyethylamine is methoxyethylamine.

[0159] In some embodiments, the method includes (e) contacting the aminated macromolecule with a compound comprising a succinate, a phthalate, a thiol, or propylsulfone. In some embodiments, the compound comprises a succinate. In some embodiments, the succinate is an C8 alkenyl succinate or an C8 alkenyl ethylamino succinate.

[0160] In some embodiments, the compound comprises a thiol. In some embodiments, the thiol is a C2 alkyl thiol.

[0161] In some embodiments, the compound comprises a phthalate. In some embodiments, the phthalate is a C1-C6 amino phthalate.

[0162] In some embodiments, the compound comprises propylsulfone. In some embodiments, the propylsulfone is dipropylsulfone ethylamine.

[0163] In some embodiments, the first monomer accounts for about 10 weight percent (wt%) to about 90 wt% of the monomer mixture. In some embodiments, the first monomer accounts for about 20 wt% to about 80 wt% of the monomer mixture. In some embodiments, the first monomer accounts for about 40 wt% to about 60 wt% of the monomer mixture. In some embodiments, the first monomer accounts for about 50 wt% of the monomer mixture. In some embodiments, the second monomer accounts for about 10 weight percent (wt%) to about 90 wt% of the monomer mixture. In some embodiments, the second monomer accounts for about 20 wt% to about 80 wt% of the monomer mixture. In some embodiments, the second monomer accounts for about 40 wt% to about 60 wt% of the monomer mixture. In some embodiments, the second monomer accounts for about 50 wt% of the monomer mixture.

[0164] In one aspect, the present disclosure describes a method for preparing a surface having repeating units of a first component and a second component, the method comprising: (a) providing a monomer mixture comprising a first monomer and a second monomer in a solvent, wherein the first monomer comprises a vinyl group and the second monomer comprises an epoxy group; (b) contacting the surface with the monomer mixture to produce a reaction mixture; (c) initiating radical polymerization to produce macromolecules immobilized on the surface; (d) contacting the macromolecules immobilized on the surface with an azide salt to produce macromolecules containing azide; and (e) optionally, contacting the macromolecules containing azide with a molecule containing an alkyne to form macromolecules containing triazole.

[0165] In some embodiments, the surface is a bead or a particle. In some embodiments, the surface is a particle. In some embodiments, the particle is a nanoparticle or a microparticle. In some embodiments, the particle has a diameter of from about 200 nanometers (nm) to about 400 nanometers. In some embodiments, the particle is a superparamagnetic iron oxide particle. In some embodiments, the particle comprises an iron oxide material. In some embodiments, the particle has an iron oxide core. In some embodiments, the particle has iron oxide crystals embedded in a polystyrene core. In some embodiments, the particle comprises an iron oxide core having a silica shell coating.

[0166] In some embodiments, the solvent is a polar solvent. In some embodiments, the solvent is acetonitrile, THF, or DMF.

[0167] In some embodiments, the radical polymerization is initiated with a radical initiator. In some embodiments, the radical initiator is AIBN.

[0168] In some embodiments, the method comprises contacting the macromolecules immobilized on the surface with a quencher after (c) and before (d). In some embodiments, when the macromolecules immobilized on the surface have a diameter of from about 300 nm to about 500 nm, the quencher is introduced into the reaction mixture. In some embodiments, the diameter is from about 325 nm to about 375 nm. In some embodiments, the quencher comprises benzoquinone.

[0169] In some embodiments, the method further comprises purifying the macromolecules immobilized on the surface. In some embodiments, the purification comprises washing the macromolecules immobilized on the surface with THF or ethanol.

[0170] In one aspect, the present disclosure describes a kit for identifying biomolecules in a biological sample, the kit comprising one or more surfaces of the present disclosure.

[0171] In one aspect, the present disclosure describes a composition for identifying biomolecules in a biological sample, the composition comprising one or more surfaces of the present disclosure and a biological sample in contact with the surface.

[0172] In some embodiments, the biological sample is plasma, serum, or blood.

[0173] In some embodiments, the one or more surfaces include at least 2 distinct surfaces, at least 3 distinct surfaces, at least 4 distinct surfaces, at least 5 distinct surfaces, at least 6 distinct surfaces, at least 7 distinct surfaces, at least 8 distinct surfaces, at least 9 distinct surfaces, at least 10 distinct surfaces, at least 11 distinct surfaces, at least 12 distinct surfaces, at least 13 distinct surfaces, at least 14 distinct surfaces, at least 15 distinct surfaces, at least 20 distinct surfaces, at least 25 surfaces, or at least 30 distinct surfaces.

[0174] In some embodiments, the one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least one physicochemical property and differ in at least one physicochemical property such that the first distinct surface and the second distinct surface are different.

[0175] In some embodiments, the one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least two physicochemical properties and differ in at least two physicochemical properties such that the first distinct surface and the second distinct surface are different.

[0176] In some embodiments, the one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least one physicochemical property and differ in at least two physicochemical properties such that the first distinct surface and the second distinct surface are different.

[0177] In some embodiments, the one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least two physicochemical properties and differ in at least one physicochemical property such that the first distinct surface and the second distinct surface are different.

[0178] In some embodiments, the physicochemical properties include size, charge, core material, shell material, porosity, or surface hydrophobicity.

[0179] In some embodiments, the size is a diameter or radius measured by dynamic light scattering, SEM, TEM, or any combination thereof.

[0180] In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface comprise a carboxylate material, wherein the first distinct particles are microparticles, and wherein the second distinct surface is a nanoparticle.

[0181] In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface have a surface charge of 0 mV to -50 mV, wherein the first distinct surface has a diameter of less than 200 nm, and wherein the second distinct surface has a diameter of greater than 200 nm.

[0182] In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface have a diameter of 100 to 400 nm, wherein the first distinct surface has a positive surface charge, and wherein the second distinct surface has a neutral surface charge.

[0183] In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface are nanoparticles, wherein the first distinct surface has a surface charge of less than -20 mV, and the second distinct surface has a surface charge of greater than -20 mV.

[0184] In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface are microparticles, wherein the first distinct surface has a negative surface charge, and wherein the second distinct surface has a positive surface charge.

[0185] In some embodiments, one or more surfaces include a subset of negatively charged nanoparticles, wherein each particle in the subset is distinct in at least one surface chemical group.

[0186] In some embodiments, one or more surfaces include a first distinct surface, a second particle, and a third distinct surface, wherein the first distinct surface, the second distinct surface, and the third distinct surface comprise an iron oxide core, a polymer shell, and have a diameter of less than about 500 nm, and wherein the first distinct surface includes a negative charge, the second distinct surface includes a positive charge, and the third distinct surface includes a neutral charge, wherein the diameter is the average diameter as measured by dynamic light scattering.

[0187] In some embodiments, at least one unique surface among one or more surfaces is a superparamagnetic iron oxide particle. In some embodiments, each surface among one or more surfaces comprises an iron oxide material. In some embodiments, at least one unique surface among one or more surfaces has an iron oxide core. In some embodiments, at least one unique surface among one or more surfaces has iron oxide crystals embedded in a polystyrene core. In some embodiments, each unique surface among one or more surfaces is a superparamagnetic iron oxide particle. In some embodiments, each unique surface among one or more surfaces comprises an iron oxide core. In some embodiments, each unique surface among one or more surfaces has iron oxide crystals embedded in a polystyrene core. In some embodiments, at least one surface among one or more surfaces comprises an iron oxide core with a silica shell coating.

[0188] In one aspect, the present disclosure describes a system for identifying biomolecules in a biological sample, the composition comprising: (i) a macromolecule immobilized on a surface of the present disclosure; (ii) a suspension solution; (iii) a biological sample comprising a certain concentration of protein; and (iv) an automated system comprising a network of units having differentiated functions in differentiating the states of complex biological samples using a plurality of surfaces having different physicochemical properties, and wherein the automated system is programmed to perform a series of steps.

[0189] In some embodiments, the network of units comprises:

[0190] The first unit comprises a multi-channel fluid transfer device for transferring fluid between units within the system;

[0191] The second unit comprises a support for storing a plurality of biological samples;

[0192] The third unit comprises a support for a sensor array plate having partitions, the partitions comprising a plurality of particles having surfaces with different physicochemical properties for detecting binding interactions between an analyte population within a complex biological sample and the plurality of particles;

[0193] The fourth unit comprises a support for storing a plurality of reagents;

[0194] The fifth unit comprises a support for storing reagents to be disposed of; and

[0195] The sixth unit comprises a support for storing consumables used by the multi-channel fluid transfer device.

[0196] In some embodiments, a series of steps includes: (i) contacting a biological sample with a designated partition of a sensor array; (ii) incubating the biological sample with a plurality of particles contained within the partition of the sensor array plate; (iii) removing all components from the partition except for the plurality of particles and the population of analytes that interact with the particles; and (iv) preparing a sample for mass spectrometry.

[0197] In some embodiments, i.-iii. are incubated at a temperature of about 20 degrees Celsius to about 100 degrees Celsius.

[0198] In some embodiments, the suspension solution contains Tris EDTA 150 mM KCl, 0.05% CHAPS buffer. In some embodiments, the suspension solution contains 10 mM Tris HCl pH 7.4, 1 mM EDTA.

[0199] In one aspect, a method for identifying proteins in a sample is described herein, the method comprising: (a) incubating one or more surfaces of the present disclosure with a biological sample containing biomolecules to form a biomolecular corona; (b) isolating at least a portion of the biomolecules in the biomolecular corona; and (c) assaying the biomolecular corona.

[0200] In some embodiments, the assay is capable of identifying 1 to 20,000 protein groups. In some embodiments, the assay is capable of identifying 1,000 to 10,000 protein groups. In some embodiments, the assay is capable of identifying 1,000 to 5,000 protein groups. In some embodiments, the assay is capable of identifying 1,200 to 2,200 protein groups.

[0201] In some embodiments, the protein group contains a peptide sequence with a minimum length of 7 amino acid residues.

[0202] In some embodiments, the assay is capable of identifying 1,000 to 10,000 proteins. In some embodiments, the assay is capable of identifying 1,800 to 5,000 proteins.

[0203] In some embodiments, the sample includes a plurality of samples. In some embodiments, the plurality of samples includes at least two or more spatially separated samples.

[0204] In some embodiments, the incubation includes contacting at least two or more spatially separated samples with one or more surfaces simultaneously.

[0205] In some embodiments, the isolation includes magnetically isolating unbound proteins in at least two or more spatially separated samples of the plurality of samples from one or more surfaces simultaneously.

[0206] In some embodiments, the assay comprises assaying a plurality of biomolecular coronas to simultaneously identify proteins in at least two or more spatially separated samples.

[0207] In some embodiments, the method further comprises repeating the method recited in any one of claims y-yy, wherein, when repeated, the incubation, isolation, and assay result in a percentile-normalized coefficient of variation (QNCV) of 20% or less, as determined by comparing peptide mass spectrometry signatures from at least three complete assay replicates for each of one or more surfaces.

[0208] In some embodiments, when repeated, the incubation, isolation, and assay result in a percentile-normalized coefficient of variation (QNCV) of 10% or less, as determined by comparing peptide mass spectrometry signatures from at least three complete assay replicates for each of one or more surfaces.

[0209] In some embodiments, the assay is capable of identifying proteins in a dynamic range of at least 7, at least 8, at least 9, or at least 10.

[0210] In some embodiments, the method further comprises washing the one or more surfaces at least once or at least twice after magnetically isolating the one or more surfaces from unbound proteins.

[0211] In some embodiments, after the assay, the method further comprises lysing the proteins in the plurality of biomolecular coronas.

[0212] In some embodiments, the method further comprises digesting the proteins in the plurality of biomolecular coronas to generate digested peptides. In some embodiments, the method further comprises purifying the digested peptides.

[0213] In some embodiments, the assay comprises identifying the proteins in the sample using mass spectrometry.

[0214] In some embodiments, the assay is performed in about 2 to about 4 hours.

[0215] In some embodiments, the method is performed in about 1 to about 20 hours. In some embodiments, the method is performed in about 2 to about 10 hours. In some embodiments, the method is performed in about 4 to about 6 hours.

[0216] In some embodiments, the isolation takes no more than about 30 minutes, no more than about 15 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 2 minutes.

[0217] In some embodiments, the plurality of samples includes at least 10 spatially isolated samples, at least 50 spatially isolated samples, at least 100 spatially isolated samples, at least 150 spatially isolated samples, at least 200 spatially isolated samples, at least 250 spatially isolated samples, or at least 300 spatially isolated samples.

[0218] In some embodiments, the plurality of samples includes at least 96 samples.

[0219] In some embodiments, the one or more surfaces include at least 2 distinct surfaces, at least 3 distinct surfaces, at least 4 distinct surfaces, at least 5 distinct surfaces, at least 6 distinct surfaces, at least 7 distinct surfaces, at least 8 distinct surfaces, at least 9 distinct surfaces, at least 10 distinct surfaces, at least 11 distinct surfaces, at least 12 distinct surfaces, at least 13 distinct surfaces, at least 14 distinct surfaces, at least 15 distinct surfaces, at least 20 distinct surfaces, at least 25 distinct surfaces, or at least 30 distinct surfaces.

[0220] In some embodiments, the one or more surfaces include at least 10 distinct surfaces.

[0221] In some embodiments, at least two spatially isolated samples differ in at least one physicochemical property.

[0222] In some embodiments, the one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least one physicochemical property and differ in at least one physicochemical property such that the first distinct surface and the second distinct surface are different.

[0223] In some embodiments, the one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least two physicochemical properties and differ in at least two physicochemical properties such that the first distinct surface and the second distinct surface are different.

[0224] In some embodiments, the one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least one physicochemical property and differ in at least two physicochemical properties such that the first distinct surface and the second distinct surface are different.

[0225] In some embodiments, the one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least two physicochemical properties and differ in at least one physicochemical property such that the first distinct surface and the second distinct surface are different.

[0226] In some embodiments, the physicochemical properties include size, charge, core material, shell material, porosity, or surface hydrophobicity.

[0227] In some embodiments, the size is a diameter or radius as measured by dynamic light scattering, SEM, TEM, or any combination thereof.

[0228] In some embodiments, one or more surfaces include a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface comprise a carboxylate material, wherein the first unique particles are microparticles, and wherein the second unique surface is a nanoparticle.

[0229] In some embodiments, one or more surfaces include a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface have a surface charge of 0 mV to -50 mV, wherein the first unique surface has a diameter less than 200 nm, and wherein the second unique surface has a diameter greater than 200 nm.

[0230] In some embodiments, one or more surfaces include a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface have a diameter of 100 to 400 nm, wherein the first unique surface has a positive surface charge, and wherein the second unique surface has a neutral surface charge.

[0231] In some embodiments, one or more surfaces include a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface are nanoparticles, wherein the first unique surface has a surface charge less than -20 mV, and the second unique surface has a surface charge greater than -20 mV.

[0232] In some embodiments, one or more surfaces include a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface are microparticles, wherein the first unique surface has a negative surface charge, and wherein the second unique surface has a positive surface charge.

[0233] In some embodiments, one or more surfaces include a subset of negatively charged nanoparticles, wherein each particle in the subset is distinct in at least one surface chemical group.

[0234] In some embodiments, one or more surfaces include a first unique surface, a second particle, and a third unique surface, wherein the first unique surface, the second unique surface, and the third unique surface comprise an iron oxide core, a polymer shell, and have a diameter less than about 500 nm, and wherein the first unique surface includes a negative charge, the second unique surface includes a positive charge, and the third unique surface includes a neutral charge, wherein the diameter is the average diameter as measured by dynamic light scattering.

[0235] In some embodiments, at least one unique surface among one or more surfaces is a nanoparticle. In some embodiments, at least one unique surface among one or more surfaces is a microparticle. In some embodiments, at least one unique surface among one or more surfaces is a superparamagnetic iron oxide particle. In some embodiments, each particle of one or more surfaces comprises an iron oxide material. In some embodiments, at least one unique surface among one or more surfaces has an iron oxide core. In some embodiments, at least one unique surface among one or more surfaces has an iron oxide crystal embedded in a polystyrene core. In some embodiments, each unique surface among one or more surfaces is a superparamagnetic iron oxide particle. In some embodiments, each unique surface among one or more surfaces comprises an iron oxide core. In some embodiments, each unique surface among one or more surfaces has an iron oxide crystal embedded in a polystyrene core. In some embodiments, at least one unique surface among one or more surfaces comprises a carboxylated polymer, an aminated polymer, a zwitterionic polymer, or any combination thereof. In some embodiments, at least one surface among one or more surfaces comprises an iron oxide core having a silica shell coating.

[0236] In some embodiments, at least one unique surface among one or more surfaces comprises a negative surface charge. In some embodiments, at least one unique surface among one or more surfaces comprises a positive surface charge. In some embodiments, at least one unique surface among one or more surfaces comprises a neutral surface charge.

[0237] In one aspect, the use of the macromolecules of the present disclosure in methods for identifying proteins in a biological sample is described herein.

[0238] In one aspect, the use of the macromolecules of the present disclosure for adsorbing proteins in a biological sample is described herein.

[0239] In one aspect, the use of the macromolecules of the present disclosure in methods for identifying proteins in a biological sample is described herein.

[0240] In one aspect, the use of the macromolecules of the present disclosure for adsorbing proteins in a biological sample is described herein.

[0241] Incorporated by reference

[0242] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0243] The novel features of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present disclosure may be better understood by reference to the following detailed description and the accompanying drawings, in which illustrative embodiments that utilize the principles of the present disclosure are described in the detailed description and shown in the drawings:

[0244] Figure 1 Shows the general design space of the epoxidized nanoparticle platform after addition of functionalized amines.

[0245] Figure 2 Shows the available transformation schemes after nanoparticle epoxidation, including reactions with glycidyl and azides to obtain different functionalities.

[0246] Figure 3 Shows a schematic diagram of nanoparticles coated with SiO2 and then with a cross-linked polymer to obtain multiple functionalities.

[0247] Figure 4 Shows the number of protein groups adhering to different surfaces and the Jaccard index indicating the difference between two surfaces in the adhering protein groups.

[0248] Figure 5 Shows the number of protein groups adhering to different surfaces and the Jaccard index indicating the difference between two surfaces in the adhering protein groups.

[0249] Figures 6A - 6C Shows Cy7-maleimide functionalizer ( Figure 6A ), relative fluorescence units as a function of concentration to determine the number of thiols per nanoparticle ( Figure 6B ) and the chemical structure of the functionalized thiolated nanoparticles ( Figure 6C ).

[0250] Figures 7A - 7C Shows Cy7-DBCO functionalizer ( Figure 7A ), relative fluorescence units as a function of concentration to determine the number of azides per nanoparticle ( Figure 7B ) and the chemical structure of the functionalized azide-modified nanoparticles ( Figure 7C ).

[0251] Figure 8 Shows the general reaction scheme for thiolated nanoparticles functionalized with peptides.

[0252] Figures 9A - 9B Shows thermogravimetric analysis (TGA) of the surface before peptide modification ( Figure 9A ) and after peptide modification ( Figure 9B ).

[0253] Figure 10 Shows the chemical structure of thiolated nanoparticles functionalized with SMCC-MagaininII.

[0254] Figure 11 Shows the reaction scheme of a peptide modified with SMCC modifying a thiolated surface.

[0255] Figure 12 Shows the reaction scheme of a peptide modified with DBCO modifying an azide-modified surface.

[0256] Figures 13A - 13B Shows a schematic diagram of modular units that can be used for peptide synthesis according to some embodiments ( Figure 13A ), and examples of binding molecules that include modular units and are configured for intermolecular or intramolecular exchange according to some embodiments ( Figure 13B ).

[0257] Figure 14 Shows examples of linear and cyclic peptides that can be coupled to the particle surface according to some embodiments.

[0258] Figure 15 Shows an example of a reaction scheme for obtaining a surface functionalized with thiol and carboxylic acid. Detailed Description

[0259] Although various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present disclosure, those skilled in the art can make various changes, alterations, and substitutions. It should be understood that various alternatives to the embodiments of the present disclosure described herein can be employed. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0260] Definitions

[0261] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications cited herein are incorporated by reference.

[0262] "Alkyl" refers to an optionally substituted straight-chain or optionally substituted branched-chain saturated hydrocarbon monovalent radical, and preferably has from one to fifteen carbon atoms (i.e., C1-C 15 alkyl). In certain embodiments, alkyl contains from one to thirteen carbon atoms (i.e., C1-C 13(alkyl). In certain embodiments, the alkyl contains one to eight carbon atoms (i.e., C1-C8 alkyl). In other embodiments, the alkyl contains one to five carbon atoms (i.e., C1-C5 alkyl). In other embodiments, the alkyl contains one to four carbon atoms (i.e., C1-C4 alkyl). In other embodiments, the alkyl contains one to three carbon atoms (i.e., C1-C3 alkyl). In other embodiments, the alkyl contains 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, 2, or 3 carbon atoms. In other embodiments, the alkyl contains one carbon atom (i.e., C1 alkyl). In other embodiments, the alkyl contains five to fifteen carbon atoms (i.e., C5-C 15 (alkyl). In other embodiments, the alkyl contains five to eight carbon atoms (i.e., C5-C8 alkyl). In other embodiments, the alkyl contains two to five carbon atoms (i.e., C2-C5 alkyl). In other embodiments, the alkyl contains three to five carbon atoms (i.e., C3-C5 alkyl). In certain embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (isopropyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). In other embodiments, 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, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, and hexyl, and longer alkyl groups such as heptyl, octyl, etc. The alkyl is connected to the rest of the molecule by a single bond. Unless otherwise expressly stated in this specification, the alkyl group is optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxy, sulfone, mercapto, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the alkyl is optionally substituted with oxo, halogen, -CN, -CF3, -OH, -OMe, -NH2, -NO2, or -C≡CH. In some embodiments, the alkyl is optionally substituted with oxo, halogen, -CN, -CF3, -OH, or -OMe. In some embodiments, the alkyl is optionally substituted with a halogen (such as F). In some embodiments, the alkyl is unsubstituted.

[0263] As used herein, C1-Cx (or C 1-x ) includes C1-C2, C1-C3... C1-C x . By way of example only, a group designated as "C1-C4" means that there are one to four carbon atoms in this part, that is, a group containing 1, 2, 3, or 4 carbon atoms. Thus, by way of example only, "C1-C4 alkyl" means that there are one to four carbon atoms in the alkyl group, that is, the alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Additionally, by way of example, C0-C2 alkylene includes direct bonds, -CH2-, and -CH2CH2- linking bonds. Additionally, by way of example, "C1-C6 hydroxy" means that there are one to six carbon atoms in the alkyl group, which is substituted by a hydroxy group (-OH).

[0264] As used herein, C1-C x (or C 1-x ) hydroxy includes C1-C x alkyl substituted by a hydroxy group (-OH). As used herein, C1-C x (or C 1-x ) heterocycloalkyl includes C1-C x alkyl substituted by a heterocycloalkyl group. As used herein, C1-C x (or C 1-x ) bicycloalkylmethane includes C1-C x alkyl substituted by a bicycloalkylmethane group. As used herein, C1-C x (or C 1-x ) alkylguanidine includes C1-C x alkyl substituted by an alkylguanidine group. As used herein, C1-C x (or C 1-x ) ether includes C1-C x alkyl in which the alkyl chain is substituted by -O- (e.g., -(CH2) x -O-(CH2) y -). As used herein, C1-C x (or C 1-x ) disulfide includes C1-C x alkyl in which the alkyl chain is substituted by -S-S- (e.g., -(CH2) x -S-S-(CH2) y -). As used herein, C1-C x (or C 1-x ) thiol includes C1-C x (or C 1-x ) alkyl substituted by a thiol group. As used herein, C1-C x (or C 1-x ) alkylamine includes C1-Cx (or C 1-x ) alkyl. As used herein, C1-C x (or C 1-x ) alkyl acetamide includes C1-C substituted by alkyl acetamide x (or C 1-x ) alkyl. As used herein, C1-C x (or C 1-x ) aminophthalate includes C1-C substituted by aminophthalate x (or C 1-x ) alkyl. As used herein, C1-C x (or C 1-x ) alkyl sulfone includes C1-C substituted by sulfone x (or C 1-x ) alkyl (e.g., -(CH2)3SOOOH or -(CH2)3S(=O)2OH). As used herein, C1-C x (or C 1-x ) alkylamine includes C1-C in which the alkyl chain is substituted by amine x (or C 1-x ) alkyl (e.g., -(CH2) x N(R)(CH2) y -), and may include secondary, tertiary or quaternary amines. As used herein, C1-C x (or C 1-x ) alkylamine also includes C1-C substituted by amine x (or C 1-x ) alkyl, e.g., -CH2CH2NH2.

[0265] "Alkoxy" refers to a radical bonded through an oxygen atom of the formula -O-alkyl, where the alkyl is an alkyl chain as defined above. Unless otherwise explicitly stated in this specification, the alkoxy group may optionally be substituted by, for example, oxo, halogen, amino, nitrile, nitro, hydroxy, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the alkoxy is optionally substituted by oxo, halogen, -CN, -CF3, -OH, -OMe, -NH2 or -NO2. In some embodiments, the alkoxy is optionally substituted by oxo, halogen, -CN, -CF3, -OH or -OMe. In some embodiments, the alkoxy is optionally substituted by halogen. In some embodiments, the alkoxy is unsubstituted.

[0266] "Alkenyl" refers to an optionally substituted straight-chain or branched-chain hydrocarbon radical group that contains at least one carbon-carbon double bond and preferably has two to twelve carbon atoms (i.e., C2-C 12(alkenyl). In certain embodiments, the alkenyl contains two to eight carbon atoms (i.e., C2-C8 alkenyl). In certain embodiments, the alkenyl contains four to eight carbon atoms (i.e., C4-C6 alkenyl). In other embodiments, the alkenyl contains six to eight carbon atoms (i.e., C6-C8 alkenyl). In certain embodiments, the alkenyl contains at least one double bond at the end of the carbon chain. In other embodiments, the alkenyl contains at least one double bond in the middle of the carbon chain. The group can be in the cis configuration or the trans configuration for the double bond, and is understood to include both isomers. Examples include but are not limited to vinyl (-CH=CH2), 1-propenyl (-CH2CH=CH2), isopropenyl [-C(CH3)=CH2], butenyl, 1,3-butadienyl, etc. Whenever a numerical range such as "C2-C6 alkenyl" appears herein, it means that the alkenyl group can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. Unless otherwise expressly stated in this specification, the alkenyl group is optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxy, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the alkenyl is optionally substituted with oxo, halogen, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkenyl is optionally substituted with oxo, halogen, -CN, -CF3, -OH, or -OMe. In some embodiments, the alkenyl is optionally substituted with halogen. The alkenyl is connected to the rest of the molecule by a single bond, such as ethenyl / vinyl, prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, pent-1,4-dienyl, etc. Unless otherwise expressly stated in this specification, the alkenyl group is optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxy, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the alkenyl is optionally substituted with oxo, halogen, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkenyl is optionally substituted with oxo, halogen, -CN, -CF3, -OH, or -OMe. In some embodiments, the alkenyl is optionally substituted with halogen. In some embodiments, the alkenyl is unsubstituted.

[0267] "Alkynyl" means an optionally substituted straight-chain or branched-chain hydrocarbon radical group that contains at least one carbon-carbon triple bond and preferably has two to twelve carbon atoms (i.e., C2-C 12alkynyl). In certain embodiments, the alkynyl contains two to eight carbon atoms (i.e., C2-C8 alkynyl). In other embodiments, the alkynyl contains two to six carbon atoms (i.e., C2-C6 alkynyl). In other embodiments, the alkynyl contains two to four carbon atoms (i.e., C2-C4 alkynyl). Whenever used herein, a numerical range such as "C2-C6 alkynyl" means that the alkynyl group can consist of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. The alkynyl is connected to the rest of the molecule by a single bond, such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, 2-propynyl, 2-butynyl, 1,3-butadiynyl, etc. Unless otherwise expressly stated in this specification, the alkynyl is optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxy, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the alkynyl is optionally substituted with oxo, halogen, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, the alkynyl is optionally substituted with oxo, halogen, -CN, -CF3, -OH, or -OMe. In some embodiments, the alkynyl is optionally substituted with halogen. In some embodiments, the alkynyl is unsubstituted.

[0268] "Alkylene" or "alkylene chain" refers to an optionally substituted straight-chain or branched-chain divalent hydrocarbon chain that connects the remainder of the molecule to a radical group containing no unsaturation and preferably has from one to twelve carbon atoms, such as methylene, ethylene, propylene, n-butylene, etc. The alkylene chain is connected to the remainder of the molecule by a single bond and to the radical group by a single bond. The points of attachment of the alkylene chain to the remainder of the molecule and to the radical group can be through any two carbon atoms within the chain. In certain embodiments, the alkylene contains from one to ten carbon atoms (i.e., C1-C8 alkylene). In certain embodiments, the alkylene contains from one to eight carbon atoms (i.e., C1-C8 alkylene). In other embodiments, the alkylene contains from one to five carbon atoms (i.e., C1-C5 alkylene). In other embodiments, the alkylene contains from one to four carbon atoms (i.e., C1-C4 alkylene). In other embodiments, the alkylene contains from one to three carbon atoms (i.e., C1-C3 alkylene). In other embodiments, the alkylene contains from one to two carbon atoms (i.e., C1-C2 alkylene). In other embodiments, the alkylene contains one carbon atom (i.e., C1 alkylene). In other embodiments, the alkylene contains from five to eight carbon atoms (i.e., C5-C8 alkylene). In other embodiments, the alkylene contains from two to five carbon atoms (i.e., C2-C5 alkylene). In other embodiments, the alkylene contains from three to five carbon atoms (i.e., C3-C5 alkylene). Unless otherwise expressly stated in this specification, the alkylene may be optionally substituted, for example, by oxo, halogen, amino, nitrile, nitro, hydroxy, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the alkylene is optionally substituted by oxo, halogen, -CN, -CF3, -OH, -OMe, -NH2 or -NO2. In some embodiments, the alkylene is optionally substituted by oxo, halogen, -CN, -CF3, -OH or -OMe. In some embodiments, the alkylene is optionally substituted by halogen. In some embodiments, the alkylene is -CH2-, -CH2CH2- or -CH2CH2CH2-. In some embodiments, the alkylene is -CH2-. In some embodiments, the alkylene is -CH2CH2-. In some embodiments, the alkylene is -CH2CH2CH2-. In some embodiments, the alkylene is unsubstituted.

[0269] "Aryl" refers to a radical derived from a hydrocarbon ring system containing at least one aromatic ring. In some embodiments, aryl contains hydrogen and 5 to 30 carbon atoms. The aryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic system, which can include a fused ring system (when fused to a cycloalkyl or heterocycloalkyl ring, the aryl is bonded through an aromatic ring atom) or a bridged ring system. In some embodiments, aryl is a 6- to 10-membered aryl. In some embodiments, aryl is a 6-membered aryl. Aryl radicals include, but are not limited to, arylene radicals derived from anthrylene, naphthylene, phenanthrylene, anthracene, azulene, benzene, aryl radicals derived from the hydrocarbon ring systems of fluoranthene, fluorene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene and benzophenanthrene. In some embodiments, aryl is phenyl. Unless otherwise expressly stated in this specification, aryl may optionally be substituted, for example, by halogen, amino, alkylamino, aminoalkyl, nitrile, nitro, hydroxy, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, -S(O)2NH-C1-C6 alkyl, etc. In some embodiments, aryl is optionally substituted by halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, -NO2, -S(O)2NH2, -S(O)2NHCH3, -S(O)2NHCH2CH3, -S(O)2NHCH(CH3)2, -S(O)2N(CH3)2 or -S(O)2NHC(CH3)3. In some embodiments, aryl is optionally substituted by halogen, methyl, ethyl, -CN, -CF3, -OH or -OMe. In some embodiments, aryl is optionally substituted by halogen. In some embodiments, aryl is substituted by alkyl, alkenyl, alkynyl, haloalkyl or heteroalkyl, where each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl is independently unsubstituted or substituted by halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2 or -NO2. In some embodiments, aryl is unsubstituted.

[0270] "Aralkyl" refers to a radical of the formula -R c -aryl, where R c is an alkylene chain as defined above, such as methylene, ethylene, etc.

[0271] "Arenyl" refers to a radical of the formula -R d -aryl, where R d is an alkenylene chain as defined above. "Arylalkynyl" refers to a radical of the formula -R e -aryl, where R e is an alkynylene chain as defined above.

[0272] "Carbocyclic ring" means a saturated, unsaturated or aromatic ring in which each atom of the ring is carbon. The carbocyclic ring may include 3- to 10-membered monocyclic rings and 6- to 12-membered bicyclic rings (such as spiro rings, fused rings or bridged rings). Each ring of the bicyclic carbocyclic ring may be selected from saturated, unsaturated and aromatic rings. An aromatic ring (e.g., phenyl) may be fused to a saturated or unsaturated ring (e.g., cyclohexane, cyclopentane or cyclohexene). Any combination of saturated, unsaturated and aromatic bicyclic rings is included in the definition of the carbocyclic ring as valence permits. In an exemplary embodiment, an aromatic ring (e.g., phenyl) may be fused to a saturated or unsaturated ring (e.g., cyclohexane, cyclopentane or cyclohexene). Any combination of saturated, unsaturated and aromatic bicyclic rings is included in the bicyclic carbocyclic ring as valence permits. The bicyclic carbocyclic ring 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 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 carbocyclic rings include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl and naphthyl. The term "unsaturated carbocyclic ring" means a carbocyclic ring having at least one degree of unsaturation and does not include aromatic carbocyclic rings. Examples of unsaturated carbocyclic rings include cyclohexadiene, cyclohexene and cyclopentene. As used herein, the term "saturated cycloalkylarylalkyl" means a saturated carbocyclic ring. Exemplary carbocyclic rings include cyclopropyl, cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl, norborane and naphthyl. The carbocyclic ring may optionally be substituted with one or more substituents (such as those described herein).

[0273] "Cycloalkyl" means a stable, partially or fully saturated, monocyclic or polycyclic carbocyclic ring, which may include a fused ring system (when fused to an aryl or heteroaryl ring, the cycloalkyl is bonded through a non-aromatic ring atom), a bridged ring system or a spiro ring system. Representative cycloalkyls include, but are not limited to, cycloalkyls having three to fifteen carbon atoms (C3-C 15 cycloalkyl), cycloalkyls having three to ten carbon atoms (C3-C 10cycloalkyl), cycloalkyl having three to eight carbon atoms (C3-C8 cycloalkyl), cycloalkyl having three to six carbon atoms (C3-C6 cycloalkyl), cycloalkyl having three to five carbon atoms (C3-C5 cycloalkyl) or cycloalkyl having three to four carbon atoms (C3-C4 cycloalkyl). In some embodiments, the cycloalkyl is a 3- to 6-membered cycloalkyl. In some embodiments, 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, decahydronaphthyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane, cis-decahydronaphthalene, trans-decahydronaphthalene, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.2]decane and 7,7-dimethyl-bicyclo[2.2.1]heptyl. Partially saturated cycloalkyls include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl. Unless otherwise expressly stated in this specification, the cycloalkyl is optionally substituted, for example, by oxo, halogen, amino, nitrile, nitro, hydroxy, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the cycloalkyl is optionally substituted by oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2 or -NO2. In some embodiments, the cycloalkyl is optionally substituted by oxo, halogen, methyl, ethyl, -CN, -CF3, -OH or -OMe. In some embodiments, the cycloalkyl is optionally substituted by halogen. In some embodiments, the cycloalkyl is unsubstituted.

[0274] "Cycloalkylalkyl" means a radical of the formula -R c -cycloalkyl, wherein R c is an alkylene chain as described above.

[0275] "Cycloalkylalkoxy" means a radical of the formula -O-R c -cycloalkyl bonded through an oxygen atom, wherein R c is an alkylene chain as described above.

[0276] "Halogenated" or "halogen" means a halogen substituent, such as bromine, chlorine, fluorine and iodine substituents.

[0277] As used herein, the term "haloalkyl" or "halogenated alkane" refers to an alkyl radical as defined above which is substituted by one or more halogen radicals, such as trifluoromethyl, dichloromethyl, bromomethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like. In some embodiments, the alkyl portion of the fluoroalkyl radical is optionally further substituted. Examples of halogen-substituted alkanes ("halogenated alkanes") include halogenated methanes (e.g., chloromethane, bromomethane, fluoromethane, iodomethane), dihalogenated methanes and trihalogenated methanes (e.g., trichloromethane, tribromomethane, trifluoromethane, triiodomethane), 1-haloethane, 2-haloethane, 1,2-dihaloethane, 1-halopropane, 2-halopropane, 3-halopropane, 1,2-dihalopropane, 1,3-dihalopropane, 2,3-dihalopropane, 1,2,3-trihalopropane, and any other suitable combination of an alkane (or substituted alkane) and a halogen (e.g., Cl, Br, F, I, etc.). When the alkyl group is substituted by more than one halogen radical, each halogen can be independently selected, such as 1-chloro,2-fluoroethane.

[0278] "Fluoroalkyl" refers to an alkyl as defined above which is substituted by one or more fluorine radicals, such as trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like.

[0279] "Hydroxyalkyl" refers to an alkyl radical as defined above which is substituted by one or more hydroxyl groups. In some embodiments, the alkyl is substituted by one hydroxyl group. In some embodiments, the alkyl is substituted by one, two, or three hydroxyl groups. Hydroxyalkyl includes, for example, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, or hydroxypentyl. In some embodiments, the hydroxyalkyl is hydroxymethyl. As used herein, "C1-C6 hydroxy" refers to a C1-C6 hydroxyalkyl.

[0280] "Aminoalkyl" refers to an alkyl radical as defined above which is substituted by one or more amines. In some embodiments, the alkyl is substituted by one amine. In some embodiments, the alkyl is substituted by one, two, or three amines. Aminoalkyl includes, for example, aminomethyl, aminoethyl, aminopropyl, aminobutyl, or aminopentyl. In some embodiments, the aminoalkyl is aminomethyl.

[0281] "Disulfide" refers to two sulfur atoms bonded to each other, where each sulfur atom contains an optionally substituted alkyl chain. In some embodiments, the disulfide can be R-S-S-R'. In some embodiments, R and R' can be the same. In some embodiments, R and R' are different. Each R and R' can independently be selected from C1-C 12 alkyl. In certain embodiments, R or R' can be substituted by an amine, a sulfone, or a carboxylic acid.

[0282] The term "heteroalkyl" refers to an alkyl group in which one or more of the skeletal atoms of the alkyl group are atoms other than carbon (e.g., oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, or combinations thereof). The heteroalkyl is attached to the remainder of the molecule at a carbon atom of the heteroalkyl. In one aspect, the heteroalkyl is a C1-C6 heteroalkyl, wherein the heteroalkyl consists of 1 to 6 carbon atoms and one or more atoms other than carbon (e.g., oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, or combinations thereof), and wherein the heteroalkyl is attached to the remainder of the molecule at a carbon atom of the heteroalkyl. Examples of such heteroalkyls are, for example, -CH2OCH3, -CH2CH2OCH3, -CH2CH2OCH2CH2OCH3, or -CH(CH3)OCH3. Unless otherwise expressly stated in this specification, the heteroalkyl is optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxy, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2, or -NO2. In some embodiments, the 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.

[0283] "Heterocycloalkyl" refers to a stable 3- to 24-membered partially or fully saturated cyclic radical that contains 2 to 23 carbon atoms and at least one ring heteroatom. In some embodiments, the heterocycloalkyl contains 1 to 8 heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur. Unless otherwise expressly stated in this specification, the heterocycloalkyl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic system, which can include a fused ring system (when fused to an aryl or heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or a bridged ring system; and the nitrogen, carbon, or sulfur atoms in the heterocycloalkyl radical can be optionally oxidized; and the nitrogen atoms can be optionally quaternized.

[0284] Representative heterocycloalkyls include, but are not limited to, heterocycloalkyls having two to fifteen carbon atoms (C2-C 15 heterocycloalkyl), heterocycloalkyls having two to ten carbon atoms (C2-C 10heterocycloalkyl), heterocycloalkyl having two to eight carbon atoms (C2-C8 heterocycloalkyl), heterocycloalkyl having two to six carbon atoms (C2-C6 heterocycloalkyl), heterocycloalkyl having two to five carbon atoms (C2-C5 heterocycloalkyl) or heterocycloalkyl having two to four carbon atoms (C2-C4 heterocycloalkyl). In some embodiments, the heterocycloalkyl is a 3- to 6-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 5- to 6-membered heterocycloalkyl. Examples of such heterocycloalkyls include, but are not limited to, aziridinyl, azetidinyl, dioxolanyl, thieno[1,3]dithiolanyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithiolanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, 1,1-dioxothiomorpholinyl, 1,3-dihydroisobenzofuran-1-yl, 3-oxo-1,3-dihydroisobenzofuran-1-yl, methyl-2-oxo-1,3-dioxol-4-yl and 2-oxo-1,3-dioxol-4-yl. The term heterocycloalkyl also includes all cyclic forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. It should be understood that when referring to the number of carbon atoms in a heterocycloalkyl, the number of carbon atoms in the heterocycloalkyl is different from the total number of atoms (including heteroatoms) that make up the heterocycloalkyl (i.e., the backbone atoms of the heterocycloalkyl ring). Unless otherwise expressly stated in this specification, the heterocycloalkyl is optionally substituted with, for example, oxo, halogen, amino, nitrile, nitro, hydroxy, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2 or -NO2. In some embodiments, the heterocycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF3, -OH or -OMe. In some embodiments, the heterocycloalkyl is optionally substituted with halogen. In some embodiments, the heterocycloalkyl is unsubstituted.

[0285] "Heterocycle" or "heterocyclic group" refers to a saturated, unsaturated or aromatic ring containing one or more ring heteroatoms. Exemplary heteroatoms include N, O, Si, P, B and S atoms. Heterocycles include, for example, 3- to 10-membered monocycles and 6- to 12-membered bicyclic rings (such as spiro, fused or bridged rings). Unless otherwise expressly stated in this specification, a heterocyclic group radical is a monocyclic, bicyclic, tricyclic or tetracyclic system, which optionally includes a fused ring, bridged ring or spiro ring system. The heteroatoms in the heterocyclic group radical are optionally oxidized. If present, one or more nitrogen atoms are optionally quaternized. The heterocyclic group radical can be partially saturated or fully saturated. The heterocyclic group is attached to the remainder of the molecule through any atom on the ring. Examples of such heterocyclic group radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithialanyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinoneyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithialanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl and 1,1-dioxothiomorpholinyl. Unless otherwise expressly stated in this specification, the term "heterocyclic group" is intended to include a heterocyclic group radical as defined above which is optionally substituted by one or more substituents. For example, the heterocyclic group can be optionally substituted by one or more substituents selected from the following: alkyl, alkenyl, alkynyl, halogen, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclic group, optionally substituted carbocyclic group alkyl, optionally substituted heterocyclic group, optionally substituted heterocyclic group alkyl, optionally substituted heteroaryl, optionally substituted heteroaryl alkyl, -R b -OR a 、-R b -OC(O)-R a 、-R b -OC(O)-OR a 、-R b -OC(O)-N(R a )2、-R b -N(R a )2、-R b -C(O)R a 、-R b -C(O)OR a 、-R b -C(O)N(R a )2、-R b -CN、-R b -O-R e -C(O)N(R a)2. -R b -N(R a )C(O)OR a 、-R b -N(R a )C(O)R a 、-R b -N(R a )S(O) t R a (where t is 1 or 2), -R b -S(O) t R a (where t is 1 or 2), -R b -S(O) t OR a (where t is 1 or 2) and -R b -S(O) t N(Ra)2 (where t is 1 or 2), where each R a is independently hydrogen, alkyl (optionally substituted by halogen, hydroxy, methoxy or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted by halogen, hydroxy, methoxy or trifluoromethyl), cycloalkylalkyl (optionally substituted by halogen, hydroxy, methoxy or trifluoromethyl), aryl (optionally substituted by halogen, hydroxy, methoxy or trifluoromethyl), arylalkyl (optionally substituted by halogen, hydroxy, methoxy or trifluoromethyl), heterocyclic group (optionally substituted by halogen, hydroxy, methoxy or trifluoromethyl), heterocyclic alkyl (optionally substituted by halogen, hydroxy, methoxy or trifluoromethyl), heteroaryl (optionally substituted by halogen, hydroxy, methoxy or trifluoromethyl) or heteroarylalkyl (optionally substituted by halogen, hydroxy, methoxy or trifluoromethyl), each R b is independently a direct bond or a straight or branched alkylene or alkenylene chain, and R e is a straight or branched alkylene or alkenylene chain, and where unless otherwise specified, each of the above substituents is unsubstituted.

[0286] "Heteroaryl" or "aromatic heterocycle" refers to a cyclic system radical containing carbon atoms and one or more ring heteroatoms (e.g., selected from nitrogen, oxygen, phosphorus, silicon, and sulfur) and at least one aromatic ring. In some embodiments, heteroaryl is a 5- to 14-membered cyclic system radical containing one to thirteen carbon atoms and one to six heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur. The heteroaryl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic system, which can include a fused ring system (when fused to a cycloalkyl or heterocycloalkyl ring, the heteroaryl is bonded through an aromatic ring atom) or a bridged ring system; and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized. In some embodiments, heteroaryl is a 5- to 10-membered heteroaryl. In some embodiments, heteroaryl is a 5- to 6-membered heteroaryl. Examples include but are not limited to azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothienyl / benzothiophenyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothienyl / benzothiophenyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothienyl / benzothiophenyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl Group, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl and thiophenyl / thienyl. Unless otherwise expressly stated in this specification, the heteroaryl is optionally substituted with, for example, halogen, amino, nitrile, nitro, hydroxy, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl and the like. In some embodiments, the heteroaryl is optionally substituted with halogen, methyl, ethyl, -CN, -CF3, -OH, -OMe, -NH2 or -NO2. In some embodiments, the 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.

[0287] The term "substituted" refers to the part in which one or more hydrogens on a carbon or a substitutable heteroatom (such as NH) in a structure are replaced by substituents. It should be understood that "substituted" or "being substituted" includes the implicit condition that such substitution is carried out according to the allowed valences of the substituted atom and the substituent, and the substitution produces a stable compound, that is, a compound that does not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc. In certain embodiments, substitution refers to the part having such a substituent that replaces two hydrogens on the same carbon atom, such as replacing two hydrogens on a single carbon with an oxo, imino or thio group. As used herein, the term "substituted" is intended to include all allowed substituents of organic compounds. Broadly speaking, allowed substituents include non-cyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For suitable organic compounds, the allowed substituents can be one or more and can be the same or different. For the purposes of this disclosure, a heteroatom (such as nitrogen) can have a hydrogen substituent and / or any allowed substituent of the organic compounds described herein that satisfies the valence of the heteroatom.

[0288] In some embodiments, the substituents can include any of the substituents described herein, for example: halogen, hydroxy, amino, oxo (=O), thio (=S), cyano (-CN), nitro (-NO2), imino (=N-H), oxime (=N-OH), hydrazino (=N-NH2), -R b -OR a 、-R b -OC(O)-R a, -R b , -OC(O)-OR a , -R b , -OC(O)-N(R a )2, -R b , -N(R a )2, -R b , -C(O)R a , -R b , -C(O)OR a , -R b , -C(O)N(R a )2, -R b , -O-R c , -C(O)N(R a )2, -R b , -N(R a )C(O)OR a , -R b , -N(R a )C(O)R a , -R b , -N(R a )S(O) t R a (where t is 1 or 2), -R b , -S(O) t R a (where t is 1 or 2), -R b , -S(O) t OR a (where t is 1 or 2) and -R b , -S(O) t , -N(R a )2(where t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl and heterocycle, any of which may optionally be substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo(=O), thioxo(=S), cyano(-CN), nitro(-NO2), imino(=N-H), oxime(=N-OH), hydrazino(=N-NH2), SF 5 , -R b , -OR a , -R b , -OC(O)-R a , -R b , -OC(O)-OR a , -R b , -OC(O)-N(R a )2, -R b , -N(R a )2, -R b , -C(O)Ra , -R b , -C(O)OR a , -R b , -C(O)N(R a ),2, -R b , -O-R c , -C(O)N(R a ),2, -R b , -N(R a ),C(O)OR a , -R b , -N(R a ),C(O)R a , -R b , -N(R a ),S(O) t R a (where t is 1 or 2), -R b , -S(O) t R a (where t is 1 or 2), -R b , -S(O) t OR a (where t is 1 or 2) and -R b , -S(O) t , -N(R a ),2(where t is 1 or 2) substituted; where each R a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl and heterocycle, where each R a , optionally, when valence allows, may be substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo(=O), thioxo(=S), cyano(-CN), nitro(-NO2), imino(=N-H), oxime(=N-OH), hydrazino(=N-NH2), -R b , -OR a , -R b , -OC(O)-R a , -R b , -OC(O)-OR a , -R b , -OC(O)-N(R a ),2, -R b , -N(R a ),2, -R b , -C(O)R a , -R b , -C(O)OR a , -R b , -C(O)N(R a ),2, -R b , -O-R c-C(O)N(R a )2, -R b -N(R a )C(O)OR a 、-R b -N(R a )C(O)R a 、-R b -N(R a )S(O) t R a (where t is 1 or 2), -R b -S(O) t R a (where t is 1 or 2), -R b -S(O) t OR a (where t is 1 or 2) and -R b -S(O) t N(R a )2 (where t is 1 or 2) substituted; where each R b is independently selected from a direct bond or a straight-chain or branched alkylene, alkenylene or alkynylene chain, and each R c is a straight-chain or branched alkylene, alkenylene or alkynylene chain.

[0289] As used in the specification and claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0290] The term "optional" or "optionally" means that the subsequent described event or circumstance may or may not occur, and the description includes both the case where the event or circumstance occurs and the case where it does not occur. For example, "optionally substituted alkyl" means "alkyl" or "substituted alkyl" as defined above. Additionally, an optionally substituted group may be unsubstituted (e.g., -CH2CH3), fully substituted (e.g., -CF2CF3), monosubstituted (e.g., -CH2CH2F) or substituted at any level between fully substituted and monosubstituted (e.g., -CH2CHF2, -CH2CF3, -CF2CH3, -CFHCHF2, etc.).

[0291] The ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 50 is to be understood as including any number, combination of numbers, or sub-range from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 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 intermediate decimal values between the above integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. For sub-ranges, “nested sub-ranges” extending from either endpoint of the range are specifically contemplated. For example, the nested sub-ranges of the exemplary range of 1 to 50 can include, in one direction, 1 to 10, 1 to 20, 1 to 30, and 1 to 40, or, in the other direction, 50 to 40, 50 to 30, 50 to 20, and 50 to 10.

[0292] The compounds and structures provided herein can be stereoisomeric. In some cases, the compounds or structures of the present disclosure can form stereoisomers. In some cases, the stereoisomers can be diastereomers (e.g., cis / trans isomers, E / Z isomers, conformational isomers, or rotamers). In some cases, the stereoisomers can be enantiomers (R, S enantiomers or + / − enantiomers). In some cases, the compounds or structures of the present disclosure can be enantiopure (e.g., 100% pure). In some cases, the compounds or structures can form a racemic mixture of enantiomers (e.g., 50% pure). In some cases, the compounds or structures of the present disclosure can be stabilized as stereoisomers, wherein the compounds or structures of the present disclosure comprise 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 the compound or structure in mixture with the corresponding stereoisomer.

[0293] The compounds and structures provided herein are intended to include all ionized forms of the compounds and structures provided herein. The compounds and structures provided herein are also intended to include all salt forms of the compounds and structures provided herein.

[0294] The compounds of the present disclosure

[0295] The present disclosure relates to macromolecules comprising one or more repeating units.

[0296] In one aspect, the present disclosure describes a macromolecule comprising a repeating unit of formula (I):

[0297]

[0298] wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl;

[0299] R1 is hydrogen, nitrogen, an optionally substituted succinate, a C1-C6 alkylsulfone, or a phthalate;

[0300] R2 is nitrogen, a C1-C6 hydroxy group, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted C3-C6 heterocycloalkyl, an optionally substituted C3-C6 bicycloalkylmethane, a C1-C6 alkylguanidine, a C1-C6 ether, an optionally substituted -C1-C6 disulfide, a C1-C6 thiol, an optionally substituted succinate, an optionally substituted C1-C6 alkylamine, a C1-C6 alkylacetamide, or a C1-C6 aminophthalate; and

[0301] q is an integer between 1 and 6,

[0302] wherein if R1 and R2 are each nitrogen, then R1 and R2 optionally together with the atoms to which they are attached form an optionally substituted heterocycle.

[0303] In one aspect, the present disclosure describes a macromolecule comprising a repeating unit of formula (I-A):

[0304]

[0305] wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl;

[0306] R1 is hydrogen, an optionally substituted succinate, a C1-C6 alkylsulfone, or a phthalate;

[0307] R2 is a C1-C6 hydroxy group, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted C3-C6 heterocycloalkyl, a C1-C 12 amine, an optionally substituted C3-C6 bicycloalkylmethane, a C1-C6 alkylguanidine, a C1-C6 ether, an optionally substituted -C1-C6 disulfide, a C1-C6 thiol, an optionally substituted succinate, an optionally substituted C1-C 12 alkylamine, a C1-C6 alkylacetamide, a C5-C 11 cycloalkyl, or a C1-C6 aminophthalate; or

[0308] R1 and R2 together with the nitrogen to which they are attached form an optionally substituted heterocycle; or

[0309] R1 and R2 together with the nitrogen to which they are attached form an azide group; and

[0310] q is an integer between 1 and 6. In some embodiments, provided herein is a polymer derived from a monomer of formula (I-A’), wherein the substituents are as defined elsewhere herein for formula (I):

[0311]

[0312] In some embodiments of formulae (I), (I-A) and (I-A’), each of Y1, Y2 and Y3 is independently selected from hydrogen or C1-C6 alkyl. In some embodiments, Y1 is hydrogen. In some embodiments, Y1 is C1-C6 alkyl. In some embodiments, Y2 is hydrogen. In some embodiments, Y2 is C1-C6 alkyl. In some embodiments, Y3 is hydrogen. In some embodiments, Y3 is C1-C6 alkyl. In some embodiments, Y1 is C1-C3 alkyl and each of Y2 and Y3 is hydrogen. In some embodiments, Y1 is C1 alkyl (e.g., -CH3) and each of Y2 and Y3 is hydrogen.

[0313] In some embodiments of formulae (I), (I-A) and (I-A’), R1 is hydrogen, nitrogen, an optionally substituted succinate, a C1-C6 alkylsulfone or a phthalate. In some embodiments of formula (I), R1 is hydrogen, an optionally substituted succinate, a C1-C6 alkylsulfone or a phthalate. In some embodiments, R1 is hydrogen. In some embodiments, R1 is nitrogen. In some embodiments, R1 is an optionally substituted succinate. In some embodiments, R1 is a C1-C6 alkylsulfone. In some embodiments, R1 is a phthalate.

[0314] In some embodiments of formulae (I), (I-A) and (I-A’), R2 is nitrogen, a C1-C6 hydroxy group, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted C3-C6 heterocycloalkyl, an optionally substituted C3-C6 bicycloalkylmethane, a C1-C6 alkylguanidine, a C1-C6 ether, an optionally substituted -C1-C6 disulfide, a C1-C6 thiol, an optionally substituted succinate, an optionally substituted C1-C6 alkylamine, a C1-C6 alkylacetamide or a C1-C6 aminophthalate. In some embodiments of formula (I), R2 is a C1-C6 hydroxy group, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted C3-C6 heterocycloalkyl, C1-C 12amines, optionally substituted C3-C6 bicycloalkylmethanes, C1-C6 alkylguanidines, C1-C6 ethers, optionally substituted -C1-C6 disulfides, C1-C6 thiols, optionally substituted succinates, optionally substituted C1-C 12 alkylamines, C1-C6 alkylacetamides, C5-C 11 cycloalkyls or C1-C6 aminophthalates. In some embodiments, R2 is nitrogen (such as azide). In some embodiments, R2 is azide (e.g., N3). In some embodiments, R2 is C1-C 12 amine. In some embodiments, R2 is C5-C 11 cycloalkyl. In some embodiments, R2 is C1-C6 hydroxy. In some embodiments, R2 is optionally substituted aryl. In some embodiments, R2 is optionally substituted heteroaryl. In some embodiments, R2 is optionally substituted C3-C6 heterocycloalkyl. In some embodiments, R2 is optionally substituted C3-C6 bicycloalkylmethane. In some embodiments, R2 is C1-C6 alkylguanidine. In some embodiments, R2 is C1-C6 ether. In some embodiments, R2 is optionally substituted -C1-C6 disulfide. In some embodiments, R2 is C1-C6 thiol. In some embodiments, R2 is optionally substituted succinate. In some embodiments, R2 is optionally substituted C1-C 12 alkylamine. In some embodiments, R2 is C1-C6 alkylacetamide. In some embodiments, R2 is C1-C6 aminophthalate. In some embodiments, together form -N3.

[0315] In some embodiments, R1 is selected from hydrogen,

[0316] In some embodiments, R2 is selected from

[0317] In some embodiments, R1 and R2 together with the nitrogen to which they are attached form an optionally substituted heterocycle (e.g., a nitrogen-containing heterocycle).

[0318] In some embodiments, R1 and R2 together with the nitrogen to which they are attached form an azide group.

[0319] In some embodiments of formulas (I), (I-A), and (I-A’), R1 is hydrogen, and R2 is selected from optionally substituted C3-C6 bicycloalkylmethane, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C3-C6 heterocycloalkyl, C1-C6 hydroxy, C1-C6 ether, optionally substituted -C1-C6 disulfide, optionally substituted succinate, optionally substituted C1-C6 alkylamine, C1-C6 alkylacetamide, or C1-C6 alkylguanidine. In some embodiments, when R2 is optionally substituted C3-C6 heterocycloalkyl, it is attached to the rest of the molecule via a substituent of the heterocycloalkyl. In some embodiments, R1 is hydrogen and R2 is optionally substituted bicyclohexylmethane. In some embodiments, R1 is hydrogen and R2 is aminobicyclohexylmethane (e.g., ). In some embodiments, R1 is hydrogen and R2 is optionally substituted aryl. In some embodiments, R1 is hydrogen and R2 is halotoluene. In some embodiments, R1 is hydrogen and R2 is 2-fluorotoluene (e.g., ). In some embodiments, R1 is hydrogen and R2 is C1-C6 hydroxy. In some embodiments, R1 is hydrogen and R2 is C3-C6 hydroxy. In some embodiments, R1 is hydrogen and R2 is -(CH2)6OH (e.g., ). In some embodiments, R1 is hydrogen and R2 is C1-C6 ether. In some embodiments, R1 is hydrogen and R2 is selected from -CH2OCH3-, -CH2CH2OCH3, -CH2CH2OCH2CH3, or -CH2OCH2CH3. In some embodiments, R1 is hydrogen and R2 is -CH2CH2OCH3. In some embodiments, R1 is hydrogen and R2 is C1-C6 alkylacetamide. In some embodiments, R1 is hydrogen and R2 is -(CH2)2 alkylacetamide (e.g., ). In some embodiments, R1 is hydrogen and R2 is optionally substituted di-C1-C6 alkyldisulfide. In some embodiments, R1 is hydrogen and R2 is -CH2CH2-S-S-CH2CH2NH2 (e.g., ). In some embodiments, R1 is hydrogen and R2 is optionally substituted succinate. In some embodiments, R1 is hydrogen and R2 is -(CH2) 1-6 NH(C=O)CH2CH2COOH. In some embodiments, R1 is hydrogen and R2 is -(CH2)6NH(C=O)CH2CH2COOH (e.g., )。In some embodiments, R1 is hydrogen and R2 is an optionally substituted heteroaryl. In some embodiments, when R2 is an optionally substituted heteroaryl, it is linked to the rest of the molecule via a substituent of the heteroaryl. In some embodiments, the optionally substituted heteroaryl is -(CH2) 1-6 imidazole. In some embodiments, R1 is hydrogen and R2 is -(CH2) 1-6 imidazole. In some embodiments, R1 is hydrogen and R2 is -(CH2)3imidazole (e.g., ); in some embodiments, R1 is hydrogen and R2 is a disubstituted C2-C4 imidazole. In some embodiments, R1 is hydrogen and R2 is dipropylimidazole (e.g., ). In some embodiments, R1 is hydrogen and R2 is an optionally substituted heterocycloalkyl. In some embodiments, R1 is hydrogen and R2 is -(CH2) 1-6 pyrrolidine. In some embodiments, R1 is hydrogen and R2 is -(CH2)2pyrrolidine (e.g., ); in some embodiments, R1 is hydrogen and R2 is an optionally substituted C1-C6 alkylamine. In some embodiments, R1 is hydrogen and R2 is -(CH2) 1-3 dimethylamine. In some embodiments, R1 is hydrogen and R2 is -(CH2)2dimethylamine (e.g., ). In some embodiments, R1 is hydrogen and R2 is a C1-C6 guanidine. In some embodiments, R1 is hydrogen and R2 is -(CH2)2guanidine (e.g., ).

[0320] In some embodiments, each of R1 and R2 is nitrogen and together they form an optionally substituted heterocycle. In some embodiments, each of R1 and R2 is nitrogen and together they form an optionally substituted heterocycle containing a triazole. In some embodiments, each of R1 and R2 is nitrogen and together they form an optionally substituted triazole containing a benzylamide. In some embodiments, each of R1 and R2 is nitrogen and together they form an optionally substituted triazole containing a benzylamide, wherein the benzylamide is halogenated. In some embodiments, R1 and R2 together with the nitrogen to which they are attached form an optionally substituted heterocycle. In some embodiments, R1 and R2 together with the nitrogen to which they are attached form an optionally substituted heteroaryl. In some embodiments, R1 and R2 together with the nitrogen to which they are attached form an azide group.

[0321] In some embodiments, R1 is an optionally substituted succinate, and R2 is selected from an optionally substituted C3-C6 bicycloalkylmethane, an optionally substituted aryl, a C1-C6 thiol, or an optionally substituted succinate. In some embodiments, R1 is a succinate, and R2 is an optionally substituted C3-C6 bicycloalkylmethane, an optionally substituted aryl, a C1-C6 thiol, or an optionally substituted succinate. In some embodiments, R1 is a succinate, and R2 is dicyclohexylmethane succinate (e.g., ). In some embodiments, R2 is an optionally substituted C3-C6 bicycloalkylmethane (e.g., ). In some embodiments, R1 is a succinate, and R2 is an optionally substituted aryl. In some embodiments, R1 is a succinate, and R2 is 2-fluorotoluene (e.g., ). In some embodiments, R1 is a succinate, and R2 is a C1-C6 thiol. In some embodiments, R1 is a succinate, and R2 is -(CH2)2SH (e.g., ). In some embodiments, R1 is a succinate, and R2 is an optionally substituted succinate. In some embodiments, R1 is a succinate, and R2 is -(CH2) 1-12 NH(C=O)CH2CH2COOH. In some embodiments, R1 is a succinate, and R2 is -(CH2)2NH(C=O)CH2CH2COOH (e.g., ). In some embodiments, R1 is a succinate, and R2 is -(CH2) 10-12 NH(C=O)CH2CH2COOH. In some embodiments, R1 is a succinate, and R2 is -(CH2) 12 NH(C=O)CH2CH2COOH (e.g., ). In some embodiments, R1 is a C2-C 12 alkenyl succinate, and R2 is a substituted succinate. In some embodiments, R1 is a C8 alkenyl succinate, and R2 is a C8 alkenyl ethylamino succinate (e.g., ).

[0322] In some embodiments, R1 is a C1-C6 alkyl sulfone, and R2 is an optionally substituted C1-C6 alkylamine. In some embodiments, the C1-C6 alkyl sulfone is -(CH2)3SOOOH. In some embodiments, R1 is -(CH2)3SOOOH (e.g., ), and R2 is selected from -(CH2) 1-6 N(CH3)2(CH2CH2CH2SOOOH) (e.g., ) or -(CH2)1-6 N(CH2CH2CH2SOOOH)2 (for example, ). In some embodiments, R1 is -(CH2)3SOOOH (for example, ), and R2 is -(CH2)2N(CH3)2(CH2CH2CH2SOOOH) (for example, ). In some embodiments, R1 is -(CH2)3SOOOH (for example, ), and R2 is -(CH2)2N(CH2CH2CH2SOOOH)2 (for example, ).

[0323] In some embodiments, R1 is a phthalate (for example, ), and R2 is a C1-C6 aminophthalate. In some embodiments, R1 is a phthalate, and R2 is a C2-C6 aminophthalate. In some embodiments, R1 is a phthalate, and R2 is a C1-C 12 aminophthalate (for example, ). In some embodiments, R1 is a phthalate, and R2 is a C6 aminophthalate (for example, ). In some embodiments, R2 is a C2 aminophthalate (for example, ).

[0324] In some embodiments of formulas (I), (I-A), and (I-A’), each of R1 and R2 is nitrogen. In some embodiments of formula (I), each of R1 and R2 is nitrogen and, together with the atoms to which they are attached, forms an optionally substituted heterocycle. In some embodiments, the optionally substituted heterocycle is

[0325] In some embodiments of formulas (I), (I-A), and (I-A’), q is an integer between 1 and 6. In some embodiments, q is an integer between 1 and 3. 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.

[0326] In one aspect, the macromolecule comprises repeating units of formula (II):

[0327]

[0328] wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl;

[0329] R4 is hydrogen or a C1-C6 mercaptan; and

[0330] R5 is a succinate, a C1-C6 mercaptan, an optionally substituted aryl, or an optionally substituted -C1-C6 disulfide.

[0331] In some embodiments, can be interchangeably referred to as

[0332] In some embodiments, provided herein is a polymer derived from a monomer of formula (II’), wherein the substituent groups are as defined in formula (II) elsewhere herein:

[0333]

[0334] In some embodiments of formulae (II) and (II’), each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1-C6 alkyl. In some embodiments, Y1 is hydrogen. In some embodiments, Y1 is a C1-C6 alkyl. In some embodiments, Y2 is hydrogen. In some embodiments, Y2 is a C1-C6 alkyl. In some embodiments, Y3 is hydrogen. In some embodiments, Y3 is a C1-C6 alkyl.

[0335] In some embodiments of formulae (II) and (II’), R4 is hydrogen or a C1-C6 mercaptan. In some embodiments, R4 is hydrogen. In some embodiments, R4 is a C1-C6 mercaptan. In some embodiments, R4 is a C1-C3 mercaptan.

[0336] In some embodiments, R5 is a succinate or an optionally substituted -C1-C6 disulfide.

[0337] In some embodiments, R5 is a succinate or an optionally substituted -C1-C6 disulfide. In some embodiments, R5 is a succinate, a C1-C6 mercaptan, or an optionally substituted -C1-C6 disulfide. In some embodiments, R5 is a succinate. In some embodiments, R5 is an optionally substituted -C1-C6 disulfide. In some embodiments, R5 is a C1-C6 mercaptan.

[0338] In some embodiments, Y1 is a C1-C3 alkyl. In some embodiments, Y1 is a C1 alkyl (e.g., -CH3). In some embodiments, each of Y2 and Y3 is hydrogen. In some embodiments, R4 is selected from hydrogen, a C1-C3 mercaptan, or -(CH2)2SH (e.g., ). In some embodiments, R5 is selected from di-C1-C6 alkyl disulfides or -CH2CH2-S-S-CH2CH2NH2 (e.g., ).

[0339] In some embodiments, R4 is hydrogen and R5 is an optionally substituted -C1-C6 disulfide. In some embodiments, R4 is hydrogen and R5 is an optionally disubstituted di-C1-C6 alkyl disulfide. In some embodiments, R4 is hydrogen and R5 is -CH2CH2-S-S-CH2CH2NH2 (e.g., ). In some embodiments, R4 is a C1-C6 thiol and R5 is a succinate. In some embodiments, R4 is a C1-C3 thiol and R5 is a succinate. In some embodiments, R4 is -(CH2)2SH (e.g., ). And R5 is a succinate.

[0340] In some embodiments, R4 is In some embodiments, R5 is

[0341] In one aspect, the macromolecule comprises a repeating unit of formula (III):

[0342]

[0343] wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl; and

[0344] each q is independently an integer between 1 and 6.

[0345] In some embodiments, is interchangeably referred to as

[0346] In some embodiments, provided herein is a macromolecule comprising a repeating unit of formula (III'):

[0347]

[0348] In some embodiments, X is O or NH. In some embodiments, X is O. In some embodiments, X is NH. In some embodiments, the repeating unit of formula (III-A) is the repeating unit of formula (III). In some embodiments, the substituents Y1, Y2, and Y3 are as defined in formula (III) herein.

[0349] In some embodiments, provided herein is a polymer derived from a monomer of formula (III'), wherein the substituents are as defined in formula (III) elsewhere herein:

[0350]

[0351] In some embodiments of formula (III), (III-A) or (III’), each of Y1, Y2 and Y3 is independently selected from hydrogen or C1-C6 alkyl. In some embodiments, Y1 is hydrogen. In some embodiments, Y1 is C1-C6 alkyl. In some embodiments, Y2 is hydrogen. In some embodiments, Y2 is C1-C6 alkyl. In some embodiments, Y3 is hydrogen. In some embodiments, Y3 is C1-C6 alkyl. In some embodiments, Y1 is C1-C3 alkyl. In some embodiments, Y1 is C1 alkyl (e.g., -CH3). In some embodiments, each of Y2 and Y3 is hydrogen. In some embodiments, each q is 2 or 3. In some embodiments, each q is 2.

[0352] In some embodiments, the macromolecule can comprise at least 5% by weight of repeating units of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’) or formula (III-A). In some embodiments, the macromolecule can comprise at least 10% by weight of repeating units of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’) or formula (III-A). In some embodiments, the macromolecule can comprise at least 25% by weight of repeating units of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’) or formula (III-A). In some embodiments, the macromolecule can comprise at least 50% by weight of repeating units of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’) or formula (III-A). In some embodiments, the macromolecule can comprise at least 75% by weight of repeating units of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’) or formula (III-A). In some embodiments, the macromolecule can comprise 5% to 95% by weight of repeating units of formula (I), formula (I-A), formula (II), formula (III) or (III-A). In some embodiments, the macromolecule can comprise 20% to 80% by weight of repeating units of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’) or formula (III-A). In some embodiments, the macromolecule can comprise 40% to 60% by weight of repeating units of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’) or formula (III-A).

[0353] In some embodiments, the macromolecules described herein are quaternized. In some embodiments, macromolecules are described that are N - quaternized derivatives of the macromolecules described herein.

[0354] In some embodiments, the repeating units of formula (I), formula (I - A), formula (I - A’), formula (II), formula (II’), formula (III), formula (III’), formula (III - A), or formula (IV) may comprise the structures shown in Table 1, where m is an integer of 2 or greater:

[0355] Table 1:

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364] In some embodiments, the macromolecule further comprises crosslinked repeating units. For example, the crosslinked repeating units can be produced by free radical polymerization of a monomer having two vinyl groups, such as divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), poly(ethylene glycol) dimethacrylate (PEGDMA) (e.g., diethylene glycol dimethacrylate (DEGDMA), triethylene glycol dimethacrylate (TEGDMA), etc.), N,N'-alkylenebisacrylamide (e.g., N,N'-methylenebisacrylamide (MBA), N,N'-ethylenebisacrylamide, N,N'-butylenebisacrylamide, etc.) or derivatives thereof. In some embodiments, the macromolecule can comprise at least 5% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise at least 25% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise at least 40% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise no more than 90% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise no more than 75% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise no more than 60% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise no more than 50% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise no more than 25% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise 5% to 95% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise 20% to 80% by weight of crosslinked repeating units. In some embodiments, the macromolecule can comprise 40% to 60% by weight of crosslinked repeating units.

[0365] In one aspect, the macromolecule comprises repeating units of a first component and a second component, wherein the first component comprises the structure of component (A) and the second component comprises the structure of component (B):

[0366]

[0367] wherein each of Y1, Y2 and Y3 is independently selected from hydrogen or C1-C6 alkyl;

[0368] Each of X1, X2 and X3 is independently selected from hydrogen or C1-C6 alkyl;

[0369] A is

[0370] R1 is hydrogen, nitrogen, an optionally substituted succinate, a C1-C6 alkylsulfone, a phthalate,

[0371] R2 is nitrogen, C1-C 12amine, C1-C6 hydroxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C3-C6 heterocycloalkyl, two or more fused 3-6 membered rings; optionally substituted C3-C6 bicycloalkylmethane, C1-C6 alkylguanidine, C1-C6 ether, optionally substituted -C1-C6 disulfide, C1-C6 thiol, optionally substituted succinate, optionally substituted C1-C6 alkylamine, C1-C6 alkylacetamide, C1-C6 aminophthalate, boric acid or monosaccharide;

[0372] R4 is hydrogen or C1-C6 thiol;

[0373] R5 is succinate or optionally substituted -C1-C6 disulfide;

[0374] B is

[0375] Z is a chain within a macromolecule;

[0376] q is an integer between 1 and 6; and

[0377] p is an integer between 1 and 20.

[0378] As used herein, refers interchangeably to As used herein, refers interchangeably to

[0379] In some embodiments, each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl. In some embodiments, Y1 is hydrogen. In some embodiments, Y1 is C1-C6 alkyl. In some embodiments, Y2 is hydrogen. In some embodiments, Y2 is C1-C6 alkyl. In some embodiments, Y3 is hydrogen. In some embodiments, Y3 is C1-C6 alkyl. In some embodiments, Y1 is C1-C3 alkyl, and each of Y2 and Y3 is hydrogen. In some embodiments, Y1 is C1 alkyl (e.g., -CH3), and each of Y2 and Y3 is hydrogen.

[0380] In some embodiments, each of X1, X2, and X3 is independently selected from hydrogen or C1-C6 alkyl. In some embodiments, X1 is hydrogen. In some embodiments, X1 is C1-C6 alkyl. In some embodiments, X2 is hydrogen. In some embodiments, X2 is C1-C6 alkyl. In some embodiments, X3 is hydrogen. In some embodiments, X3 is C1-C6 alkyl. In some embodiments, X1 is C1-C3 alkyl, and each of X2 and X3 is hydrogen. In some embodiments, X1 is C1 alkyl (e.g., -CH3), and each of X2 and X3 is hydrogen.

[0381] In some embodiments, Z is a chain within a macromolecule. In some embodiments, the chain may comprise units of monomer (A). In some embodiments, the chain may comprise units of monomer (B).

[0382] In some embodiments, q is an integer between 1 and 6. In some embodiments, q is an integer between 1 and 3. 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.

[0383] In some embodiments, p is an integer between 1 and 20. 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. In some embodiments, p is 7. In some embodiments, p is 8. In some embodiments, p is 9. In some embodiments, p is 10. In some embodiments, p is 11. In some embodiments, p is 12. In some embodiments, p is 13. In some embodiments, p is 14. In some embodiments, p is 15. In some embodiments, p is 16. In some embodiments, p is 17. In some embodiments, p is 18. In some embodiments, p is 19. In some embodiments, p is 20.

[0384] In some embodiments, B is and A is In some embodiments, B is and A is In some embodiments, B is and A is In some embodiments, B is and A is

[0385] In some embodiments, B is and A is In some embodiments, B is and A is In some embodiments, B is and A is In some embodiments, B is and A is In some embodiments, B is and A is

[0386] In some embodiments, B is and A is In some embodiments, B is and A is In some embodiments, B is and A is In some embodiments, B is and A is

[0387] In some embodiments, the combination of R1, R2, R4, and R5 is as described elsewhere herein or as disclosed in Table 1. In some embodiments, B is A is p is 1, R1 is hydrogen, and R2 is an optionally substituted C1-C6 alkylamine (e.g., alkyl-substituted dimethylamine or -(CH2)3 dimethylamine). In some embodiments, B is A is R1 is hydrogen, and R2 is C 1-12 alkylamine (e.g., -CH2CH2NH2 or -(CH2)6NH2). In some embodiments, B is A is R1 is hydrogen, and R2 is C 1-12 alkylamine (e.g., -CH2CH2NH2 or -(CH2)6NH2). In some embodiments, B is A is R1 is hydrogen, and R2 is three fused 6-membered cycloalkyl rings. In some embodiments, B is A is R1 is hydrogen, and R2 is three fused 6-membered cycloalkyl rings. In some embodiments, B is A is R1 is hydrogen, and R2 is -(CH2) 1-6 pyridine (e.g., -(CH2)pyridine). In some embodiments, B is A is R1 is hydrogen, and R2 is -(CH2) 1-6 pyridine (e.g., -(CH2)pyridine). In some embodiments, B is A is R1 is hydrogen, and R2 is a C1-C6 hydroxyl group (e.g., -(CH2)2OH). In some embodiments, B is A is R1 is hydrogen, and R2 is a C1-C6 hydroxyl group (e.g., -(CH2)2OH). In some embodiments, B is A is R1 is hydrogen, and R2 is C 1-12Alkylamine (e.g., -(CH2) 12 NH2). In some embodiments, B is A is R1 is hydrogen, and R2 is C 1-12 Alkylamine (e.g., -(CH2) 12 NH2). In some embodiments, B is A is R1 is hydrogen, and R2 is boronic acid (e.g., phenylboronic acid). In some embodiments, B is A is R1 is hydrogen, and R2 is boronic acid (e.g., phenylboronic acid). In some embodiments, B is A is R1 is hydrogen, and R2 is C1-C6 hydroxy (e.g., -(CH2)2OH). In some embodiments, B is A is R1 is hydrogen, and R2 is C1-C6 hydroxy (e.g., -(CH2)2OH).

[0388] In one aspect, the macromolecule comprises repeating units of a first component and a second component, wherein the first component comprises the structure of component (A), and the second component comprises the structure of component (B):

[0389]

[0390] As provided herein, is interchangeably used with to refer to.

[0391] In some embodiments, each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl. In some embodiments, Y1 is hydrogen. In some embodiments, Y1 is C1-C6 alkyl. In some embodiments, Y2 is hydrogen. In some embodiments, Y2 is C1-C6 alkyl. In some embodiments, Y3 is hydrogen. In some embodiments, Y3 is C1-C6 alkyl. In some embodiments, Y1 is C1-C3 alkyl, and each of Y2 and Y3 is hydrogen. In some embodiments, Y1 is C1 alkyl (e.g., -CH3), and each of Y2 and Y3 is hydrogen.

[0392] In some embodiments, each of X1, X2, and X3 is independently selected from hydrogen or C1-C6 alkyl. In some embodiments, X1 is hydrogen. In some embodiments, X1 is C1-C6 alkyl. In some embodiments, X2 is hydrogen. In some embodiments, X2 is C1-C6 alkyl. In some embodiments, X3 is hydrogen. In some embodiments, X3 is C1-C6 alkyl. In some embodiments, X1 is C1-C3 alkyl and each of X2 and X3 is hydrogen. In some embodiments, X1 is C1 alkyl (e.g., -CH3) and each of X2 and X3 is hydrogen.

[0393] In some embodiments, A is In some embodiments, A is In some embodiments, A is In some embodiments, A is In some embodiments, the substituents of A (e.g., R1, R2, R4, R5) are described elsewhere herein. In some embodiments B is In some embodiments, B is In some embodiments, B is In some embodiments, B is

[0394] In some embodiments, B is In some embodiments, B is

[0395] In some embodiments, q is an integer between 1 and 6. In some embodiments, q is an integer between 1 and 3. 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.

[0396] In some embodiments, p is an integer between 1 and 20. 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. In some embodiments, p is 7. In some embodiments, p is 8. In some embodiments, p is 9. In some embodiments, p is 10. In some embodiments, p is 11. In some embodiments, p is 12. In some embodiments, p is 13. In some embodiments, p is 14. In some embodiments, p is 15. In some embodiments, p is 16. In some embodiments, p is 17. In some embodiments, p is 18. In some embodiments, p is 19. In some embodiments, p is 20.

[0397] In some embodiments, the macromolecule may further comprise a second structure of component (A). In some embodiments, the first structure of component (A) and the second structure of component (A) may be different. In some embodiments, the first structure of component (A) may be In some embodiments, the first structure of component (A) may be In some embodiments, the first structure of component (A) may comprise R1 as hydrogen and R2 as -CH2CH2NH2. In some embodiments, the second structure of component (A) may comprise R1 as hydrogen and R2 as a C1-C6 hydroxy group (e.g., -CH2CH2OH), an optionally substituted aryl group (e.g., 2-fluorotoluene), an optionally substituted heteroaryl group (e.g., 1-propylimidazole), two or more fused 5- to 6-membered rings (e.g., three fused 6-membered rings), or a monosaccharide (e.g., d-glucose).

[0398] The present disclosure also provides macromolecules comprising repeating units of component (A) such as repeating units of poly(ethylene glycol) dimethacrylate.

[0399] In some embodiments, component (A) can be present in the monomer mixture in a weight percentage of from about 10% to about 90%. In some embodiments, component (A) can be present in the monomer mixture in a weight percentage of from about 20% to about 80%. In some embodiments, component (A) can be present in the monomer mixture in a weight percentage of from about 40% to about 60%. In some embodiments, component (A) can be present in the monomer mixture in a weight percentage of about 50%. In some embodiments, component (B) or component (B’) can be present in the monomer mixture in a weight percentage of from about 10% to about 90%. In some embodiments, component (B) or component (B’) can be present in the monomer mixture in a weight percentage of from about 20% to about 80%. In some embodiments, component (B) or component (B’) can be present in the monomer mixture in a weight percentage of from about 40% to about 60%. In some embodiments, component (B) or component (B’) can be present in the monomer mixture in a weight percentage of about 50%.

[0400] As used herein, respectively indicate that when two vinyl groups have undergone a polymerization reaction to crosslink the macromolecular chain, divinylbenzene, ethylene glycol dimethacrylate, and methylene bis(acrylamide) are incorporated into the macromolecular chain. Thus, as shown in Tables 1-4, represents crosslinked divinylbenzene. Thus, as shown in Tables 1-4, represents crosslinked N,N'-methylenebis(acrylamide). Thus, as shown in Tables 1-4, represents crosslinked ethylene glycol dimethacrylate.

[0401] In some embodiments, the incorporation of refers to the incorporation of component (B) as a crosslinking agent into the macromolecule.

[0402] In some embodiments, the macromolecule may comprise repeating units as shown in Table 2, where n, m, x, and y denote the repeating units. In some embodiments, m is an integer greater than 2. In some embodiments, m is an integer between 2 and 50,000 (e.g., between 10 and 10,000, between 25 and 2,500, or between 50 and 1,000). In some embodiments, n is an integer greater than 2. In some embodiments, n is an integer between 2 and 50,000 (e.g., between 10 and 10,000, between 25 and 2,500, or between 50 and 1,000). In some embodiments, the repeating units represented by n and m are randomly copolymerized. In some embodiments, x is an integer greater than 2. In some embodiments, x is an integer between 2 and 50,000 (e.g., between 10 and 10,000, between 25 and 2,500, or between 50 and 1,000). In some embodiments, y is an integer greater than 2. In some embodiments, y is an integer between 2 and 50,000 (e.g., between 10 and 10,000, between 25 and 2,500, or between 50 and 1,000).

[0403] Table 2:

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415] As used herein, denotes the incorporation of divinylbenzene into the macromolecular chain when two vinyl groups have undergone a polymerization reaction to crosslink the macromolecular chain. In some embodiments, is represented as or a mixture thereof. In some embodiments, is represented as As used herein is represented as when two vinyl groups have undergone a polymerization reaction to crosslink the macromolecular chain, N,N'-methylenebisacrylamide (MBA) is incorporated into the macromolecular chain. In some embodiments is represented as or a mixture thereof. In some embodiments is represented as As used herein, when p is 1 is represented as when two vinyl groups have undergone a polymerization reaction to crosslink the macromolecular chain, ethylene glycol dimethacrylate (EGDMA) is incorporated into the macromolecular chain.

[0416] In any of the embodiments provided herein may refer to (polymerized) divinylbenzene. In some cases, the divinylbenzene is p-divinylbenzene. In some cases, the divinylbenzene is o-divinylbenzene. In some cases, the divinylbenzene is m-divinylbenzene. In some cases The structure of may interchangeably refer to In some cases, as used herein may refer to In some cases, as used herein may interchangeably refer to

[0417] In one aspect, the present disclosure describes a surface comprising a moiety of formula (IV):

[0418]

[0419] wherein Z is a linking moiety comprising a straight chain having 2 to 20 atoms selected from carbon, oxygen, and nitrogen, and optional substituents on the carbon and any nitrogen atoms in the straight chain;

[0420] R1 is hydrogen or a succinate ester; and

[0421] R2 is an optionally substituted -C1-C6 disulfide or C1-C6 thiol.

[0422] In some embodiments, Z is a straight chain having from 2 to 20 atoms. In some embodiments, Z is a straight chain having from 2 to 12 atoms. In some embodiments, Z is a straight chain having from 2 to 6 atoms. In some embodiments, Z is a straight chain having 2 atoms. In some embodiments, Z is a straight chain having 3 atoms. In some embodiments, Z is a straight chain having 4 atoms. In some embodiments, Z is a straight chain having 5 atoms. In some embodiments, Z is a straight chain having 6 atoms. In some embodiments, Z contains only carbon. In some embodiments, Z is a C2-C6 alkyl chain. In some embodiments, Z is a C3 alkyl. In some embodiments, Z contains oxygen, nitrogen, carbon, or a combination thereof. In some embodiments, Z contains substituents on the straight chain.

[0423] In some embodiments, Z contains an optionally substituted C2-C8 heteroalkyl (e.g., substituted by a hydroxyl group). In some embodiments, Z contains an optionally substituted C2-C8 alkoxy (e.g., substituted by a hydroxyl group).

[0424] In some embodiments, R1 is hydrogen, an optionally substituted succinate, an optionally substituted glutarate, an optionally substituted adipate, an optionally substituted pimelate, an optionally substituted suberate, an optionally substituted azelate, or an optionally substituted sebacate. In some embodiments, R1 is an optionally substituted glutarate. In some embodiments, R1 is an optionally substituted adipate. In some embodiments, R1 is an optionally substituted pimelate. In some embodiments, R1 is an optionally substituted suberate. In some embodiments, R1 is an optionally substituted azelate. In some embodiments, R1 is an optionally substituted sebacate. In some embodiments, R1 is hydrogen. In some embodiments, R1 is an optionally substituted C1-C6 alkyl. In some embodiments, R1 is substituted by one or more oxo and -COOH. In some embodiments, R1 is a succinate. In some embodiments, R1 is an optionally substituted succinate. In some embodiments, R2 is an optionally substituted -C1-C6 disulfide. In some embodiments, R2 is a C1-C6 thiol. In some embodiments, R2 is a substituted -C1-C6 alkyl disulfide. In some embodiments, R2 is a substituted di-C1-C6 alkyl disulfide. In some embodiments, R2 is -CH2CH2-S-S-CH2CH2NH2. In some embodiments, R2 is a C2 thiol (e.g., -(CH2)2SH). In some embodiments, Z is attached to a surface. In some embodiments, Z is covalently attached to a surface.

[0425] In some embodiments, the structure of formula (IV) comprises (Compound 508). In some embodiments, the structure of formula (IV) comprises (Compound 507). In some embodiments, the structure of formula (IV) comprises (Compound 509). Figure 15 Shows the reaction schemes for preparing Compounds 508 and 509, as well as the particle size and surface potential.

[0426] In one aspect, described herein is a surface that comprises a macromolecule or moiety of the present disclosure as described elsewhere herein, wherein the macromolecule or moiety is immobilized on the surface. In some embodiments, the structures disclosed in Tables 3 and 4 are immobilized on the surface, where n, m, x, y, and the surface are described elsewhere herein.

[0427] Table 3

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438] Table 4:

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451] In one aspect, the present disclosure describes a surface that comprises a macromolecule or portion of the present disclosure as described elsewhere herein, wherein the macromolecule or portion is immobilized on the surface. In some embodiments, the structure is immobilized on the surface and is described in Table 5, where n, m, and the surface are described elsewhere herein.

[0452] Table 5:

[0453]

[0454]

[0455] In some embodiments, as represented in the compounds herein (e.g., such as in Tables 1-5) represents a point of attachment of a unit of component (A) or component (B). In some embodiments, as represented in the compounds herein (e.g., such as in Tables 1-5) represents a point of attachment of a unit of component (A) or component (B). In some embodiments, represents a point of attachment for crosslinking with the monomer.

[0456] As used herein, represents incorporation of divinylbenzene into the macromolecular chain when two vinyl groups have undergone polymerization to crosslink the macromolecular chain. In some embodiments, represents as or a mixture thereof. In some embodiments, represents as As used herein, represents incorporation of N,N'-methylenebisacrylamide (MBA) into the macromolecular chain when two vinyl groups have undergone polymerization to crosslink the macromolecular chain. In some embodiments, represents as or a mixture thereof. In some embodiments, represents as As used herein, when p is 1, It represents that when two vinyl groups have undergone a polymerization reaction to crosslink the macromolecular chain, ethylene glycol dimethacrylate (EGDMA) is incorporated into the macromolecular chain.

[0457] In any of the embodiments provided herein, can refer to (polymerized) divinylbenzene. In some cases, the divinylbenzene is p-divinylbenzene. In some cases, the divinylbenzene is o-divinylbenzene. In some cases, the divinylbenzene is m-divinylbenzene. In some cases, The structure of can interchangeably refer to In some cases, as used herein can refer to In some cases, as used herein can interchangeably refer to

[0458] In some embodiments, the macromolecule or moiety is covalently coupled to the surface. In some embodiments, the macromolecule or moiety is electrostatically coupled to the surface. In some embodiments, the macromolecule or moiety is coupled to the surface via a polymerization event. In some embodiments, the polymerization event can include reacting with vinyl groups on the surface. In some embodiments, the surface comprises silica and a silane coupling agent is used to link the vinyl groups to the surface. In some embodiments, the surface can be a bead. In some embodiments, the surface can be a particle. In some embodiments, the particle can be a nanoparticle or microparticle having the dimensions and properties described elsewhere herein. In some embodiments, the particle can comprise features and properties as described elsewhere herein (e.g., comprising iron oxide).

[0459] In some embodiments of formula (I), formula (I-A), formula (I-A'), formula (II), formula (II'), formula (III), formula (III'), or formula (III-A) and formula (IV), each of R1, R2, R4, R5 is independently selected from hydrogen, nitrogen,

[0460]

[0461]

[0462] In some embodiments, R1, R2, R4, and R5 may contain a charge. In some embodiments, R1, R2, R4, and R5 may contain a positive charge. In such cases, R2, R2, R4, and R5 may contain a negatively charged counterion. In some cases, the negatively charged counterion may be a halogen (e.g., fluoride, chloride, bromide, or iodide). In some embodiments, R1, R2, R4, and R5 may contain a negative charge. In such cases, R1, R2, R4, and R5 may contain a positively charged counterion (e.g., sodium ion, lithium ion, etc.).

[0463] In some embodiments, the surface is the surface of compound 450 (e.g., the compounds in Table 4).

[0464] In some embodiments, the surface is the surface of compound 424 (e.g., the compounds in Table 4).

[0465] In some embodiments, the surface is the surface of the compound: of.

[0466] In some embodiments, the surface is the surface of compound 418 (e.g., the compounds in Table 4).

[0467] In some embodiments, the surface is the surface of compound 413 (e.g., the compounds in Table 4).

[0468] In some embodiments, the surface is the surface of compound 428 (e.g., the compounds in Table 4).

[0469] In some embodiments, the surface is the surface of compound 425 (e.g., the compounds in Table 4).

[0470] In some embodiments, the surface is of.

[0471] In some embodiments, the surface is the surface of compound 449 (e.g., the compounds in Table 4).

[0472] In some embodiments, the surface is the surface of compound 413 (e.g., the compounds in Table 4).

[0473] In some embodiments, the surface is the surface of compound 441 (e.g., the compounds in Table 4).

[0474] In some embodiments, the surface is the surface of compound 425 (e.g., the compounds in Table 4).

[0475] In some embodiments, the surface is the surface of compound 424 (e.g., the compounds in Table 4).

[0476] In some embodiments, the surface is the surface of compound 431 (e.g., a compound in Table 4).

[0477] In some embodiments, the surface is the surface of compound 428 (e.g., a compound in Table 4).

[0478] In some embodiments, the surface is the surface of compound 507.

[0479] In some embodiments, Z represents the point of attachment of the units of component (A) or component (B).

[0480] In some embodiments, the macromolecule (e.g., bound to the surface) is an iron oxide nanoparticle.

[0481] In some embodiments, the macromolecule is a silica-coated iron oxide nanoparticle.

[0482] In some cases, multiple macromolecules are used in combination. In some cases, one or more unique macromolecules (or macromolecules bound to the surface) are used to enhance the surface's ability to, for example, identify proteins, peptides, or groups of proteins.

[0483] In some embodiments, the surfaces provided herein can be used in combination with one or more other unique surfaces provided herein, such as in any of the methods provided herein. In some cases, when used in combination, the surfaces can exhibit enhanced performance, such as in the identification of proteins, peptides, or groups of proteins. In some embodiments, two or more surfaces provided herein can be used in combination, such as in any of the methods provided herein. In some embodiments, three or more surfaces provided herein can be used in combination, such as in any of the methods provided herein. In some embodiments, two unique surfaces provided herein can be provided in combination, such as in any of the methods provided herein.

[0484] In some embodiments, the surfaces of compound 450 and compound 424 (e.g., compounds in Table 4) can be provided together in the methods provided herein.

[0485] In some embodiments, the surfaces of compound 424 (e.g., a compound in Table 4) and compound 509 can be provided together in the methods provided herein.

[0486] In some embodiments, the surfaces of compound 418 and compound 424 (e.g., compounds in Table 4) can be provided together in the methods provided herein.

[0487] In some embodiments, the surfaces of compound 428 and compound 424 (e.g., compounds in Table 4) can be provided together in the methods provided herein.

[0488] In some embodiments, the surfaces of compound 425 and compound 424 (e.g., the compounds in Table 4) can be provided together in the methods provided herein.

[0489] In some embodiments, the surfaces of compound 413 and compound 424 (e.g., the compounds in Table 4) can be provided together in the methods provided herein.

[0490] In some embodiments, the surfaces of compound 413 and compound 507 can be provided together in the methods provided herein.

[0491] In some embodiments, the surfaces of compound 508 and compound 424 can be provided together in the methods provided herein.

[0492] Some embodiments disclosed herein include surfaces for adsorbing biomolecules from biological samples, wherein the surfaces are functionalized with carboxylic acids and thiols, and wherein the functionalization promotes the adsorption of biomolecules upon contact with the biological sample. As non-limiting examples, the surfaces can be compound 328, compound 508, or compound 509. Such surfaces can be advantageous, for example, to adsorb a large number of different proteins in a biological sample, they effectively compress the dynamic range of biomolecules in the biological sample, exhibit optimal adsorption properties at biological pH, and / or provide improved reproducibility for biological assays.

[0493] In some embodiments, the molar ratio of carboxylic acid to thiol functionalization can be from about 3:1 to about 1:3. In some embodiments, the molar ratio of carboxylic acid to thiol functionalization can be from about 2:1 to about 1:2. In some embodiments, the molar ratio of carboxylic acid to thiol functionalization can be from about 3:2 to about 2:3. In some embodiments, the molar ratio of carboxylic acid to thiol functionalization can be from about 4:3 to about 3:4.

[0494] In some embodiments, the carboxylic acid and thiol are covalently coupled to the surface. In some embodiments, the surface comprises a polymer functionalized with carboxylic acid and thiol. In some embodiments, the polymer comprises repeating units functionalized with both carboxylic acid and thiol.

[0495] In some embodiments, the surface is functionalized with the following moieties: wherein R 1 is an optionally substituted succinate, an optionally substituted glutarate, an optionally substituted adipate, an optionally substituted pimelate, an optionally substituted suberate, an optionally substituted azelate, or an optionally substituted sebacate; and R 2 is an optionally substituted C1-C6 thiol. In some embodiments, R 1 is succinate, and R 2is ethyl mercaptan. A suitable tethering moiety can be covalently attached to the nitrogen atom, thereby covalently coupling the moiety to the surface.

[0496] In some embodiments, the surface is functionalized with the following moieties: A suitable tethering moiety can be covalently attached to the nitrogen atom, thereby covalently coupling the moiety to the surface.

[0497] Surfaces functionalized with carboxylic acid and thiol can be used in any of the systems, methods, and kits disclosed herein.

[0498] Surface

[0499] In some embodiments, the surfaces disclosed herein can be used to identify multiple proteins, peptides, or protein groups using the Proteograph TM workflow (corresponding to MS analysis of the biomolecular corona of unique surfaces in the surface), which is incorporated herein by reference in its entirety. Feature intensity refers to the intensity of discrete spikes (“features”) seen on a plot of mass-to-charge ratio versus intensity in a mass spectrometry run of a sample. These features can correspond to variable ionization fragments of peptides and / or proteins. Using the data analysis methods described herein, feature intensities can be classified into protein groups. A protein group refers to two or more proteins identified by shared peptide sequences. Alternatively, a protein group can refer to one protein identified using a unique identification sequence. For example, if a shared peptide sequence is determined between two proteins (Protein 1: XYZZX and Protein 2: XYZYZ) in a sample, the protein group can be the “XYZ protein group” with two members (Protein 1 and Protein 2). Alternatively, if the peptide sequence is unique to a single protein (Protein 1), the protein group can be the “ZZX” protein group with one member (Protein 1). Each protein group can be supported by more than one peptide sequence. Proteins detected or identified according to the present disclosure can refer to unique proteins detected in a sample (e.g., proteins unique relative to other proteins detected using mass spectrometry). Thus, analysis of the proteins present in the unique corona corresponding to the unique surface in the surface results in a large number of feature intensities. This number decreases as the feature intensities are processed into unique peptides, further decreases as the unique peptides are processed into unique proteins, and further decreases as the peptides are grouped into protein groups (two or more proteins sharing unique peptide sequences).

[0500] The surface disclosed herein can be used to identify at least 100 proteins, at least 200 proteins, at least 300 proteins, at least 400 proteins, at least 500 proteins, at least 600 proteins, at least 700 proteins, at least 800 proteins, at least 900 proteins, at least 1000 proteins, at least 1100 proteins, at least 1200 proteins, at least 1300 proteins, at least 1400 proteins, at least 1500 proteins, at least 1600 proteins, at least 1700 proteins, at least 1800 proteins, at least 1900 proteins, at least 2000 proteins, at least 2100 proteins, at least 2200 proteins, at least 2300 proteins, at least 2400 proteins, at least 2500 proteins, at least 2600 proteins, at least 2700 proteins, at least 2800 proteins, at least 2900 proteins, at least 3000 proteins, at least 3100 proteins, at least 3200 proteins, at least 3300 proteins, at least 3400 proteins, at least 3500 proteins, at least 3600 proteins, at least 3700 proteins, at least 3800 proteins, at least 3900 proteins, at least 4000 proteins, at least 4100 proteins, at least 4200 proteins, at least 4300 proteins, at least 4400 proteins, at least 4500 proteins, at least 4600 proteins, at least 4700 proteins, at least 4800 proteins, at least 4900 proteins, at least 5000 proteins, at least 10000 proteins, at least 20000 proteins, at least 50000 proteins, at least 100000 proteins, from 100 to 5000 proteins, from 200 to 4700 proteins, from 300 to 4400 proteins, from 400 to 4100 proteins, from 500 to 3800 proteins, from 600 to 3500 proteins, from 700 to 3200 proteins, from 800 to 2900 proteins, from 900 to 2600 proteins, from 1000 to 2300 proteins, from 1000 to 3000 proteins, from 3000 to 4000 proteins, from 4000 to 5000 proteins, from 5000 to 6000 proteins, from 6000 to 7000 proteins, from 7000 to 8000 proteins, from 8000 to 9000 proteins, from 9000 to 10000 proteins, from 10000 to 11000 proteins, from 11000 to 12000 proteins, from 12000 to 13000 proteins, from 13000 to 14000 proteins, from 14000 to 15000 proteins, from 15000 to 16000 proteins, from 16000 to 17000 proteins, from 17000 to 18000 proteins, from 18000 to 19000 proteins,19,000 to 20,000 proteins, 20,000 to 25,000 proteins, 25,000 to 30,000 proteins, 10,000 to 20,000 proteins, 10,000 to 50,000 proteins, 20,000 to 100,000 proteins, 2,000 to 20,000 proteins, 1,800 to 20,000 proteins or 10,000 to 100,000 proteins.

[0501] The surface disclosed in this article can be used to identify at least 100 protein groups, at least 200 protein groups, at least 300 protein groups, at least 400 protein groups, at least 500 protein groups, at least 600 protein groups, at least 700 protein groups, at least 800 protein groups, at least 900 protein groups, at least 1000 protein groups, at least 1100 protein groups, at least 1200 protein groups, at least 1300 protein groups, at least 1400 protein groups, at least 1500 protein groups, at least 1600 protein groups, at least 1700 protein groups, at least 1800 protein groups, at least 1900 protein groups, at least 2000 protein groups, at least 2100 protein groups, at least 2200 protein groups, at least 2300 protein groups, at least 2400 protein groups, at least 2500 protein groups, at least 2600 protein groups, at least 2700 protein groups, at least 2800 protein groups, at least 2900 protein groups, at least 3000 protein groups, at least 3100 protein groups, at least 3200 protein groups, at least 3300 protein groups, at least 3400 protein groups, at least 3500 protein groups, at least 3600 protein groups, at least 3700 protein groups, at least 3800 protein groups, at least 3900 protein groups, at least 4000 protein groups, at least 4100 protein groups, at least 4200 protein groups, at least 4300 protein groups, at least 4400 protein groups, at least 4500 protein groups, at least 4600 protein groups, at least 4700 protein groups, at least 4800 protein groups, at least 4900 protein groups, at least 5000 protein groups, at least 10000 protein groups, at least 20000 protein groups, at least 100000 protein groups, 100 to 5000 protein groups, 200 to 4700 protein groups, 300 to 4400 protein groups, 400 to 4100 protein groups, 500 to 3800 protein groups, 600 to 3500 protein groups, 700 to 3200 protein groups, 800 to 2900 protein groups, 900 to 2600 protein groups, 1000 to 2300 protein groups, 1000 to 3000 protein groups, 3000 to 4000 protein groups, 4000 to 5000 protein groups, 5000 to 6000 protein groups, 6000 to 7000 protein groups, 7000 to 8000 protein groups, 8000 to 9000 protein groups, 9000 to 10000 protein groups, 10000 to 11000 protein groups,11,000 to 12,000 protein groups, 12,000 to 13,000 protein groups, 13,000 to 14,000 protein groups, 14,000 to 15,000 protein groups, 15,000 to 16,000 protein groups, 16,000 to 17,000 protein groups, 17,000 to 18,000 protein groups, 18,000 to 19,000 protein groups, 19,000 to 20,000 protein groups, 20,000 to 25,000 protein groups, 25,000 to 30,000 protein groups, 10,000 to 20,000 protein groups, 10,000 to 50,000 protein groups, 20,000 to 100,000 protein groups, 2,000 to 20,000 protein groups, 1,800 to 20,000 protein groups, or 10,000 to 100,000 protein groups. For example, Figure 4 shows the number of protein groups with different surface adhesions of the present disclosure and the Jaccard index representing the difference between two surfaces in terms of the adhered protein groups. In another example, Figure 5 shows the number of protein groups adhered to different surfaces of the present disclosure and the Jaccard index representing the difference between two surfaces in terms of the adhered protein groups; NP-1 to NP-5 are commercially available nanoparticles found in the Proteograph TM V1.2 kit, and P-073 is a dextran-functionalized nanoparticle.

[0502] The surfaces disclosed herein can be used to identify the number of unique proteins disclosed herein, and / or any specific protein disclosed herein, over a wide dynamic range. For example, a surface comprising a unique surface disclosed herein can enrich proteins in a sample, which can be identified across the entire dynamic range of proteins present in the sample (e.g., a plasma sample) using a Proteograph workflow. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 2. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 3. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 4. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 5. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 6. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 7. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 8. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 9. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 10. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 11. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 12. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 13. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 14. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of at least 15. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of 2 to 15. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of 6 to 15. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of 8 to 12. In some embodiments, a surface comprising any number of unique surfaces disclosed herein enriches and identifies proteins over a dynamic range of 2 to 5.In some embodiments, surfaces that include any number of the unique surfaces disclosed herein enrich and identify proteins within a dynamic range of 5 to 10.

[0503] In some embodiments, a panel can have more than one surface. Increasing the number of surfaces in a panel can be a way to increase the number of proteins that can be identified in a given sample. For example, a panel size of one particle type can identify at least 400 unique proteins, a panel size of two surfaces can identify at least 550 proteins, a panel size of three surfaces can identify at least 700 proteins, a panel size of four surfaces can identify at least 800 proteins, a panel size of five surfaces can identify at least 900 proteins, a panel size of six surfaces can identify at least 1000 proteins, a panel size of seven surfaces can identify at least 1050 proteins, a panel size of eight surfaces can identify at least 1100 proteins, a panel size of nine surfaces can identify at least 1175 proteins, a panel size of ten surfaces can identify 1200 proteins, a panel size of eleven surfaces can identify at least 1250 proteins, and a panel size of twelve surfaces can identify at least 1300 proteins. The surfaces can include nano-surfaces. In some embodiments, the surfaces are particles, such as microparticles and nanoparticles.

[0504] In some embodiments, a panel size of one surface type can identify 400 to 100 unique proteins. In some embodiments, a panel size of two surfaces can identify 800 to 1500 unique proteins. In some embodiments, a panel size of three surfaces can identify 1000 to 2000 unique proteins. In some embodiments, a panel size of four surfaces can identify 1500 to 3500 unique proteins. In some embodiments, a panel size of five surfaces can identify 2500 to 4500 unique proteins. In some embodiments, a panel size of six surfaces can identify 4000 to 5500 unique proteins. In some embodiments, a panel size of seven surfaces can identify 4500 to 6000 unique proteins. In some embodiments, a panel size of eight surfaces can identify 5000 to 6500 unique proteins. In some embodiments, a panel size of nine surfaces can identify 5500 to 7000 unique proteins. In some embodiments, a panel size of ten surfaces can identify 6000 to 7500 unique proteins.

[0505] The present disclosure provides unique surfaces that include at least one physicochemical property that differs between a first surface and a second surface. For example, the present disclosure provides surfaces having at least 2 unique surfaces, at least 3 unique surfaces, at least 4 unique surfaces, at least 5 unique surfaces, at least 6 unique surfaces, at least 7 unique surfaces, at least 8 unique surfaces, at least 9 unique surfaces, at least 10 unique surfaces, at least 11 unique surfaces, at least 12 unique surfaces, at least 13 unique surfaces, at least 14 unique surfaces, at least 15 unique surfaces, at least 20 unique surfaces, at least 25 unique surfaces, at least 30 unique surfaces, at least 35 unique surfaces, at least 40 unique surfaces, at least 45 unique surfaces, at least 50 unique surfaces, at least 100 unique surfaces, at least 150 unique surfaces, at least 200 unique surfaces, at least 250 unique surfaces, at least 300 unique surfaces, at least 350 unique surfaces, at least 400 unique surfaces, at least 450 unique surfaces, at least 500 unique surfaces, from 2 to 500 unique surfaces, from 2 to 5 unique surfaces, from 5 to 10 unique surfaces, from 10 to 15 unique surfaces, from 15 to 20 unique surfaces, from 20 to 40 unique surfaces, from 40 to 60 unique surfaces, from 60 to 80 unique surfaces, from 80 to 100 unique surfaces, from 100 to 500 unique surfaces, from 4 to 15 unique surfaces, or from 2 to 20 unique surfaces. The surfaces can include nano-surfaces.

[0506] In some embodiments, the present disclosure provides a kit size of at least 1 unique type of particle, at least 2 unique surfaces, at least 3 unique surfaces, at least 4 unique surfaces, at least 5 unique surfaces, at least 6 unique surfaces, at least 7 unique surfaces, at least 8 unique surfaces, at least 9 unique surfaces, at least 10 unique surfaces, at least 11 unique surfaces, at least 12 unique surfaces, at least 13 unique surfaces, at least 14 unique surfaces, at least 15 unique surfaces, at least 16 unique surfaces, at least 17 unique surfaces, at least 18 unique surfaces, at least 19 unique surfaces, at least 20 unique surfaces, at least 25 unique surfaces, at least 30 unique surfaces, at least 35 unique surfaces, at least 40 unique surfaces, at least 45 unique surfaces, at least 50 unique surfaces, at least 55 unique surfaces, at least 60 unique surfaces, at least 65 unique surfaces, at least 70 unique surfaces, at least 75 unique surfaces, at least 80 unique surfaces, at least 85 unique surfaces, at least 90 unique surfaces, at least 95 unique surfaces, at least 100 unique surfaces, from 1 to 5 unique surfaces, from 5 to 10 unique surfaces, from 10 to 15 unique surfaces, from 15 to 20 unique surfaces, from 20 to 25 unique surfaces, from 25 to 30 unique surfaces, from 30 to 35 unique surfaces, from 35 to 40 unique surfaces, from 40 to 45 unique surfaces, from 45 to 50 unique surfaces, from 50 to 55 unique surfaces, from 55 to 60 unique surfaces, from 60 to 65 unique surfaces, from 65 to 70 unique surfaces, from 70 to 75 unique surfaces, from 75 to 80 unique surfaces, from 80 to 85 unique surfaces, from 85 to 90 unique surfaces, from 90 to 95 unique surfaces, from 95 to 100 unique surfaces, from 1 to 100 unique surfaces, from 20 to 40 unique surfaces, from 5 to 10 unique surfaces, from 3 to 7 unique surfaces, from 2 to 10 unique surfaces, from 6 to 15 unique surfaces or from 10 to 20 unique surfaces. In certain embodiments, the present disclosure provides a kit size of from 3 to 10 surfaces. In certain embodiments, the present disclosure provides a kit size of from 4 to 11 unique surfaces. In certain embodiments, the present disclosure provides a kit size of from 5 to 15 unique surfaces. In certain embodiments, the present disclosure provides a kit size of from 5 to 15 unique surfaces. In certain embodiments, the present disclosure provides a kit size of from 8 to 12 unique surfaces. In certain embodiments, the present disclosure provides a kit size of from 9 to 13 unique surfaces. In certain embodiments, the present disclosure provides a kit size of from 2 to 20 unique surfaces. In certain embodiments, the present disclosure provides a kit size of 10 unique surfaces. Surfaces can include nano-surfaces. In some embodiments, the surfaces are particles, such as microparticles and nanoparticles.

[0507] For example, the present disclosure provides a surface having at least 2 distinct surfaces, at least 3 different surface chemistries, at least 4 different surface chemistries, at least 5 different surface chemistries, at least 6 different surface chemistries, at least 7 different surface chemistries, at least 8 different surface chemistries, at least 9 different surface chemistries, at least 10 different surface chemistries, at least 11 different surface chemistries, at least 12 different surface chemistries, at least 13 different surface chemistries, at least 14 different surface chemistries, at least 15 different surface chemistries, at least 20 different surface chemistries, at least 25 different surface chemistries, at least 30 different surface chemistries, at least 35 different surface chemistries, at least 40 different surface chemistries, at least 45 different surface chemistries, at least 50 different surface chemistries, at least 100 different surface chemistries, at least 150 different surface chemistries, at least 200 different surface chemistries, at least 250 different surface chemistries, at least 300 different surface chemistries, at least 350 different surface chemistries, at least 400 different surface chemistries, at least 450 different surface chemistries, at least 500 different surface chemistries, from 2 to 500 different surface chemistries, from 2 to 5 different surface chemistries, from 5 to 10 different surface chemistries, from 10 to 15 different surface chemistries, from 15 to 20 different surface chemistries, from 20 to 40 different surface chemistries, from 40 to 60 different surface chemistries, from 60 to 80 different surface chemistries, from 80 to 100 different surface chemistries, from 100 to 500 different surface chemistries, from 4 to 15 different surface chemistries, or from 2 to 20 different surface chemistries.

[0508] The present disclosure provides a surface having at least 2 different surface chemistries, at least 3 different surface chemistries, at least 4 different surface chemistries, at least 5 different surface chemistries, at least 6 different surface chemistries, at least 7 different surface chemistries, at least 8 different surface chemistries, at least 9 different surface chemistries, at least 10 different surface chemistries, at least 11 different surface chemistries, at least 12 different surface chemistries, at least 13 different surface chemistries, at least 14 different surface chemistries, at least 15 different surface chemistries, at least 20 different surface chemistries, at least 25 different surface chemistries, at least 30 different surface chemistries, at least 35 different surface chemistries, at least 40 different surface chemistries, at least 45 different surface chemistries, at least 50 different surface chemistries, at least 100 different surface chemistries, at least 150 different surface chemistries, at least 200 different surface chemistries, at least 250 different surface chemistries, at least 300 different surface chemistries, at least 350 different surface chemistries, at least 400 different surface chemistries, at least 450 different surface chemistries, at least 500 different surface chemistries, from 2 to 500 different surface chemistries, from 2 to 5 different surface chemistries, from 5 to 10 different surface chemistries, from 10 to 15 different surface chemistries, from 15 to 20 different surface chemistries, from 20 to 40 different surface chemistries, from 40 to 60 different surface chemistries, from 60 to 80 different surface chemistries, from 80 to 100 different surface chemistries, from 100 to 500 different surface chemistries, from 4 to 15 different surface chemistries, or from 2 to 20 different surface chemistries.

[0509] In some embodiments, the kit includes a kit having particles of different sizes. For example, the kit can include a first particle having a diameter of about 100 nm, a second particle having a diameter of about 3 microns, and a third particle having a diameter of about 4 microns. Additionally, the particles can have the same surface chemistry. In some embodiments, the different particle sizes in the kit can be incubated separately with a biological fluid. In some embodiments, the different particle sizes in the kit can be incubated with a biological fluid in a single mixture. As used herein, a particle mixture having a polydispersity index (PDI) greater than 0.5 (e.g., greater than 0.75) should be considered to have two or more different particle sizes.

[0510] The present disclosure provides a surface having at least 2 different physical properties, at least 3 different physical properties, at least 4 different physical properties, at least 5 different physical properties, at least 6 different physical properties, at least 7 different physical properties, at least 8 different physical properties, at least 9 different physical properties, at least 10 different physical properties, at least 11 different physical properties, at least 12 different physical properties, at least 13 different physical properties, at least 14 different physical properties, at least 15 different physical properties, at least 20 different physical properties, at least 25 different physical properties, at least 30 different physical properties, at least 35 different physical properties, at least 40 different physical properties, at least 45 different physical properties, at least 50 different physical properties, at least 100 different physical properties, at least 150 different physical properties, at least 200 different physical properties, at least 250 different physical properties, at least 300 different physical properties, at least 350 different physical properties, at least 400 different physical properties, at least 450 different physical properties, at least 500 different physical properties, from 2 to 500 different physical properties, from 2 to 5 different physical properties, from 5 to 10 different physical properties, from 10 to 15 different physical properties, from 15 to 20 different physical properties, from 20 to 40 different physical properties, from 40 to 60 different physical properties, from 60 to 80 different physical properties, from 80 to 100 different physical properties, from 100 to 500 different physical properties, from 4 to 15 different physical properties, or from 2 to 20 different physical properties.

[0511] In some embodiments, the kit comprises a kit having particles of different sizes. For example, the kit can include a first particle having a diameter of about 100 nm, a second particle having a diameter of about 3 microns, and a third particle having a diameter of about 4 microns. Additionally, these particles can have the same surface chemistry. In some embodiments, the different particle sizes in the kit can be incubated with a biological fluid separately. In some embodiments, the different particle sizes in the kit can be incubated with a biological fluid in a single mixture. As used herein, a particle mixture having a polydispersity index (PDI) greater than 0.5 (e.g., greater than 0.75, greater than 1.0, greater than 1.5, or greater than 2.0) should be considered to have two or more different particle sizes.

[0512] In some embodiments, the panel that identifies proteins and correlates biomarkers with diseases includes a panel selected from the surfaces described in Tables 3 and 4. Surfaces that are particularly suitable for identifying a large number of proteins (e.g., greater than 1500 proteins) in a sample include 5 to 10 unique surfaces in an assay. The number of unique surfaces included in the surface can be adjusted for a specific application (e.g., detecting a subset of specific proteins or detecting a group of markers associated with a specific disease). In some embodiments, the panel having physicochemically unique surfaces (optimally for identifying proteins and correlating biomarkers with diseases) includes silica-coated SPIONs, acrylamide-based SPIONs, and acrylate-based SPIONs. For example, the panel of surfaces disclosed herein that generates information-rich proteomic data via their protein corona (which can be correlated with biomarkers and diseases with high sensitivity and specificity) includes silica-coated SPIONs (SP-003), poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA)-coated SPIONs (SP-007), and poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)-coated SPIONs (SP-011).

[0513] In some embodiments, the entire assay time (including sample preparation and LC-MS) from a single pooled plasma can be about 8 hours. In some embodiments, the entire assay time (including sample preparation and LC-MS) from a single pooled plasma can be about at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, at least 5 minutes to 10 minutes, at least 10 minutes to 20 minutes, at least 20 minutes to 30 minutes, at least 30 minutes to 40 minutes, at least 40 minutes to 50 minutes, at least 50 minutes to 60 minutes, at least 1 hour to 1.5 hours, at least 1.5 hours to 2 hours, at least 2 hours to 2.5 hours, at least 2.5 hours to 3 hours, at least 3 hours to 3.5 hours, at least 3.5 hours to 4 hours, at least 4 hours to 4.5 hours, at least 4.5 hours to 5 hours, at least 5 hours to 5.5 hours, at least 5.5 hours to 6 hours, at least 6 hours to 6.5 hours, at least 6.5 hours to 7 hours, at least 7 hours to 7.5 hours, at least 7.5 hours to 8 hours, at least 8 hours to 8.5 hours, at least 8.5 hours to 9 hours, at least 9 hours to 9.5 hours, or at least 9.5 hours to 10 hours.

[0514] Biological sample

[0515] The surfaces of the present disclosure can be used to generate proteomic data from the protein corona and subsequently correlate it with any of the biological states described herein. Samples consistent with the present disclosure include biological samples from a subject. The subject can be a human or a non-human animal. The biological sample can be a biological fluid. For example, the biological fluid can be plasma, serum, CSF, urine, tears, cell lysate, tissue lysate, cell homogenate, tissue homogenate, nipple aspirate, fecal sample, synovial fluid, and whole blood or saliva. The biological sample can be a culture medium from cell culture. The sample can also be a non-biological sample, such as water, milk, a solvent, or any sample homogenized into a fluid state. In some embodiments, the biological sample can be a cell-free biological sample (e.g., plasma or serum). In some embodiments, the biological sample can contain extracellular vesicles. The biological sample can contain multiple proteins or proteomic data, and the proteins or proteomic data can be analyzed after adsorbing the proteins onto the surfaces of various surfaces in the kit and subsequently digesting the protein corona. The proteomic data can include nucleic acids, peptides, or proteins. Any sample herein can contain multiple different analytes, which can be analyzed using the compositions and methods disclosed herein. The analytes can be proteins, peptides, small molecules, nucleic acids, metabolites, lipids, or any molecule that can bind to or interact with the surface of the particle type.

[0516] Compositions and methods for multi-omics analysis are disclosed herein. "Multi-omics" or "multi-omic" can refer to an analytical method for large-scale analysis of biomolecules, where the data set is multiple groups, such as proteome, genome, transcriptome, lipidome, and metabolome. Non-limiting examples of multi-omics data include proteomic data, genomic data, lipidomic data, glycomic data, transcriptomic data, or metabolomic data. "Biomolecule" in "biomolecular corona" can refer to any molecule or biological component produced by or present in an organism. Non-limiting examples of biomolecules include proteins (protein corona), polypeptides, polysaccharides, sugars, lipids, lipoproteins, metabolites, oligonucleotides, nucleic acids (DNA, RNA, microRNA, plasmid, single-stranded nucleic acid, double-stranded nucleic acid), metabolome, and small molecules, such as primary metabolites, secondary metabolites, and other natural products, or any combination thereof. In some embodiments, the biomolecule is selected from proteins, nucleic acids, lipids, and metabolome.

[0517] In some embodiments, the sample of the present disclosure can be a plurality of samples. At least two samples among the plurality of samples can be spatially isolated. Spatially isolated means samples contained in separate volumes. For example, spatially isolated samples can refer to samples in separate wells in a plate or in separate tubes. Spatially isolated samples can refer to samples in separate wells in a plate or in separate tubes and assayed together on the same device. In some embodiments, the present disclosure provides a surface compatible with analyzing a plurality of samples, such as at least 2 spatially isolated samples, at least 5 spatially isolated samples, at least 10 spatially isolated samples, at least 15 spatially isolated samples, at least 20 spatially isolated samples, at least 25 spatially isolated samples, at least 30 spatially isolated samples, at least 35 spatially isolated samples, at least 40 spatially isolated samples, at least 45 spatially isolated samples, at least 50 spatially isolated samples, at least 55 spatially isolated samples, at least 60 spatially isolated samples, at least 65 spatially isolated samples, at least 70 spatially isolated samples, at least 75 spatially isolated samples, at least 80 spatially isolated samples, at least 85 spatially isolated samples, at least 90 spatially isolated samples, at least 95 spatially isolated samples, at least 96 spatially isolated samples, at least 100 spatially isolated samples, at least 120 spatially isolated samples, at least 140 spatially isolated samples, at least 160 spatially isolated samples, at least 180 spatially isolated samples, at least 200 spatially isolated samples, at least 220 spatially isolated samples, at least 240 spatially isolated samples, at least 260 spatially isolated samples, at least 280 spatially isolated samples, at least 300 spatially isolated samples, at least 320 spatially isolated samples, at least 340 spatially isolated samples, at least 360 spatially isolated samples, at least 380 spatially isolated samples, at least 400 spatially isolated samples, at least 420 spatially isolated samples, at least 440 spatially isolated samples, at least 460 spatially isolated samples, at least 480 spatially isolated samples, at least 500 spatially isolated samples, at least 600 spatially isolated samples, at least 700 spatially isolated samples, at least 800 spatially isolated samples, at least 900 spatially isolated samples, at least 1000 spatially isolated samples, at least 1100 spatially isolated samples, at least 1200 spatially isolated samples, at least 1300 spatially isolated samples, at least 1400 spatially isolated samples, at least 1500 spatially isolated samples, at least 1600 spatially isolated samples, at least 1700 spatially isolated samples, at least 1800 spatially isolated samples,At least 1,900 spatially isolated samples, at least 2,000 spatially isolated samples, at least 5,000 spatially isolated samples, at least 10,000 spatially isolated samples, 2 to 10 spatially isolated samples, 2 to 100 spatially isolated samples, 2 to 200 spatially isolated samples, 2 to 300 spatially isolated samples, 50 to 150 spatially isolated samples, 10 to 20 spatially isolated samples, 20 to 30 spatially isolated samples, 30 to 40 spatially isolated samples, 40 to 50 spatially isolated samples, 50 to 60 spatially isolated samples, 60 to 70 spatially isolated samples, 70 to 80 spatially isolated samples, 80 to 90 spatially isolated samples, 90 to 100 spatially isolated samples, 100 to 150 spatially isolated samples, 150 to 200 spatially isolated samples, 200 to 250 spatially isolated samples, 250 to 300 spatially isolated samples, 300 to 350 spatially isolated samples, 350 to 400 spatially isolated samples, 400 to 450 spatially isolated samples, 450 to 500 spatially isolated samples, 500 to 600 spatially isolated samples, 600 to 700 spatially isolated samples, 700 to 800 spatially isolated samples, 800 to 900 spatially isolated samples, 900 to 1,000 spatially isolated samples, 1,000 to 2,000 spatially isolated samples, 2,000 to 3,000 spatially isolated samples, 3,000 to 4,000 spatially isolated samples, 4,000 to 5,000 spatially isolated samples, 5,000 to 6,000 spatially isolated samples, 6,000 to 7,000 spatially isolated samples, 7,000 to 8,000 spatially isolated samples, 8,000 to 9,000 spatially isolated samples, or 9,000 to 10,000 spatially isolated samples.

[0518] The methods disclosed herein include isolating a surface from one or more samples. The surface can be superparamagnetic to allow for rapid isolation or separation from the sample by application of a magnetic field. Additionally, multiple spatially isolated samples can be processed in parallel. Thus, the methods disclosed herein isolate or separate the surface from unbound proteins in multiple spatially isolated sets using a magnet simultaneously. For example, the surface can be incubated with multiple spatially isolated samples, where each spatially isolated sample is in a well of a microtiter plate (e.g., a 96-well plate). After incubation, by placing the entire plate on a magnet, the surface in each well of the microtiter plate can be separated from the unbound proteins present in the spatially isolated samples. This simultaneously pulls down the superparamagnetic particles in the surface. The supernatant in each well can be removed to remove the unbound proteins. These steps (incubation, pull-down using a magnet) can be repeated to effectively wash the particles and thereby remove residual background unbound proteins that may be present in the sample. This is one example, but those skilled in the art can envision many other scenarios where superparamagnetic particles are rapidly isolated from one or more spatially isolated samples simultaneously.

[0519] In some embodiments, the kits of the present disclosure provide for the identification and measurement of specific proteins in a biological sample by processing proteomics data via a corona formed on the surface of the digestion particles. Examples of proteins that can be identified and measured include high-abundance proteins, medium-abundance proteins, and low-abundance proteins. Low-abundance proteins can be present in the sample at a concentration equal to or less than about 10 ng / mL. High-abundance proteins can be present in the sample at a concentration equal to or greater than about 10 μg / mL. Medium-abundance proteins can be present in the sample at a concentration between about 10 ng / mL and about 10 μg / mL. Examples of high-abundance proteins include albumin, IgG, and the top 14 proteins that account for 95% of the plasma mass. Additionally, any protein that can be purified using a conventional depletion column can be directly detected in the sample using the surfaces disclosed herein. Examples of proteins can be any protein listed in the disclosed databases, such as Keshishian et al. (Mol Cell Proteomics. September 2015;14(9):2375-93. Doi:10.1074 / mcp.M114.046813. Epub February 27, 2015), Farr et al. (J Proteome Res. January 3, 2014;13(1):60-75. Doi:10.1021 / pr4010037. Epub December 6, 2013), or Pernemalm et al. (Expert Rev Proteomics. August 2014;11(4):431-48. Doi:10.1586 / 14789450.2014.901157. Epub March 24, 2014).

[0520] In some embodiments, examples of proteins whose surface measurement and identification are disclosed herein include albumin, IgG, lysozyme, CEA, HER-2 / neu, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA125, CA19.9, CA 15.3, leptin, prolactin, osteopontin, IGF-II, CD98, desmin, sPigR, 14-3-3eta, troponin I, B-type natriuretic peptide, BRCA1, c-Myc, IL-6, fibrinogen, EGFR, gastrin, PH, G-CSF, desmin, NSE, FSH, VEGF, P21, PCNA, calcitonin, PR, CA125, LH, somatostatin, S100, insulin, alpha-prolactin, ACTH, Bcl-2, ERα, Ki-67, p53, cathepsin D, beta-catenin, VWF, CD15, k-ras, caspase 3, EPN, CD10, FAS, BRCA2, CD30L, CD30, CGA, CRP, prothrombin, CD44, APEX, transferrin, GM-CSF, E-cadherin, IL-2, Bax, IFN-γ, beta-2-MG, TNFα, c-erbB-2, trypsin, cyclin D1, MG B, XBP-1, HG-1, YKL-40, S-γ, NESP-55, netrin-1, twinfilin, GADD45A, CDK-6, CCL21, BrMS1, 17βHDI, PDGFRA, Pcaf, CCL5, MMP3, occludin-4, and occludin-3. In some embodiments, other examples of proteins whose surface measurement and identification are disclosed herein are any protein or group of proteins listed in an open target database for a particular disease indication of interest (such as prostate cancer, lung cancer, or Alzheimer's disease).

[0521] Method

[0522] In one aspect, a method of preparing a surface having repeating units of a first component and a second component is described herein, wherein the method can include (a) providing a monomer mixture comprising a first monomer and a second monomer in a solvent, wherein the first monomer can comprise a vinyl group and wherein the second monomer can comprise an epoxy group; (b) contacting the surface with the monomer mixture to produce a reaction mixture; (c) initiating radical polymerization to produce macromolecules immobilized on the surface; (d) contacting the macromolecules immobilized on the surface with an amine to produce aminated macromolecules; and (e) optionally, contacting the aminated macromolecules with a compound comprising a succinate, a phthalate, or a propane sulfone. In one aspect, a method of preparing a surface having repeating units of a first component and a second component is described herein, wherein the method can include: (a) providing a monomer mixture comprising a first monomer and a second monomer in a solvent, wherein the first monomer can comprise a vinyl group and wherein the second monomer can comprise an epoxy group; (b) contacting the surface with the monomer mixture to produce a reaction mixture; (c) initiating radical polymerization to produce macromolecules immobilized on the surface; (d) contacting the macromolecules immobilized on the surface with an azide salt to produce macromolecules containing azide; and (e) optionally, contacting the macromolecules containing azide with an alkyne-containing molecule to form triazole-containing macromolecules. In some embodiments, the surface having repeating units of a first component and a second component is a macromolecule immobilized on a surface of the present disclosure as described elsewhere herein. In some embodiments, the surface having repeating units of a first component and a second component is a macromolecule immobilized on a surface of the present disclosure as described elsewhere herein.

[0523] The surface can include features or properties as described elsewhere herein. For example, the surface can be a particle comprising an iron oxide core and a silica layer. In some embodiments, the solvent can be a polar solvent. In some embodiments, the solvent can be a nonpolar solvent. In some embodiments, the solvent can include ethanol, water, acetonitrile, tetrahydrofuran, dimethylformamide, or a combination thereof. In some embodiments, a radical initiator can be used to initiate the polymerization. In some embodiments, the radical initiator is azobisisobutyronitrile (AIBN).

[0524] In some embodiments, the method can include contacting the surface - immobilized macromolecule with a quencher after (c) and before (d). The quencher can contact the macromolecule at any suitable time to obtain the desired size of the macromolecule and / or the surface. In some embodiments, when the surface - immobilized macromolecule has a diameter of about 100 nanometers (nm) to about 600 nm, the quencher can be introduced into the reaction mixture. In some embodiments, when the surface has a diameter of about 200 nm to about 500 nm, the reaction can be quenched. In some embodiments, when the surface has a diameter of about 250 nm to about 400 nm, the reaction can be quenched. In some embodiments, when the surface has a diameter of about 250 nm to about 350 nm, the reaction can be quenched. In some embodiments, when the surface has a diameter of about 325 nm to about 375 nm, the reaction can be quenched. In some embodiments, the quencher can include ethylene glycol or acrylate. In some embodiments, the quencher can include benzoquinone.

[0525] In some embodiments, the method can further include purifying the surface - immobilized macromolecule. In some embodiments, purification can include washing the surface - immobilized macromolecule with a solvent. In some embodiments, the solvent can comprise ethanol, water, acetonitrile, tetrahydrofuran, dimethylformamide, or a combination thereof. In some embodiments, the solvent can comprise ethanol or tetrahydrofuran.

[0526] In some embodiments, the first monomer can comprise a vinyl group. In some embodiments, the first monomer can comprise at least two vinyl groups. In some embodiments, the first monomer can comprise two vinyl groups. In some embodiments, the first monomer is a cross - linker. In some embodiments, the first monomer can be divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), poly(ethylene glycol) dimethacrylate (PEGDMA) (e.g., diethylene glycol dimethacrylate (DEGDMA), triethylene glycol dimethacrylate (TEGDMA), etc.), N,N’ - alkylenebisacrylamide (e.g., N,N’ - methylenebisacrylamide (MBA), N,N’ - ethylenebisacrylamide, N,N’ - butylenebisacrylamide, etc.) or a derivative thereof. In some embodiments, the second monomer comprises a vinyl group. In some embodiments, the second monomer can comprise glycidyl methacrylate or glycidyl acrylate. In some embodiments, the amine can be a C1 - C 12 alkylamine, a C1 - C6 hydroxyamine, or a C1 - C6 alkoxyethylamine. In some embodiments, the amine can be diethylamine, ethanolamine, hexanolamine, methoxyethylamine, or any other amine of the present disclosure. In some embodiments, the amine can be diethylamine. In some embodiments, the amine is a diamine, such as a C1 - C 20Alkylenediamine. Non-limiting examples of suitable diamines include ethylenediamine, propylenediamine, butylenediamine, hexamethylenediamine, laurylenediamine, etc. In some embodiments, the diamine can react with two epoxide-containing monomer units of a macromolecule. In some embodiments, the diamine can react with two monomer units of a macromolecule (both containing epoxides) and crosslink. In some embodiments, the diamine can react with one epoxide-containing monomer unit of a macromolecule. In some embodiments, the diamine can react with one epoxide-containing monomer unit, leaving unreacted terminal amine groups. In some embodiments, a portion of the diamine reacts with one epoxide-containing monomer unit, and a portion of the diamine reacts with two epoxide-containing monomer units. In some embodiments, the azide salt is an alkali metal salt. In some embodiments, the azide salt is sodium azide. In some embodiments, the azide salt is lithium azide.

[0527] In some embodiments, the first monomer can account for about 10% to about 90% by weight of the monomer mixture. In some embodiments, the first monomer can account for about 20 to about 80% by weight of the monomer mixture. In some embodiments, the first monomer can account for about 40% to about 60% by weight of the monomer mixture. In some embodiments, the first monomer can account for about 50% by weight of the monomer mixture. In some embodiments, the second monomer can account for about 10% to about 90% by weight of the monomer mixture. In some embodiments, the second monomer can account for about 20 to about 80% by weight of the monomer mixture. In some embodiments, the second monomer can account for about 40% to about 60% by weight of the monomer mixture. In some embodiments, the second monomer can account for about 50% by weight of the monomer mixture.

[0528] In some embodiments, the method includes (e) contacting the aminated macromolecule with a compound comprising a succinate, a phthalate, a thiol, or a propylsulfone. In some embodiments, the compound can comprise a succinate (e.g., C8 alkenyl succinate or C8 alkenyl ethylamino succinate). In some embodiments, the compound can comprise a thiol (e.g., C2 alkyl thiol). In some embodiments, the compound can comprise a phthalate (e.g., C1-C6 amino phthalate). In some embodiments, the compound can comprise a propylsulfone (e.g., dipropylsulfone ethylamine).

[0529] In some embodiments, the method can produce surface - immobilized macromolecules as described herein. For example, the method can produce macromolecules immobilized on a surface (e.g., a particle), where the macromolecule comprises repeating units according to formula (I), formula (I - A), formula (I - A’), formula (II), formula (II’), formula (III), formula (III’), or formula (III - A) or any of the repeating units in Table 1. As another example, the method can produce surface - immobilized macromolecules, where the macromolecule comprises repeating units of a first component and a second component, or any of the repeating units in Table 2. As an example, Figure 1 shows the general design space of the epoxidized nanoparticle platform after addition of a functionalized amine, while Figure 2 shows the available conversion schemes after nanoparticle epoxidation, including reactions with glycidyl and azide to obtain different functionalities.

[0530] In one aspect, a method for identifying a protein in a sample is described herein, the method comprising (a) incubating one or more surfaces with a biological sample comprising a biomolecule to form a biomolecular corona; (b) isolating at least a portion of the biomolecules in the biomolecular corona; and (c) assaying the biomolecular corona.

[0531] In some embodiments, the one or more surfaces are selected from the surfaces disclosed elsewhere herein. In some embodiments, the surface is selected from Table 3 and / or Table 4.

[0532] In some embodiments, determining the biomolecular corona can identify 1 to 50,000 protein groups or proteins. In some embodiments, 1 to 20,000 protein groups or proteins can be identified. In some embodiments, at least 100 protein groups can be identified. In some embodiments, at least 300 protein groups can be identified. In some embodiments, at least 500 protein groups can be identified. In some embodiments, 1,000 to 10,000 protein groups or proteins can be identified. In some embodiments, 1,000 to 5,000 protein groups or proteins can be identified. In some embodiments, 1,800 to 5,000 protein groups or proteins can be identified. In some embodiments, 1,200 to 2,200 protein groups or proteins can be identified. In some embodiments, the protein groups or proteins can comprise peptide sequences with a minimum length of 2 amino acid residues. In some embodiments, the protein groups can comprise peptide sequences with a minimum length of 2 amino acid residues. In some embodiments, the protein groups can comprise peptide sequences with a minimum length of 5 amino acid residues. In some embodiments, the protein groups can comprise peptide sequences with a minimum length of 7 amino acid residues. In some embodiments, the protein groups can comprise peptide sequences with a minimum length of 8 amino acid residues. In some embodiments, the protein groups can comprise peptide sequences with a minimum length of 9 amino acid residues. In some embodiments, the protein groups can comprise peptide sequences with a minimum length of 10 amino acid residues. In some embodiments, the method can further include lysing the proteins of the biomolecular corona. In some embodiments, the method can further include digesting the proteins of the biomolecular corona. In some embodiments, the digested proteins can be purified. In some embodiments, the particles and the proteins of the biomolecular corona are incubated with a protease to digest the proteins.

[0533] In some embodiments, the method further comprises repeating the method described herein, wherein, when repeated, the incubation, isolation, and determination yield a percentile normalized coefficient of variation (QNCV) of 30% or less, as determined by comparing the peptide mass spectrometry signatures of at least three complete assay replicates from each of one or more surfaces. In some embodiments, when repeated, the incubation, isolation, and determination yield a percentile normalized coefficient of variation (QNCV) of 25% or less, as determined by comparing the peptide mass spectrometry signatures of at least three complete assay replicates from each of one or more surfaces. In some embodiments, when repeated, the incubation, isolation, and determination yield a percentile normalized coefficient of variation (QNCV) of 20% or less, as determined by comparing the peptide mass spectrometry signatures of at least three complete assay replicates from each of one or more surfaces. In some embodiments, the assay is capable of identifying proteins within a dynamic range of at least 7, at least 8, at least 9, or at least 10. In some embodiments, the assay is capable of identifying proteins within a dynamic range of no more than 12, no more than 11, no more than 10, no more than 9, or no more than 8.

[0534] In some embodiments, the method may further comprise washing the one or more surfaces at least once after isolating the one or more surfaces from unbound proteins. In some embodiments, the method may further comprise washing the one or more surfaces at least twice after isolating the one or more surfaces from unbound proteins. In some embodiments, the method may further comprise washing the one or more surfaces at least three times after isolating the one or more surfaces from unbound proteins. Isolation can be performed, for example, using magnetic isolation or centrifugation.

[0535] In some embodiments, the method may further comprise solubilizing the proteins of the biomolecular corona. In some embodiments, the method may further comprise denaturing the proteins of the biomolecular corona.

[0536] In some embodiments, the assay comprises identifying a protein in a sample using mass spectrometry. In some embodiments, the assay comprises using tandem mass spectrometry. In some embodiments, the assay comprises using liquid chromatography tandem mass spectrometry. In some embodiments, the assay may comprise ELISA, Edman degradation, immunoaffinity techniques, single molecule protein sequencing, etc. In some embodiments, the assay is performed in about 2 to about 4 hours. In some embodiments, the method is performed in about 1 to about 20 hours. In some embodiments, the method is performed in about 2 to about 10 hours. In some embodiments, the method is performed in about 4 to about 6 hours. In some embodiments, the isolation takes no more than about 30 minutes, no more than about 15 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 2 minutes. In some embodiments, a plurality of spatially isolated samples are processed according to the method. In some embodiments, the plurality of samples comprises at least 10 spatially isolated samples, at least 50 spatially isolated samples, at least 100 spatially isolated samples, at least 150 spatially isolated samples, at least 200 spatially isolated samples, at least 250 spatially isolated samples, or at least 300 spatially isolated samples. In a further embodiment, the plurality of samples comprises at least 96 samples.

[0537] In some embodiments, one or more surfaces comprise a first unique surface and a second unique surface, wherein each of the first unique surface and the second unique surface comprises a macromolecule as described herein (e.g., comprising a repeating macromolecule represented by Formula (I), Formula (I-A), Formula (I-A’), Formula (II), Formula (II’), Formula (III), Formula (III’), Formula (III-A), Formula (IV) or described in Tables 1-4). In some embodiments, one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface comprises a macromolecule as described herein and the second unique surface is a non-polymer surface. In some embodiments, the non-polymer surface is a non-polymer surface containing a carboxyl group. In some embodiments, the non-polymer surface is a non-polymer surface containing an amine. In some embodiments, the non-polymer surface is a silica surface. In some embodiments, the non-polymer surface comprises a moiety of Formula (IV).

[0538] In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein each of the first distinct surface and the second distinct surface comprises a macromolecule selected from Table 1 and / or Table 2. In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface comprises a macromolecule selected from Table 1 and / or Table 2, and the second distinct surface is a non-polymer surface. In some embodiments, the non-polymer surface is a non-polymer surface containing a carboxyl group. In some embodiments, the non-polymer surface is a non-polymer surface containing an amine. In some embodiments, the non-polymer surface is a silica surface. In some embodiments, the non-polymer surface comprises a moiety of formula (IV).

[0539] In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein each of the first distinct surface and the second distinct surface comprises a surface selected from Table 3 and / or Table 4. In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface comprises a surface selected from Table 3 and / or Table 4, and wherein the second distinct surface is non-polymeric. In some embodiments, the non-polymer surface is a non-polymer surface containing a carboxyl group. In some embodiments, the non-polymer surface is a non-polymer surface containing an amine. In some embodiments, the non-polymer surface is a silica surface. In some embodiments, the non-polymer surface comprises a moiety of formula (IV).

[0540] In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least one physicochemical property and differ in at least one physicochemical property such that the first distinct surface and the second distinct surface are different. By way of example, the first surface may include macromolecule S-337 and the second surface may include macromolecule S-370. Both surfaces may have polymeric properties and may exhibit different surface charges as measured by zeta potential analysis. In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least two physicochemical properties and differ in at least two physicochemical properties such that the first distinct surface and the second distinct surface are different. In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, wherein the first distinct surface and the second distinct surface share at least one physicochemical property and differ in at least two physicochemical properties such that the first distinct surface and the second distinct surface are different.

[0541] In some embodiments, one or more surfaces include a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface share at least two physicochemical properties and differ in at least one physicochemical property such that the first unique surface and the second unique surface are different. In further embodiments, the physicochemical properties include size, charge, core material, shell material, porosity, or surface hydrophobicity. In further embodiments, the size is a diameter or radius as measured by dynamic light scattering, SEM, TEM, or any combination thereof.

[0542] In some embodiments, the surface can be a particle. Particles consistent with the present disclosure can be manufactured in a wide range of sizes and used in methods for forming a protein corona after incubation in a biological fluid. For example, the particles disclosed herein can have at least 10 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1000 nm, at least 1100 nm, at least 1200 nm, at least 1300 nm, at least 1400 nm, at least 1500 nm, at least 1600 nm, at least 1700 nm, at least 1800 nm, at least 1900 nm, at least 2000 nm, at least 2100 nm, at least 2200 nm, at least 2300 nm, at least 2400 nm, at least 2500 nm, at least 2600 nm, at least 2700 nm, at least 2800 nm, at least 2900 nm, at least 3000 nm, at least 3100 nm, at least 3200 nm, at least 3300 nm, at least 3400 nm, at least 3500 nm, at least 3600 nm, at least 3700 nm, at least 3800 nm, at least 3900 nm, at least 4000 nm, at least 4100 nm, at least 4200 nm, at least 4300 nm, at least 4400 nm, at least 4500 nm, at least 4600 nm, at least 4700 nm, at least 4800 nm, at least 4900 nm, at least 5000 nm, at least 5100 nm, at least 5200 nm, at least 5300 nm, at least 5400 nm, at least 5500 nm, at least 5600 nm, at least 5700 nm, at least 5800 nm, at least 5900 nm, at least 6000 nm, at least 6100 nm, at least 6200 nm, at least 6300 nm, at least 6400 nm, at least 6500 nm, at least 6600 nm, at least 6700 nm, at least 6800 nm, at least 6900 nm, at least 7000 nm, at least 7100 nm, at least 7200 nm, at least 7300 nm, at least 7400 nm, at least 7500 nm, at least 7600 nm, at least 7700 nm, at least 7800 nm, at least 7900 nm, at least 8000 nm, at least 8100 nm, at least 8200 nm, at least 8300 nm, at least 8400 nm, at least 8500 nm, at least 8600 nm, at least 8700 nm, at least 8800 nm, at least 8900 nm, at least 9000 nm, at least 9100 nm, at least 9200 nm, at least 9300 nm, at least 9400 nm, at least 9500 nm, at least 9600 nm, at least 9700 nm, at least 9800 nm, at least 9900 nm, at least 10000 nm or from 10 nm to 50 nm, from 50 nm to 100 nm,100 nm to 150 nm, 150 nm to 200 nm, 200 nm to 250 nm, 250 nm to 300 nm, 300 nm to 350 nm, 350 nm to 400 nm, 400 nm to 450 nm, 450 nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, 600 nm to 650 nm, 650 nm to 700 nm, 700 nm to 750 nm, 750 nm to 800 nm, 800 nm to 850 nm, 850 nm to 900 nm, 100 nm to 300 nm, 150 nm to 350 nm, 200 nm to 400 nm, 250 nm to 450 nm, 300 nm to 500 nm, 350 nm to 550 nm, 400 nm to 600 nm, 450 nm to 650 nm, 500 nm to 700 nm, 550 nm to 750 nm, 600 nm to 800 nm, 650 nm to 850 nm, 700 nm to 900 nm or 10 nm to 900 nm, 10 to 100 nm, 100 to 200 nm, 200 to 300 nm, 300 to 400 nm, 400 to 500 nm, 500 to 600 nm, 600 to 700 nm, 700 to 800 nm, 800 to 900 nm, 900 to 1000 nm, 1000 to 1100 nm, 1100 to 1200 nm, 1200 to 1300 nm, 1300 to 1400 nm, 1400 to 1500 nm, 1500 to 1600 nm, 1600 to 1700 nm, 1700 to 1800 nm, 1800 to 1900 nm, 1900 to 2000 nm, 2000 to 2100 nm, 2100 to 2200 nm, 2200 to 2300 nm, 2300 to 2400 nm, 2400 to 2500 nm, 2500 to 2600 nm, 2600 to 2700 nm, 2700 to 2800 nm, 2800 to 2900 nm, 2900 to 3000 nm, 3000 to 3100 nm, 3100 to 3200 nm, 3200 to 3300 nm, 3300 to 3400 nm, 3400 to 3500 nm, 3500 to 3600 nm, 3600 to 3700 nm, 3700 to 3800 nm, 3800 to 3900 nm, 3900 to 4000 nm, 4000 to 4100 nm, 4100 to 4200 nm, 4200 to 4300 nm, 4300 to 4400 nm, 4400 to 4500 nm, 4500 to 4600 nm, 4600 to 4700 nm, 4700 to 4800 nm, 4800 to 4900 nm, 4900 to 5000 nm, 5000 to 5100 nm, 5100 to 5200 nm, 5200 to 5300 nm, 5300 to 5400 nm, 5400 to 5500 nmDiameters from 5500 to 5600 nm, 5600 to 5700 nm, 5700 to 5800 nm, 5800 to 5900 nm, 5900 to 6000 nm, 6000 to 6100 nm, 6100 to 6200 nm, 6200 to 6300 nm, 6300 to 6400 nm, 6400 to 6500 nm, 6500 to 6600 nm, 6600 to 6700 nm, 6700 to 6800 nm, 6800 to 6900 nm, 6900 to 7000 nm, 7000 to 7100 nm, 7100 to 7200 nm, 7200 to 7300 nm, 7300 to 7400 nm, 7400 to 7500 nm, 7500 to 7600 nm, 7600 to 7700 nm, 7700 to 7800 nm, 7800 to 7900 nm, 7900 to 8000 nm, 8000 to 8100 nm, 8100 to 8200 nm, 8200 to 8300 nm, 8300 to 8400 nm, 8400 to 8500 nm, 8500 to 8600 nm, 8600 to 8700 nm, 8700 to 8800 nm, 8800 to 8900 nm, 8900 to 9000 nm, 9000 to 9100 nm, 9100 to 9200 nm, 9200 to 9300 nm, 9300 to 9400 nm, 9400 to 9500 nm, 9500 to 9600 nm, 9600 to 9700 nm, 9700 to 9800 nm, 9800 to 9900 nm, 9900 to 10000 nm. The diameters can be measured by dynamic light scattering (DLS) as an indirect measurement of size. The DLS measurement can be an “intensity weighted” average, meaning that the size distribution used to calculate the mean can be weighted by the sixth power of the radius. This can be referred to herein as the “z-average” or “intensity average”.

[0543] Alternatively, the particles disclosed herein can have at least 10 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, at least 1000 nm, at least 1100 nm, at least 1200 nm, at least 1300 nm, at least 1400 nm, at least 1500 nm, at least 1600 nm, at least 1700 nm, at least 1800 nm, at least 1900 nm, at least 2000 nm, at least 2100 nm, at least 2200 nm, at least 2300 nm, at least 2400 nm, at least 2500 nm, at least 2600 nm, at least 2700 nm, at least 2800 nm, at least 2900 nm, at least 3000 nm, at least 3100 nm, at least 3200 nm, at least 3300 nm, at least 3400 nm, at least 3500 nm, at least 3600 nm, at least 3700 nm, at least 3800 nm, at least 3900 nm, at least 4000 nm, at least 4100 nm, at least 4200 nm, at least 4300 nm, at least 4400 nm, at least 4500 nm, at least 4600 nm, at least 4700 nm, at least 4800 nm, at least 4900 nm, at least 5000 nm, at least 5100 nm, at least 5200 nm, at least 5300 nm, at least 5400 nm, at least 5500 nm, at least 5600 nm, at least 5700 nm, at least 5800 nm, at least 5900 nm, at least 6000 nm, at least 6100 nm, at least 6200 nm, at least 6300 nm, at least 6400 nm, at least 6500 nm, at least 6600 nm, at least 6700 nm, at least 6800 nm, at least 6900 nm, at least 7000 nm, at least 7100 nm, at least 7200 nm, at least 7300 nm, at least 7400 nm, at least 7500 nm, at least 7600 nm, at least 7700 nm, at least 7800 nm, at least 7900 nm, at least 8000 nm, at least 8100 nm, at least 8200 nm, at least 8300 nm, at least 8400 nm, at least 8500 nm, at least 8600 nm, at least 8700 nm, at least 8800 nm, at least 8900 nm, at least 9000 nm, at least 9100 nm, at least 9200 nm, at least 9300 nm, at least 9400 nm, at least 9500 nm, at least 9600 nm, at least 9700 nm, at least 9800 nm, at least 9900 nm, at least 10000 nm or from 10 nm to 50 nm, 50 nm to 100 nm, 100 nm to 150 nm, 150 nm to 200 nm, 200 nm to 250 nm, 250 nm to 300 nm, 300 nm to 350 nm,350 nm to 400 nm, 400 nm to 450 nm, 450 nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, 600 nm to 650 nm, 650 nm to 700 nm, 700 nm to 750 nm, 750 nm to 800 nm, 800 nm to 850 nm, 850 nm to 900 nm, 100 nm to 300 nm, 150 nm to 350 nm, 200 nm to 400 nm, 250 nm to 450 nm, 300 nm to 500 nm, 350 nm to 550 nm, 400 nm to 600 nm, 450 nm to 650 nm, 500 nm to 700 nm, 550 nm to 750 nm, 600 nm to 800 nm, 650 nm to 850 nm, 700 nm to 900 nm or 10 nm to 900 nm, 10 to 100 nm, 100 to 200 nm, 200 to 300 nm, 300 to 400 nm, 400 to 500 nm, 500 to 600 nm, 600 to 700 nm, 700 to 800 nm, 800 to 900 nm, 900 to 1000 nm, 1000 to 1100 nm, 1100 to 1200 nm, 1200 to 1300 nm, 1300 to 1400 nm, 1400 to 1500 nm, 1500 to 1600 nm, 1600 to 1700 nm, 1700 to 1800 nm, 1800 to 1900 nm, 1900 to 2000 nm, 2000 to 2100 nm, 2100 to 2200 nm, 2200 to 2300 nm, 2300 to 2400 nm, 2400 to 2500 nm, 2500 to 2600 nm, 2600 to 2700 nm, 2700 to 2800 nm, 2800 to 2900 nm, 2900 to 3000 nm, 3000 to 3100 nm, 3100 to 3200 nm, 3200 to 3300 nm, 3300 to 3400 nm, 3400 to 3500 nm, 3500 to 3600 nm, 3600 to 3700 nm, 3700 to 3800 nm, 3800 to 3900 nm, 3900 to 4000 nm, 4000 to 4100 nm, 4100 to 4200 nm, 4200 to 4300 nm, 4300 to 4400 nm, 4400 to 4500 nm, 4500 to 4600 nm, 4600 to 4700 nm, 4700 to 4800 nm, 4800 to 4900 nm, 4900 to 5000 nm, 5000 to 5100 nm, 5100 to 5200 nm, 5200 to 5300 nm, 5300 to 5400 nm, 5400 to 5500 nm, 5500 to 5600 nm, 5600 to 5700 nm, 5700 to 5800 nm, 5800 to 5900 nm, 5900 to 6000 nm,Radii from 6000 to 6100 nm, 6100 to 6200 nm, 6200 to 6300 nm, 6300 to 6400 nm, 6400 to 6500 nm, 6500 to 6600 nm, 6600 to 6700 nm, 6700 to 6800 nm, 6800 to 6900 nm, 6900 to 7000 nm, 7000 to 7100 nm, 7100 to 7200 nm, 7200 to 7300 nm, 7300 to 7400 nm, 7400 to 7500 nm, 7500 to 7600 nm, 7600 to 7700 nm, 7700 to 7800 nm, 7800 to 7900 nm, 7900 to 8000 nm, 8000 to 8100 nm, 8100 to 8200 nm, 8200 to 8300 nm, 8300 to 8400 nm, 8400 to 8500 nm, 8500 to 8600 nm, 8600 to 8700 nm, 8700 to 8800 nm, 8800 to 8900 nm, 8900 to 9000 nm, 9000 to 9100 nm, 9100 to 9200 nm, 9200 to 9300 nm, 9300 to 9400 nm, 9400 to 9500 nm, 9500 to 9600 nm, 9600 to 9700 nm, 9700 to 9800 nm, 9800 to 9900 nm, 9900 to 10000 nm.,

[0544] In some examples, the particles disclosed herein have a diameter of 100 nm to 400 nm. In other examples, the particles disclosed herein have a radius of 100 nm to 400 nm. Particle size can be determined by a variety of techniques, such as dynamic light scattering or electron microscopy (e.g., SEM, TEM). The particles disclosed herein can be nanoparticles or microparticles.

[0545] In addition, the particles can have a uniform size distribution or a non-uniform size distribution. The polydispersity index (PDI) is a measure of the size distribution and can be measured by techniques such as dynamic light scattering. A low PDI indicates a more uniform size distribution, while a higher PDI indicates a more non-uniform size distribution. For example, the particles disclosed herein can have a PDI less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.15, or less than 0.1. In certain embodiments, the particles disclosed herein have a PDI less than 0.1. In some embodiments, the particles can have a PDI of at least 0.5, at least 0.75, at least 1.0, at least 1.5, or at least 2.0.

[0546] The particles disclosed herein can have different surface charge ranges. The particles can be negatively charged, positively charged, or neutrally charged. In some embodiments, the particles have a surface charge of -150 mV to -100 mV, -100 mV to -90 mV, -90 mV to -80 mV, -80 mV to -70 mV, -70 mV to -60 mV, -60 mV to -50 mV, -50 mV to -40 mV, -40 mV to -30 mV, -30 mV to -20 mV, -20 mV to -10 mV, -10 mV to 0 mV, 0 mV to 10 mV, 10 mV to 20 mV, 20 mV to 30 mV, 30 mV to 40 mV, 40 mV to 50 mV, 50 mV to 60 mV, 60 mV to 70 mV, 70 mV to 80 mV, 80 mV to 90 mV, 90 mV to 100 mV, 100 mV to 110 mV, 110 mV to 120 mV, 120 mV to 130 mV, 130 mV to 140 mV, 140 mV to 150 mV, -150 mV to -100 mV, -100 mV to 0 mV, 0 mV to 100 mV, 100 mV to 150 mV. In a particular example, the particles disclosed herein have a surface charge of -60 mV to 60 mV. The surface charge can be evaluated by zeta potential analysis using an appropriate buffer at neutral pH.

[0547] In some embodiments, one or more surfaces include a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface comprise a carboxylate material, and wherein the first unique surface, the second unique surface, or both are nanoparticles. In some embodiments, one or more surfaces include a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface have a surface charge of 0 mV to -50 mV, and wherein the first unique surface, the second unique surface, or both have a diameter of less than 400 nm. In some embodiments, one or more surfaces include a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface have a diameter of 100 to 400 nm, wherein the first unique surface has a positive surface charge, and wherein the second unique surface has a negative surface charge.

[0548] In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, where the first distinct surface and the second distinct surface are nanoparticles, where the first distinct surface has a surface charge of less than -20 mV, and the second distinct surface has a surface charge of greater than 20 mV. In some embodiments, one or more surfaces include a first distinct surface and a second distinct surface, where the first distinct surface and the second distinct surface are microparticles, where the first distinct surface has a negative surface charge, and where the second distinct surface has a positive surface charge. In some embodiments, one or more surfaces include a subset of negatively charged nanoparticles, where each surface of the subset is distinct in at least one surface chemical group. In some embodiments, one or more surfaces include a first distinct surface, a second distinct surface, and a third distinct surface, where the first distinct surface, the second distinct surface, and the third distinct surface include an iron oxide core, and have a diameter of less than about 500 nm, and where the first distinct surface includes a negative charge of less than -40 mV, the second distinct surface includes a positive charge of greater than 20 mV, and the third distinct surface includes a negative charge of -20 mV to -40 mV.

[0549] In some embodiments, three or more distinct magnetic surfaces include nanoparticles. In some embodiments, three or more distinct magnetic surfaces include microparticles. In some embodiments, at least one distinct surface among the three or more distinct magnetic surfaces is a superparamagnetic iron oxide particle. In some embodiments, at least one distinct surface among the three or more distinct magnetic surfaces comprises an iron oxide material. In some embodiments, at least one distinct surface among the three or more distinct magnetic surfaces has an iron oxide core. In some embodiments, at least one distinct surface among the three or more distinct magnetic surfaces has an iron oxide crystal embedded in a polystyrene core.

[0550] In some embodiments, each distinct surface among the three or more distinct magnetic surfaces is a superparamagnetic iron oxide particle. In some embodiments, each distinct surface among the three or more distinct magnetic surfaces includes an iron oxide core. In some embodiments, each distinct surface among the three or more distinct magnetic surfaces has an iron oxide crystal embedded in a polystyrene core. In some embodiments, at least one surface among the three or more distinct magnetic surfaces includes a polymer coating.

[0551] In some embodiments, three or more distinct magnetic surfaces include a carboxylated polymer, an aminated polymer, or any combination thereof. In some embodiments, at least one of the three or more distinct magnetic surfaces includes an iron oxide core having a silica shell coating. In some embodiments, at least one of the three or more distinct magnetic surfaces includes an iron oxide core having a poly(N-(3-(dimethylamino)propyl)methacrylamide) (PDMAPMA) coating. In some embodiments, at least one of the three or more distinct magnetic surfaces includes an outer surface containing carboxyl groups. In some embodiments, at least one of the three or more distinct magnetic surfaces includes an outer surface containing amines. In some embodiments, at least one of the three or more distinct magnetic surfaces includes an iron oxide core having a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) coating.

[0552] In some embodiments, at least one of the three or more distinct magnetic surfaces includes a negative surface charge. In some embodiments, at least one of the three or more distinct magnetic surfaces includes a positive surface charge. In some embodiments, at least one of the three or more distinct magnetic surfaces includes a neutral surface charge.

[0553] In some aspects, the present disclosure provides a method for determining the biological state of a sample from a subject, comprising: exposing a biological sample to a kit comprising a plurality of surfaces, thereby generating a plurality of protein coronas; generating proteomics data from the plurality of protein coronas; determining the protein profile of the plurality of protein coronas; and correlating the protein profile with the biological state, wherein the kit comprises at least two different surfaces. The surface can be any surface described herein. For example, the surface can be a magnetic nanoparticle having an outer layer containing any of the macromolecules in Table 1 and / or Table 2. In one example, Figure 3 Schematic illustration of nanoparticles coated with SiO2 and then with a cross-linked polymer to obtain multiple functionalities.

[0554] In some embodiments, the kit comprises at least three different surfaces. In some embodiments, the method correlates the protein profile with the biological state with an accuracy of at least 90%. In some embodiments, the plurality of surfaces includes at least one iron oxide nanoparticle.

[0555] Peptide-Decorated Macromolecules and Surfaces

[0556] In some aspects, some of the macromolecules and surfaces described herein may also comprise a peptide as described in PCT / US2022 / 027080, which is incorporated herein by reference in its entirety. In some embodiments, the macromolecules of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’), formula (III-A) or formula (IV) may be modified to further comprise a peptide. In some embodiments, the peptide may be bound to the surface via specific or non-specific interactions. In some embodiments, the macromolecule of formula (I) may further comprise a peptide. In some embodiments, the macromolecule of formula (I-A) may further comprise a peptide. In some embodiments, the macromolecule of formula (II) may further comprise a peptide. In some embodiments, the macromolecule of formula (III) may further comprise a peptide. In some embodiments, the macromolecule of formula (III-A) may further comprise a peptide. In some embodiments, the macromolecule of formula (IV) may further comprise a peptide.

[0557] In some aspects, the macromolecules of formula (I), formula (I-A), formula (I-A’), formula (II), formula (II’), formula (III), formula (III’), formula (III-A) or formula (IV) may further comprise a peptide, and the peptide may be covalently bonded to the macromolecule via a linker. In some aspects, a macromolecule is disclosed herein that comprises repeating units of a first component and crosslinked repeating units, wherein the first component comprises the structure of component (A’):

[0558]

[0559] wherein

[0560] each of Y1, Y2 and Y3 is independently selected from hydrogen or C1-C6 alkyl;

[0561] each of X1, X2 and X3 is independently selected from hydrogen or C1-C6 alkyl;

[0562] A is

[0563] G’ or W’ comprises Q’;

[0564] Q’ is a peptide.

[0565] The structure of may interchangeably refer to

[0566] In some embodiments, each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1-C6 alkyl group. In some embodiments, Y1 is hydrogen or a C1-C6 alkyl group. In some embodiments, Y1 is hydrogen. In some embodiments, Y1 is a C1-C6 alkyl group. In some embodiments, Y2 is hydrogen or a C1-C6 alkyl group. In some embodiments, Y2 is hydrogen. In some embodiments, Y2 is a C1-C6 alkyl group. In some embodiments, Y3 is hydrogen or a C1-C6 alkyl group. In some embodiments, Y3 is hydrogen. In some embodiments, Y3 is a C1-C6 alkyl group.

[0567] In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is

[0568] In some embodiments, G’ or W’ comprises Q’. In some embodiments, G’ comprises Q’. In some embodiments, W’ comprises Q’. In some embodiments, Q’ comprises a peptide. In some embodiments, W’ is In some embodiments, G’ is

[0569] In some embodiments, the crosslinked repeating unit may comprise a polymerizable unit. In some embodiments, the crosslinked repeating units may be randomly distributed throughout the macromolecule. In some embodiments, the crosslinked repeating units may be distributed throughout the macromolecule in a controlled manner. In some embodiments, the crosslinked repeating units may be crosslinked with another crosslinked repeating unit. In some embodiments, the crosslinked repeating units may be crosslinked with the component (A’).

[0570] In some aspects, there is provided a macromolecule comprising repeating units of a first component and a second component, wherein the first component comprises the structure of the component (A’), and the second component comprises the structure of the component (B):

[0571]

[0572] wherein

[0573] each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1-C6 alkyl group;

[0574] Each of X1, X2, and X3 is independently selected from hydrogen or C1-C6 alkyl;

[0575] A is

[0576] G’ or W’ contains Q’;

[0577] Q’ is a peptide.

[0578] B is

[0579] Z is a unit of monomer (A’) or monomer (B);

[0580] q is an integer between 1 and 6; and

[0581] p is an integer between 1 and 20.

[0582] In some aspects, the present disclosure provides a macromolecule comprising repeating units of a first component and a second component, wherein the first component comprises the structure of component (A’), and the second component comprises the structure of component (B’):

[0583]

[0584] In some embodiments, each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl. In some embodiments, Y1 is hydrogen or C1-C6 alkyl. In some embodiments, Y1 is hydrogen. In some embodiments, Y1 is C1-C6 alkyl. In some embodiments, Y2 is hydrogen or C1-C6 alkyl. In some embodiments, Y2 is hydrogen. In some embodiments, Y2 is C1-C6 alkyl. In some embodiments, Y3 is hydrogen or C1-C6 alkyl. In some embodiments, Y3 is hydrogen. In some embodiments, Y3 is C1-C6 alkyl. In some embodiments, X1, X2, and X3 are independently selected from hydrogen or C1-C6 alkyl. In some embodiments, X1 is hydrogen or C1-C6 alkyl. In some embodiments, X1 is hydrogen. In some embodiments, X1 is C1-C6 alkyl. In some embodiments, X2 is hydrogen or C1-C6 alkyl. In some embodiments, X2 is hydrogen. In some embodiments, X2 is C1-C6 alkyl. In some embodiments, X3 is hydrogen or C1-C6 alkyl. In some embodiments, X3 is hydrogen. In some embodiments, X3 is C1-C6 alkyl.

[0585] In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is In some embodiments, A’ is

[0586] In some embodiments, G’ or W’ comprises Q’. In some embodiments, G’ comprises Q’. In some embodiments, W’ comprises Q’. In some embodiments, Q’ comprises a peptide.

[0587] In some embodiments, B is In some embodiments, B is In some embodiments, B is In some embodiments, B is In some embodiments, Z is a unit of monomer (A’) or monomer (B). In some embodiments, Z is a unit of monomer (A’). In some embodiments, Z is a unit of monomer (B). In some embodiments, B is In some embodiments, B is In some embodiments, B is In some embodiments, B is

[0588] In some embodiments, B is In some embodiments, B is

[0589] In some embodiments, q is an integer between 1 and 6. In some embodiments, p is an integer between 1 and 20. In some embodiments, the macromolecule is immobilized on a surface.

[0590] In some embodiments, W’ is In some embodiments, G’ is

[0591] In some aspects, provided herein is a macromolecule comprising repeating units of a first component, wherein the first component comprises the structure of component (A’) as described above. In some embodiments, the macromolecule may further comprise crosslinked repeating units. For example, the crosslinked repeating units can be produced by free radical polymerization of a monomer having two vinyl groups, such as divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), poly(ethylene glycol) dimethacrylate (PEGDMA) (e.g., diethylene glycol dimethacrylate (DEGDMA), triethylene glycol dimethacrylate (TEGDMA), etc.), N,N’-alkylenebisacrylamide (e.g., N,N’-methylenebisacrylamide (MBA), N,N’-ethylenebisacrylamide, N,N’-butylenebisacrylamide, etc.) or derivatives thereof. In some embodiments, the macromolecule may comprise at least 5% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise at least 25% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise at least 40% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise no more than 90% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise no more than 75% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise no more than 60% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise no more than 50% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise no more than 25% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise 5% to 95% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise 20% to 80% by weight of crosslinked repeating units. In some embodiments, the macromolecule may comprise 40% to 60% by weight of crosslinked repeating units.

[0592] In some embodiments, the macromolecule may comprise at least 5% by weight of repeating units of the first component. In some embodiments, the macromolecule may comprise at least 25% by weight of repeating units of the first component. In some embodiments, the macromolecule may comprise at least 40% by weight of repeating units of the first component.

[0593] In some aspects, provided herein is a surface comprising a moiety of formula (IV’):

[0594]

[0595] wherein

[0596] Z is a linking moiety comprising a straight chain having 2 to 20 atoms selected from carbon, oxygen and nitrogen, and optional substituents on any carbon and any nitrogen atoms in the straight chain;

[0597] R1’ is hydrogen or a succinate ester; and

[0598] R2’ is C1-C6 alkyl-G’;

[0599] G’ contains Q’;

[0600] Q’ is a peptide;

[0601] wherein the peptide does not contain cysteine.

[0602] In some embodiments, Z is a linking moiety that comprises a straight chain having 2 to 20 atoms selected from carbon, oxygen, and nitrogen, and optional substituents on any of the carbon and any nitrogen atoms in the straight chain. In some embodiments, R1’ is hydrogen or a succinate ester. In some embodiments, R1’ is hydrogen. In some embodiments, R1’ is a succinate ester. In some embodiments, R2’ is C1-C6 alkyl-G’. In some embodiments, G’ contains Q’. In some embodiments, Q’ is a peptide. In specific embodiments, the peptide does not contain cysteine. In some embodiments, G’ is

[0603] In some embodiments, Z is a straight chain having 2 to 20 atoms. In some embodiments, Z is a straight chain having 2 to 12 atoms. In some embodiments, Z is a straight chain having 2 to 6 atoms. In some embodiments, Z is a straight chain having 2 atoms. In some embodiments, Z is a straight chain having 3 atoms. In some embodiments, Z is a straight chain having 4 atoms. In some embodiments, Z is a straight chain having 5 atoms. In some embodiments, Z is a straight chain having 6 atoms. In some embodiments, Z contains only carbon. In some embodiments, Z is a C2-C6 alkyl chain. In some embodiments, Z is a C3 alkyl. In some embodiments, Z contains oxygen, nitrogen, carbon, or a combination thereof. In some embodiments, Z contains substituents on the straight chain.

[0604] In some embodiments, the peptide contains up to about 40 amino acids. In some embodiments, the peptide contains at least about 20 amino acids. In some embodiments, the peptide contains a synthetic sequence. In some embodiments, the peptide contains non-natural amino acids.

[0605] In some aspects, the present disclosure provides macromolecules modified with peptides. In some embodiments, the macromolecules that can be modified comprise thiols or azides. In some aspects, a surface is described herein that comprises a macromolecule modified with a peptide immobilized on the surface. In some embodiments, the macromolecule is covalently coupled to the surface. In some embodiments, the macromolecule is electrostatically coupled to the surface. In some embodiments, the macromolecule is coupled to the surface through a polymerization event. In some embodiments, the polymerization event comprises reacting with vinyl groups on the surface. In some aspects, a peptide-functionalized surface is described herein. In some embodiments, the peptide comprises up to about 40 amino acids. In some aspects, the surface is a bead or a particle. In some embodiments, the surface is a particle. In some aspects, the particle comprises an iron oxide core with a silica shell coating.

[0606] Macromolecules modified with peptides can be used in systems, methods, and compositions in the same manner as other macromolecules disclosed herein. Systems and methods are provided herein for using macromolecules modified with peptides and such macromolecules immobilized on a surface for binding or enriching biomolecules (e.g., proteins). In some aspects, a system is provided herein that comprises a surface, a macromolecule modified with a peptide (wherein the peptide comprises a binding site), and a protein that interacts with the peptide at the binding site. A method is provided herein for identifying at least a plurality of biomolecules or portions thereof from a surface, the method comprising contacting a biological sample with a surface comprising a macromolecule modified with a peptide, wherein the peptide is configured to bind to a protein, releasing a plurality of biomolecules or portions thereof from the surface, and identifying at least a plurality of biomolecules or portions thereof from the surface. In some embodiments, the biomolecule is a protein.

[0607] In some embodiments, the methods provided herein comprise contacting a biological sample with at least two unique surfaces (e.g., macromolecules bound to a surface) provided elsewhere herein. In some embodiments, the methods provided herein comprise contacting a biological sample with two unique surfaces (e.g., two unique macromolecules bound to a surface).

[0608] In some embodiments, the at least two unique surfaces comprise a structure In some embodiments, the at least two unique surfaces comprise a structure In some embodiments, the at least two unique surfaces comprise a structure In some embodiments, the at least two unique surfaces comprise a structure In some embodiments, the at least two unique surfaces comprise a structure In some embodiments, the at least two unique surfaces comprise a structure In some embodiments, A junction point of a unit representing component (A) or component (B) as described elsewhere herein. In some embodiments, A junction point of a unit representing component (A) or component (B) as described elsewhere herein. In some embodiments, provided herein are compositions comprising at least two unique surfaces as provided elsewhere herein (such as, a combination of two unique surfaces as provided herein).

[0609] In some aspects, provided herein is a system comprising a surface, a macromolecule coupled to the surface comprising a peptide (wherein the peptide comprises a binding site), and a protein that interacts with the peptide at the binding site. In some embodiments, the surface is a particle. In some embodiments, the particle is a superparamagnetic iron oxide nanoparticle. In some embodiments, the particle comprises an iron oxide material. In some embodiments, the particle has an iron oxide core. In some embodiments, the particle has an iron oxide crystal embedded in a polystyrene core. In some embodiments, the particle comprises an iron oxide core having a silica shell coating.

[0610] In some embodiments, the peptide is coupled to the surface at a density of at least 1 peptide per 5 square nanometers, at least 1 peptide per 50 square nanometers, or at least 1 peptide per 500 square nanometers. In some embodiments, the surface further comprises a plurality of peptides coupled thereto, wherein each peptide in the plurality of peptides is configured to bind to at least three different proteins. In some embodiments, at least two different proteins specifically bind to the peptide. In some embodiments, the system further comprises a plurality of biomolecules adsorbed on the surface. In some embodiments, the plurality of biomolecules comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 proteins that non-specifically bind to the peptide. In some embodiments, the plurality of biomolecules comprises a dynamic range of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the surface provided in solution has at least about 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 cm per μL of solution 2Surface area. In some embodiments, a plurality of biomolecules are captured on the surface such that for at least two biomolecules among the plurality of biomolecules captured on the surface, the abundance ratio between at least two biomolecules among the plurality of biomolecules in solution is altered. In some embodiments, the visibility of the plurality of biomolecules in a downstream assay is increased. In some embodiments, the visibility of the biomolecules among the plurality of biomolecules can be measured by the intensity measurable by mass spectrometry. In some embodiments, the protein comprises a target protein. In some embodiments, the protein includes a vacuolar protein, lysosomal lumen, spliceosome tri-snRNP complex, U4 / U6 xU5 tri-snRNP complex, secretory granule lumen, intracellular organelle lumen, membrane raft, spliceosome snRNP complex, sperm proteasome complex, or Golgi lumen protein.

[0611] In some aspects, provided herein is a method of contacting a biological sample with a surface as described herein, wherein the surface comprises a peptide and the peptide is configured to bind to a protein, releasing a plurality of biomolecules or a portion thereof from the surface, and identifying at least the plurality of biomolecules or a portion thereof from the surface, wherein the plurality of biomolecules or a portion thereof comprises one or more biomolecules among at least three different biomolecules in the biological sample. In some embodiments, the biomolecule comprises a protein. In some embodiments, the biomolecule comprises a target protein. In some embodiments, the protein comprises a vacuolar lumen, lysosomal lumen, spliceosome tri-snRNP complex, U4 / U6 xU5 tri-snRNP complex, secretory granule lumen, intracellular organelle lumen, membrane raft, spliceosome snRNP complex, sperm proteasome complex, or Golgi lumen protein.

[0612] Oligopeptide functionalization

[0613] The binding molecule can comprise a peptide. Peptides are a collection of widespread and diverse biomolecules that can encompass a wide range of physical and chemical properties. Depending on their composition, sequence, and chemical modification, peptides can be hydrophilic, hydrophobic, amphiphilic, lipophilic, lipophobic, positively charged, negatively charged, zwitterionic, neutral, chaotropic, antichaotropic, reactive, redox-active, inert, acidic, basic, rigid, flexible, or any combination thereof. Thus, peptide surface functionalization can confer a range of physicochemical properties to the particles.

[0614] The particle can comprise a single peptide surface functionalization or multiple peptide surface functionalizations. A single peptide surface functionalization can comprise a plurality of identical or sequence-sharing peptides that bind to the particle in a uniform manner. For example, a particle comprising a single peptide surface functionalization can comprise approximately 3x10 5A peptide having the sequence alanine-valine-tyrosine-proline-histidine-phosphotyrosine-hydroxyproline-phenylalanine-tryptophan-alanine-arginine, with each peptide coupled to the particle surface via its C-terminal arginine.

[0615] Multiple peptide surface functionalizations can include multiple peptides that share a common sequence but bind to the particle in multiple ways. For example, multiple identical peptides sharing the common sequence alanine-lysine-alanine-lysine-alanine-lysine-proline can provide multiple surface functionalizations for a single particle when coupled to the particle separately through any one of the lysine residues. Multiple peptide surface functionalizations can include multiple peptides that share a common sequence but carry different chemical modifications. For example, the particle can include multiple peptide surface functionalizations that share a common sequence but differ in N-terminal functionalization. Multiple peptide surface functionalizations can include peptides having different lengths or sequences.

[0616] The particle can include any number of peptide surface functionalizations. The particle can include a single peptide surface functionalization. The particle can include at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 80, at least 100, at least 150, at least 200, at least 250, at least 500, at least 10 3 at least 2x10 3 at least 5x10 3 at least 10 4 at least 5x10 4 at least 10 5 at least 5x10 5 at least 10 6 or at least 10 6 at most 10 5 at most 5x10 5 at most 10 4 at most 5x10 4 at most 10 3 at most 5x10 3, up to 500, up to 250, up to 200, up to 150, up to 100, up to 80, up to 50, up to 40, up to 30, up to 25, up to 20, up to 15, up to 12, up to 10, up to 8, up to 6, up to 5, up to 4, up to 3, or up to 2 types of peptide surface functionalizations. In some embodiments, each peptide surface functionalization of the particle is unique. For example, such diversity of surface functionalizations can be achieved by relatively easy combinatorial peptide synthesis. Since peptide sequence diversity grows exponentially with peptide length, even relatively short oligopeptide libraries can contain sufficient diversity to statistically ensure unique peptide functionalizations on the particle surface. For example, a peptide of ten amino acids selected from the set of 20 proteinogenic amino acids can contain more than 10 13 different sequences, such that the chance that a particle containing 10 6 random sequence peptides of length 10 contains two identical peptides is less than 10-2%.

[0617] Peptide surface functionalizations can be distributed on the particle surface in a random or ordered manner. In some embodiments, multiple peptide surface functionalizations on a single particle can be spatially separated such that a first region of the particle contains a first peptide surface functionalization and a second region of the particle contains a second surface functionalization.

[0618] Peptide surface functionalization can include a range of peptide masses or lengths. In some embodiments, peptide surface functionalization is amino acid dimer surface functionalization, amino acid trimer surface functionalization, oligopeptide surface functionalization (e.g., having a length between about 2 and about 30 amino acids), polypeptide surface functionalization (e.g., having a length greater than about 30 amino acids), or protein surface functionalization (e.g., a peptide having a defined structure). Multiple peptide surface functionalizations can include peptides of the same length. Multiple peptide surface functionalizations include peptides of different lengths. Multiple peptide surface functionalizations can be multiple oligopeptide surface functionalizations. Multiple peptide surface functionalizations can include peptides having a length between 5 and 12 amino acids. Multiple peptide surface functionalizations can include peptides having a length between 4 and 8, between 4 and 10, between 4 and 12, between 4 and 15, between 4 and 20, between 4 and 25, between 5 and 8, between 5 and 10, between 5 and 12, between 5 and 15, between 5 and 20, between 5 and 25, between 6 and 8, between 6 and 10, between 6 and 12, between 6 and 15, between 6 and 20, between 6 and 25, between 7 and 10, between 7 and 12, between 7 and 15, between 7 and 20, between 7 and 25, between 8 and 10, between 8 and 12, between 8 and 15, between 8 and 20, between 8 and 25, between 10 and 12, between 10 and 15, between 10 and 20, between 10 and 25, between 12 and 15, between 12 and 20, 12 and 25, between 15 and 20, between 15 and 25, between 20 and 25, or between 20 and 30 amino acids.

[0619] Peptide surface functionalization may include a subset of amino acid types. Since different amino acid types confer different properties to the peptide, the properties of peptide surface functionalization can be determined at least in part by constructing peptide surface functionalization from a limited number of amino acid types. For example, glutamic acid and aspartic acid tend to lower the isoelectric point of the peptide to which they are attached, while histidine, lysine, and arginine tend to raise the isoelectric point of the peptide while providing nucleophilic characteristics. The properties of peptide surface functionalization can be regulated by controlling the types of constituent amino acids and the ratios between the types, thereby regulating the physicochemical surface properties of the particles. In many cases, peptide surface functionalization will lack cysteine residues to prevent thiol nucleophilic behavior, redox activity, and cross-linking. In many cases, peptide surface functionalization will include a defined ratio or range of ratios of acidic and basic side chains as a means of controlling the isoelectric point and charge. Peptide surface functionalization may be free of at least one of cysteine, methionine, tryptophan, tyrosine, phenylalanine, and their derivatives. Peptide functionalization may be free of at least two of cysteine, methionine, tryptophan, tyrosine, phenylalanine, and their derivatives. Peptide functionalization may be free of at least three of cysteine, methionine, tryptophan, tyrosine, phenylalanine, and their derivatives. Peptide functionalization may be free of at least four of cysteine, methionine, tryptophan, tyrosine, phenylalanine, and their derivatives. Peptide functionalization may be free of cysteine, methionine, tryptophan, tyrosine, phenylalanine, and their derivatives. The peptide may comprise an amino acid selected from alanine, arginine, aspartic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, proline, serine, asparagine, threonine, valine, and their derivatives. The peptide may comprise an amino acid selected from alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, asparagine, threonine, tryptophan, tyrosine, valine, and their derivatives. Multiple peptides may comprise up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, or up to 20 types of amino acids. The peptide may comprise up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, or up to 20 types of amino acids.The peptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 types of amino acids. Multiple peptides may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 types of amino acids.

[0620] Peptide functionalization may comprise non-protein amino acids. The non-protein amino acids may be chemically modified forms of protein amino acids. Such chemical modifications may comprise acylation, alkylation, amidation, deamidation, carbamylation, carbonylation, carboxylation, decarboxylation, citrullination, flavination, glycosylation, halogenation, hydroxylation, nitrosylation, oxidation, phosphorylation, prenylation, racemization, reduction, succinylation, sulfation or any combination thereof. The non-protein amino acids may comprise post-translational modifications.

[0621] Peptide functionalization can provide a specific isoelectric point for the particle. The particle may comprise an isoelectric point within 2 pH units, within 1.5 pH units, within 1 pH unit, within 0.75 pH unit, within 0.5 pH unit, within 0.25 pH unit or within 0.1 pH unit of the peptide functionalization coupled to its surface. A method may comprise forming a biomolecular corona at a certain pH, where the particle and the analyte have opposite charges, or where both the particle and the analyte are neutral. A method may comprise separating a plurality of particles by the isoelectric point of the particle (e.g., by isoelectric focusing). The peptide functionalization may comprise an isoelectric point between 4 and 10. The peptide functionalization may comprise an isoelectric point between 7 and 10. The peptide functionalization may comprise an isoelectric point between 4 and 7. The peptide functionalization may comprise an isoelectric point between 5 and 10. The peptide functionalization may comprise an isoelectric point between 6 and 8. The particle may comprise multiple peptide functionalizations having different isoelectric points. One peptide functionalization or multiple peptide functionalizations may provide multiple pka values for the particle.

[0622] The peptide functionalization or the peptide functionalization library may be constructed from modular units. The modular units may include individual amino acids, oligomeric units (such as oligopeptides comprising between 2 and 6 amino acid units) or non-peptide chemical or material units (such as succinyl linkers). For example, multiple modular units may comprise oligopeptides having about 1 to about 6 amino acids. The modular units may be biodegradable (e.g., configured for metabolism and mineralization by biological systems). Figure 13AA schematic diagram of a modular unit structure consistent with the present disclosure is provided. The modular unit may include amino acids, oligopeptides, non-peptide moieties (e.g., nucleotides), or any combination thereof 1301. The modular unit can be linear or branched. The modular unit may include a first reactive handle 1302 and a second reactive handle 1303. Each reactive handle may include a different coupling specificity. For example, the first reactive handle may be configured to couple only with a third type of reactive handle, while the second reactive handle may be configured to couple only with a fourth type of reactive handle. Conversely, the two reactive handles may share a specificity. For example, the first reactive handle may be configured to couple with a third type or a fourth type of reactive handle, while the second reactive handle may be configured to couple with a fourth type of reactive handle and a fifth type of reactive handle. In other cases, the first reactive handle is the same as the second reactive handle.

[0623] The modular unit can be individually addressed from a plurality of modular units (e.g., chemical reactivity or physical properties unique to a modular unit that may include a peptide). Two or more modular units can be connected by a linking element. Such a linking element can be non-peptidic and can include, for example, sugars, lipids, nucleic acids, or an alkyl backbone for incorporation into peptide functionalization. Individual modular units can be configured to undergo positional exchange such that a first modular unit from a first peptide functionalization exchanges positions with a second modular unit from a first peptide functionalization or from a second peptide functionalization. Additionally, a single modular unit can be configured for removal, modification (e.g., functional group coupling, oxidation, or reduction), or replacement by a separate modular unit. Modifiable and replaceable modular unit building blocks can be used to rapidly generate diverse peptide functionalization libraries. Such peptide functionalizations can contain between 7 and 20 amino acid residues.

[0624] Figure 13B Examples of such modular peptide binding molecules are provided. The peptide binding molecule 1304 can be coupled to a particle 1305. The peptide binding molecule can contain multiple modular units 1306. The unit is capable of intermolecular (e.g., with another peptide binding molecule) or intramolecular exchange 1307, thereby providing a mechanism for generating a second peptide binding molecule 1309 that contains a common modular unit from a first peptide binding molecule 1308.

[0625] Figure 14 Examples of peptide binding molecules with different structural configurations are provided. The peptide binding molecule can contain a linear structure, such as the structure of 1401. The peptide binding molecule can also contain a branched or cyclic structure, as shown in 1402 and 1403.

[0626] Analysis and automation system

[0627] In one aspect, a system for identifying biomolecules in a biological sample is described herein, wherein the system can include (i) a macromolecule immobilized on one or more surfaces as described elsewhere herein; (ii) a suspension solution; (iii) a biological sample containing proteins; and (iv) an automated system that includes a network of units with differentiated functions for isolating proteins adsorbed to one or more surfaces, and wherein the automated system is programmed to perform a series of steps.

[0628] In some embodiments, the macromolecule immobilized on a surface (or the surfaces of the present disclosure) as described elsewhere herein, the suspension solution, and the biological sample containing a certain concentration of proteins can be incubated at a temperature of about 10 degrees Celsius (°C) to about 100 °C. In some embodiments, one or more components in the composition can be incubated at a temperature of about 20 °C to about 90 °C. In some embodiments, one or more components in the composition can be incubated at a temperature of about 20 °C to about 50 °C. In some embodiments, the incubation can last for at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or at least 45 minutes. In some embodiments, the incubation can last for no more than 3 hours, no more than 2 hours, no more than 90 minutes, or no more than 1 hour. In some embodiments, the incubation can last for about 20 to about 90 minutes.

[0629] In some embodiments, the suspension can contain one or more buffers. For example, the pH can be modified as disclosed in PCT / US2023 / 068457 filed on June 14, 2023, which is hereby incorporated by reference in its entirety. The pH can be, for example, about 5, about 6, about 7, about 8, about 9, about 10, about 5 to about 7, or about 9 to about 10. In some embodiments, the suspension solution can contain Tris, EDTA, and CHAPS buffers. For example, the suspension solution can be Tris, EDTA in 150 millimolar (mM) KCl, and 0.05% CHAPS buffer. In another example, the suspension solution can be 10 mM Tris HCl pH 7.4, 1 mM EDTA. In some embodiments, the suspension contains tris(hydroxymethyl)aminomethane. In some embodiments, the suspension contains tris(hydroxymethyl)aminomethane with a pH of about 9.5.

[0630] In some aspects, the present disclosure provides an automated system that includes a network of units as described in WO2021 / 026172, which is incorporated herein by reference in its entirety. In some embodiments, the network of units can include differential functionality in the following aspects: using a plurality of particles having surfaces with different physicochemical properties to distinguish the states of complex biological samples, wherein: the first unit includes a multi-channel fluid transfer device for transferring fluids between the units within the system; the second unit includes a support for storing a plurality of biological samples; the third unit includes a support for a sensor array plate having partitions, the partitions including a plurality of particles having surfaces with different physicochemical properties for binding an analyte population in the complex biological sample; the fourth unit includes a support for storing a plurality of reagents; the fifth unit includes a support for storing reagents to be disposed of; the sixth unit includes a support for storing consumables used by the multi-channel fluid transfer device; and wherein the system is programmed to perform a series of steps that include: contacting the complex biological sample with a designated partition of the sensor array; incubating the complex biological sample with the plurality of particles contained within the partition of the sensor array plate; removing components from the partition other than the plurality of particles and the analyte population that interacts with the particles; and optionally preparing the analyte population for analysis (such as mass spectrometry).

[0631] In some embodiments, the first unit includes a degree of mobility such that it can access all other units within the system. In some embodiments, the first unit includes the ability to perform a pipetting function.

[0632] In some embodiments, the supports of the second unit and / or the third unit include supports for single plates, 6-well plates, 12-well plates, 96-well plates, or microtube racks. In some embodiments, the second and / or unit includes a thermal unit capable of regulating the temperature of the support and the sample. In some embodiments, the second unit and / or the third unit includes a rotational unit capable of physically agitating and / or mixing the sample.

[0633] In some embodiments, a plurality of particles having surfaces with different physicochemical properties for binding an analyte population within the biological sample are immobilized on the surface within the partition of the sensor array. In some embodiments, the plurality of particles includes a plurality of magnetic nanoparticles having different physicochemical properties for binding an analyte population within the complex biological sample. In some embodiments, the system includes a step in which the sensor array plate is transferred to an additional seventh unit and incubated for an additional amount of time, the seventh unit including a magnetized support and a thermal unit capable of regulating the temperature of the support and the sample.

[0634] In some embodiments, the fourth unit includes a reagent set for: generating a sensor array plate; washing unbound samples; and / or preparing samples for mass spectrometry. In some embodiments, contacting a biological sample with a designated partition of the sensor array includes pipetting a designated volume of the biological sample into the designated partition of the sensor array. In some embodiments, contacting a biological sample with a designated partition of the sensor array includes pipetting a volume corresponding to a ratio of multiple particles in solution to the biological sample of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, or 1:20.

[0635] In some embodiments, contacting a biological sample with a designated partition of the sensor array includes pipetting at least 10 microliters, at least 20 microliters, at least 50 microliters, at least 100 microliters, at least 250 microliters, at least 500 microliters, or at least 1000 microliters of the biological sample into the designated partition of the sensor array. In some embodiments, contacting the biological sample with the designated partition of the sensor array includes pipetting a volume not exceeding 1000 microliters, not exceeding 500 microliters, not exceeding 250 microliters, not exceeding 150 microliters, not exceeding 100 microliters, not exceeding 75 microliters, not exceeding 50 microliters, or not exceeding 30 microliters.

[0636] In some embodiments, incubating a biological sample with a plurality of particles contained within a partition of a sensor array plate includes an incubation time of at least about 10 seconds, at least about 15 seconds, at least about 20 seconds, at least about 25 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 60 seconds, at least about 90 seconds, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 45 minutes, at least about 50 minutes, at least about 60 minutes, at least about 90 minutes, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, or at least about 24 hours. In some embodiments, incubating a biological sample with a plurality of particles contained within a partition of a sensor array plate includes an incubation time of no more than 24 hours, no more than 12 hours, no more than 6 hours, no more than 3 hours, no more than 2 hours, no more than 90 minutes, no more than 75 minutes, no more than 60 minutes, no more than 45 minutes, or no more than 30 minutes. In some embodiments, incubating a biological sample with a plurality of particles contained within a partition of a sensor array plate includes an incubation time of 30 minutes to 3 hours.

[0637] In some embodiments, incubating a biological sample with a plurality of particles contained within a partition of a substrate includes an incubation temperature between about 4°C and about 40°C. Incubating a biological sample with a plurality of particles contained within a partition of a substrate can include an incubation temperature between about 4°C and about 37°C. Incubating a biological sample with a plurality of particles contained within a partition of a substrate can include an incubation temperature between about 20°C and about 50°C. Incubating a biological sample with a plurality of particles contained within a partition of a substrate can include an incubation temperature between about 4°C and about 100°C.

[0638] In some embodiments, removing all components from the partition except for the plurality of particles and the population of analytes that interact with the particles includes a series of washing steps.

[0639] In some embodiments, a second unit can facilitate transfer of a sample for mass spectrometry to a mass spectrometry unit.

[0640] In some aspects, the present disclosure provides an automated device for identifying proteins in a biological sample, the automated device comprising: a sample preparation unit; a substrate comprising a plurality of channels; a plurality of pipettes; a plurality of solutions; a plurality of surfaces as described herein; and wherein the automated device is configured to form a protein corona and digest the protein corona.

[0641] In some embodiments, the automated device further comprises a magnetic source. In some embodiments, the automated device is configured for BCA, gel, or trypsin digestion of the protein corona.

[0642] In some embodiments, the automated device is enclosed. In some embodiments, the automated device is sterilized prior to use. In some embodiments, the automated device is configured for mass spectrometry. In some embodiments, the automated device is temperature controlled.

[0643] A variety of different analytical techniques can be used to identify, measure, and quantify proteomic data of a sample. For example, SDS-PAGE or any gel-based separation technique can be used to analyze proteomic data. Peptides and proteins can also be identified, measured, and quantified using immunoassays such as ELISA. Alternatively, mass spectrometry, high performance liquid chromatography, LC-MS / MS, Edman degradation, immunoaffinity techniques, and the methods disclosed in EP3548652, WO2019083856, WO2019133892 (each of which is incorporated herein by reference in its entirety), and other protein separation techniques can be used to identify, measure, and quantify proteomic data.

[0644] In some aspects, the present disclosure provides an automated device for generating a subset of biomolecules from a biological sample, comprising: a substrate comprising a plurality of partitions, a first unit containing the biological sample, and a loading unit that is movable along the substrate and capable of transferring volumes (e.g., volumes of buffer) between different units of the device. In some cases, the substrate is a multi-well plate.

[0645] The plurality of partitions may include a plurality of sensor elements. The plurality of sensor elements may include surfaces. The plurality of sensor elements may be surfaces as disclosed herein (e.g., particles). For example, the sensor elements may include a first unique nanoparticle and a second unique nanoparticle, wherein the first unique nanoparticle comprises a surface selected from Table 3 and / or Table 4.

[0646] The partitions in the multiple partitions may include from 1 to 100 types of sensor elements (e.g., unique surfaces). The partitions in the multiple partitions may include from 2 to 50 types of sensor elements. The partitions in the multiple partitions may include from 2 to 20 types of sensor elements. The partitions in the multiple partitions may include from 2 to 5 types of sensor elements. The partitions in the multiple partitions may include from 3 to 8 types of sensor elements. The partitions in the multiple partitions may include from 4 to 10 types of sensor elements. The partitions in the multiple partitions may include from 5 to 12 types of sensor elements. The partitions in the multiple partitions may include from 6 to 15 types of sensor elements. The partitions in the multiple partitions may include from 8 to 20 types of sensor elements. The partitions in the multiple partitions may include 2 types of sensor elements. The partitions in the multiple partitions may include 3 types of sensor elements. The partitions in the multiple partitions may include 4 types of sensor elements.

[0647] Two or more of the partitions in the multiple partitions may include different numbers of sensor elements. Two or more of the partitions in the multiple partitions may include different types of sensor elements. Two or more of the partitions in the multiple partitions may include a combination of types and / or numbers of sensor elements that are different from other partitions in the multiple partitions. Subsets of the partitions in the multiple partitions may each contain a unique combination of sensor elements that is different from other partitions in the multiple partitions.

[0648] The sensor elements may be stored in a dry form inside or within the partitions. The dry sensor elements may be reconstituted or rehydrated before use. The sensor elements may also be stored in a solution. For example, a substrate partition may include a solution containing a high-concentration surface.

[0649] The partitions in the plurality of partitions include sensor elements at different concentrations or amounts (e.g., mass / mole amount per unit volume of the sample). The partitions in the plurality of partitions can include sensor elements from 1 pM to 100 nM. The partitions in the plurality of partitions can include sensor elements from 1 pM to 500 pM. The partitions in the plurality of partitions can include sensor elements from 10 pM to 1 nM. The partitions in the plurality of partitions can include sensor elements from 100 pM to 10 nM. The partitions in the plurality of partitions can include sensor elements from 500 pM to 100 nM. The partitions in the plurality of partitions can include sensor elements from 50 μg / ml to 300 μg / ml. The partitions in the plurality of partitions can include sensor elements from 100 μg / ml to 500 μg / ml. The partitions in the plurality of partitions can include sensor elements from 250 μg / ml to 750 μg / ml. The partitions in the plurality of partitions can include sensor elements from 400 μg / ml to 1 mg / ml. The partitions in the plurality of partitions can include sensor elements from 600 μg / ml to 1.5 mg / ml. The partitions in the plurality of partitions can include sensor elements from 800 μg / ml to 2 mg / ml. The partitions in the plurality of partitions can include sensor elements from 1 mg / ml to 3 mg / ml. The partitions in the plurality of partitions can include sensor elements from 2 mg / ml to 5 mg / ml. The partitions in the plurality of partitions can include sensor elements greater than 5 mg / ml.

[0650] The loading unit can be configured to move and transfer volumes (e.g., volumes of solutions or powders) between any unit, compartment, or partition within the device. The loading unit can be configured to move precise volumes (e.g., within 0.1%, 0.01%, 0.001% of a specified volume). The loading unit can be configured to collect a volume from a substrate or from a compartment or partition within the substrate, and dispense the volume back into the substrate or into a compartment or partition within the substrate, or dispense the volume or a portion of the volume into different units, compartments, or partitions. The loading unit can be configured to move multiple volumes simultaneously, such as 2 to 400 individual volumes. The loading unit can include a plurality of pipette tips.

[0651] The loading unit can be configured to move a volume of liquid. The volume can be about 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 0.5 μl, 0.6 μl, 0.7 μl, 0.8 μl, 0.9 μl, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 12 μl, 15 μl, 20 μl, 25 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 120 μl, 150 μl, 180 μl, 200 μl, 250 μl, 300 μl, 400 μl, 500 μl, 600 μl, 800 μl, 1 ml or greater than 1 ml. The liquid can be a biological sample or a solution.

[0652] In some cases, the solution includes a wash solution, a resuspension solution, a denaturing solution, a buffer, a reagent (e.g., a reducing reagent), or any combination thereof. In some cases, the solution contains a biological sample.

[0653] In part by virtue of these functions, the loading unit can be capable of partitioning the sample. In some embodiments, this includes partitioning the sample into multiple partitions. The sample can be partitioned 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 sample can be partitioned into 96, 192 or 384 partitions. The automated device can include multiple substrates containing partitions. The automated device can include 1, 2, 3, 4, 5 or more substrates containing partitions. In some cases, the loading unit loads different volumes of biological samples into different partitions. In some cases, the loading unit loads the same volume into two or more partitions. The volume of the biological sample loaded into the partition can be about 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, 0.5 μl, 0.6 μl, 0.7 μl, 0.8 μl, 0.9 μl, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 12 μl, 15 μl, 20 μl, 25 μl, 30 μl, 40 μl, 50 μl, 60 μl, 70 μl, 80 μl, 90 μl, 100 μl, 120 μl, 150 μl, 180 μl, 200 μl, 250 μl, 300 μl, 400 μl, 500 μl, 600 μl, 800 μl, 1 ml or greater than 1 ml. The volume of the biological sample loaded into the partition can be about 10 μl to 400 μl. The volume of the biological sample loaded into the partition can be about 5 μl to 150 μl. The volume of the biological sample loaded into the partition can be about 35 μl to 80 μl. In some cases, the loading unit can partition two or more biological samples. For example, the sample storage unit can contain two biological samples, and the system partitions them in a microplate. In some embodiments, the loading unit can facilitate the transfer of the sample for mass spectrometry to the mass spectrometry unit.

[0654] The system can be configured to dilute a sample or a sample partition. The sample or sample partition can be diluted with a buffer, water (e.g., purified water), a non-aqueous solvent, or any combination thereof. The diluent can be stored in an automated device before being dispensed into the substrate partition. The automated device can store multiple diluents that differ in terms of pH, salinity, osmolality, viscosity, dielectric constant, or any combination thereof. The diluent can be used to adjust the chemical properties of the sample or sample partition. The automated device can dilute the sample or sample partition by a factor of 2, 3, 4, 5, 6, 8, 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or more. The automated device can perform different dilutions on two samples or sample partitions. The system can perform different dilutions on each of multiple partitions. For example, the system can perform different dilutions on each of 96 sample partitions in a 96-well plate. In some cases, different dilutions include different degrees of dilution (e.g., 2-fold vs. 4-fold). In some cases, different dilutions include diluting with different solutions (e.g., different buffers). In some cases, two sample partitions can be made to differ in one or more chemical properties such as pH, salinity, or viscosity.

[0655] In some cases, the system can modify the chemical composition of the sample or sample partition. The system can modify or adjust the pH, salinity, osmolality, dielectric constant, viscosity, buffer type, salt type, sugar type, detergent type, or any combination thereof for the sample or sample partition. Such modification or adjustment can include mixing a reagent from a fourth unit with the sample or sample partition. The system can modify the chemical composition of two samples or sample partitions differently.

[0656] The system or automated device of the present disclosure may also include an incubation element. The incubation element may contact, support, or hold another component (e.g., a substrate or unit) of the automated device. The incubation unit may contact, support, or hold multiple components of the automated device. The incubation element may contact the substrate to facilitate heat transfer between the incubation element and the substrate. The incubation unit may be configured to control the temperature of one or more components of the automated device, such as by heating or cooling. The incubation element may enable the components of the device to be cooled from 20°C to 1°C. The incubation element may enable the components of the device to be heated from 25°C to 100°C. The incubation element may be able to set the temperature of the components of the device from 4°C to 37°C. The incubation element may be configured to heat or cool different parts of the components of the automated device to different temperatures. For example, the incubation element may keep the first partition in the substrate at 30°C while keeping the second partition in the substrate at 35°C. The incubation element may control the temperature of the sample or partition. The incubation element may include a temperature sensor (e.g., a thermocouple) for detecting the temperature inside the partition or container. The incubation element may calibrate its heating or cooling based on the readings from the temperature sensor.

[0657] The incubation element may be configured to physically agitate the components of the automated device. The agitation may be in the form of shaking or rotating, vibrating, rocking, sonication, or any combination thereof. The incubation element may be capable of providing multiple agitation intensities and / or frequencies. For example, the incubation element may include multiple settings for shaking at different frequencies and amplitudes. The incubation element may also be able to stir and / or mix a volume (e.g., a portion of a biological sample).

[0658] The automated device may include a unit containing a resuspension solution. The loading unit may be capable of transferring a volume of the resuspension solution to a partition among a plurality of partitions of a substrate. In some cases, this results in dilution of the sample present in the partition and may further result in desorption of a plurality of biomolecules from the biomolecular corona deposited on the sensor element within the partition. The number of biomolecules desorbed from the biomolecular corona may depend on the volume of the resuspension solution added to the partition, the temperature of the partition, the composition of the resuspension solution (e.g., salinity, osmolality, viscosity, dielectric constant, or pH), the volume of the biological sample within the partition, and the type of sensor element and the composition of the biomolecules in the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in less than 5% of the biomolecules being desorbed from the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in 10% to 20% of the biomolecules being desorbed from the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in 20% to 30% of the biomolecules being desorbed from the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in 30% to 40% of the biomolecules being desorbed from the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in 40% to 50% of the biomolecules being desorbed from the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in 50% to 60% of the biomolecules being desorbed from the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in 60% to 70% of the biomolecules being desorbed from the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in 70% to 80% of the biomolecules being desorbed from the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in 80% to 90% of the biomolecules being desorbed from the biomolecular corona. Transfer of the volume of the resuspension solution to the partition may result in greater than 90% of the biomolecules being desorbed from the biomolecular corona.

[0659] In some cases, multiple rounds of desorption are performed. In each round, the supernatant containing the desorbed biomolecules may be collected, analyzed, or discarded. The type and abundance of the biomolecules in the supernatant may vary between desorption rounds. The automated device may perform one or more desorption and discard cycles (i.e., washes), followed by one or more desorption cycles including sample collection and / or analysis.

[0660] The resuspension solution can be customized to optimize the enrichment of specific biomarkers. The resuspension solution can contain buffers such as Tris-EDTA (TE), CHAPS, PBS, citrate, HEPES, MES, CHES, or other biological buffers. The resuspension solution can contain 150 mM KCl, 0.05% CHAPS buffer in Tris EDTA (TE). The resuspension solution can contain 10 mM TrisHCl pH 7.4, 1 mM EDTA. The resuspension solution can also contain highly purified water (e.g., distilled water or deionized water). Biomolecule desorption can be enhanced by heating or agitating the incubation element. The supernatant can be transferred to a new partition after desorption. The resuspension solution can be used to dilute the sample.

[0661] The automated device can include a unit containing a denaturing solution. The denaturing solution can contain proteases. The denaturing solution can contain chemicals capable of peptide cleavage (e.g., cyanogen bromide, formic acid or hydroxylamine, 2-nitro-5-thiocyanatobenzoic acid). The denaturing solution can contain chemical denaturants 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 can contain reducing agents such as 2-mercaptoethanol, dithiothreitol, or tris(2-carboxyethyl)phosphine. The protease can be trypsin and / or LysC. The denaturing solution can be added to the partition after desorption. The denaturing solution can be added to the partition containing the biomolecular corona.

[0662] The automated device can include a magnet or a magnet array. The automated device can be capable of moving the substrate to and from the magnet or magnet array. The magnet array can be configured such that a plurality of magnets from the magnet array can be placed directly beneath a plurality of partitions from the substrate. The magnet can be capable of immobilizing magnetic sensor elements (e.g., magnetic particles such as superparamagnetic iron oxide nanoparticles with or without a coating) within the partitions on the substrate. For example, the magnet can prevent magnetic nanoparticles from being removed from the partition during the washing step. The magnet can also cause agglomeration of a batch of magnetic particles. The magnet can produce particle agglomeration in less than 10 minutes. The magnet can produce particle agglomeration in less than 5 minutes. The particle agglomeration can include particles having a biomolecular corona.

[0663] The automated device may include a purification unit. The purification unit may include a plurality of partitions containing adsorbent or resin. The purification unit may include a solid phase extraction array or plate. The solid phase extraction array or plate may contain a polar stationary phase material. The solid phase extraction array or plate may contain a non-polar stationary phase material. The solid phase extraction array or plate may contain a C18 stationary phase material (e.g., octadecyl silica gel). The automated device may include a unit containing a conditioning solution for the purification unit (e.g., a conditioning solution for the solid phase extraction material). The automated device may include a unit having an elution solution for removing biomolecules from the purification unit.

[0664] In some embodiments, components other than the plurality of sensor elements and the analyte population (e.g., proteins, metabolites, lipids, etc.) that interact with the plurality of sensor elements are removed from the partition (i.e., the washing step). In some cases, the automated device may perform a series of washing steps. The washing step may remove biomolecules that are not bound to the sensor elements within the partition. The washing step may desorb a subset of the biomolecules bound to the sensor elements within the partition. For example, the washing step may result in the desorption and removal of a subset of the soft corona analytes while leaving most of the hard corona analytes bound to the sensor elements.

[0665] In some aspects, the present disclosure provides an automated device for identifying proteins in a biological sample, the automated device including: a sample preparation unit; a substrate including a plurality of channels; a plurality of pipettes; a plurality of solutions, a plurality of surfaces, and wherein the automated device is configured to form a protein corona and digest the protein corona.

[0666] In some aspects, the present disclosure provides an automated device for identifying proteins in a biological sample, the automated device including: a sample preparation unit; a substrate including a plurality of channels; a plurality of pipettes; a plurality of solutions, a plurality of nanoparticles, wherein the automated device is configured to form a protein corona and digest the protein corona, and wherein at least one of the solutions is a TE 150 mM KCl 0.05% CHAPS buffer.

[0667] In some embodiments, the sample preparation unit is configured to add a plurality of nanoparticles to the substrate using a plurality of pipettes. In some embodiments, wherein the sample preparation unit is configured to add a biological sample to the substrate using a plurality of pipettes. In some embodiments, the sample preparation unit is configured to incubate the plurality of nanoparticles and the biological sample to form a protein corona.

[0668] In some embodiments, the sample preparation unit is configured to separate the protein corona from the supernatant to form protein corona pellets. In some embodiments, the sample preparation unit is configured to reconstitute the protein corona pellets with a TE 150 mM KCl 0.05% CHAPS buffer.

[0669] In some embodiments, the automated device further includes a magnetic source. In some embodiments, the automated device is configured for BCA, gel, or trypsin digestion of the protein corona.

[0670] In some embodiments, the automated device is enclosed. In some embodiments, the automated device is sterilized prior to use. In some embodiments, the automated device is configured for mass spectrometry. In some embodiments, the automated device is temperature-controlled.

[0671] Kit

[0672] In one aspect, the present disclosure describes a kit for identifying biomolecules in a biological sample, wherein the kit can include one or more surfaces of the present disclosure as described elsewhere herein. In some embodiments, the kit can be used to perform the methods for identifying proteins in a sample as disclosed herein.

[0673] The kits of the present disclosure can include one or more surfaces (e.g., particles) for interrogating a sample. In some embodiments, the kit includes one or more surfaces (e.g., particles) as provided elsewhere herein. In some instances, the surface is capable of binding multiple proteins in a biological fluid to produce a protein corona.

[0674] In some embodiments, the protein corona includes a first protein from the biological fluid. In some embodiments, the protein corona includes a second protein from the biological fluid. In some embodiments, the concentration of the second protein present in the biological fluid is 6 orders of magnitude higher than the concentration of the first protein (e.g., 2, 3, 4, 5, 7, 8, 10 orders of magnitude).

[0675] The kit can be pre-packaged into discrete aliquots. In another example, the kit can include multiple different surface types (e.g., particles with different surface chemistries) that can be used to interrogate a sample. Multiple particle types can be pre-packaged, where each particle type among the multiple particles is individually packaged. Alternatively, multiple particle types can be packaged together to include a combination of particle types in a single package. In some embodiments, the kit includes two or more packages containing different particles, where at least one package includes two or more different particles. In some embodiments, the particles can be lyophilized and stored in a sealed container. In some embodiments, the particles can be stored in a fluid, such as water with a suitable preservative (e.g., sodium azide).

[0676] In some embodiments, the kit further comprises a denaturing agent. In some embodiments, the denaturing agent comprises at least one of the following: sodium dodecyl sulfate, acetic acid, trichloroacetic acid, sulfosalicylic acid, sodium bicarbonate, ethanol, formaldehyde, glutaraldehyde, urea, guanidine chloride, lithium perchlorate, 2-mercaptoethanol, dithiothreitol, tris(2-carboxyethyl)phosphine (TCEP), or any combination thereof.

[0677] In some embodiments, the kit further comprises a reducing agent. In some embodiments, the reducing agent comprises TCEP, dithiothreitol, β-mercaptoethanol, glutathione, cysteine, or any combination thereof.

[0678] In some embodiments, the kit further comprises an alkylating agent. In some cases, the alkylating agent is configured to alkylate proteins. In some embodiments, the alkylating agent comprises iodoacetamide, iodoacetic acid, acrylamide, chloroacetamide, or any combination thereof.

[0679] In some embodiments, the kit further comprises a digesting agent. In some cases, the digesting agent is an enzymatic agent. In some cases, the digesting agent is configured to digest proteins. In some embodiments, the digesting agent comprises trypsin, lysin, serine protease, or any combination thereof. In some embodiments, the digesting agent comprises trypsin. In some embodiments, the digesting agent comprises trypsin and LysC.

[0680] In some embodiments, the kit further comprises a terminating agent, which is configured to terminate the digesting agent (e.g., the enzymatic age...

Claims

1. A macromolecule comprising repeating units of formula (I-A): wherein each of Y1, Y2 and Y3 is independently selected from hydrogen or C1-C6 alkyl; R1 is hydrogen, an optionally substituted succinate, a C1-C6 alkylsulfone or a phthalate; R2 is C1-C6 hydroxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C3-C6 heterocycloalkyl, C1-C 12 amine, optionally substituted C3-C6 bicycloalkylmethane, C1-C6 alkylguanidine, C1-C6 ether, optionally substituted C1-C6 disulfide, C1-C6 thiol, optionally substituted succinate, optionally substituted C1-C 12 alkylamine, C1-C6 alkylacetamide, C5-C 11 optionally substituted cycloalkyl or C1-C6 aminophthalate; or R1 and R2 together with the nitrogen to which they are attached form an optionally substituted heterocycle; or R1 and R2 together with the nitrogen to which they are attached form an azide group; and q is an integer between 1 and 6.

2. The macromolecule according to claim 1, wherein Y1 is C1-C3 alkyl.

3. The macromolecule according to claim 1 or 2, wherein Y1 is C1 alkyl.

4. The macromolecule according to any one of claims 1-3, wherein each of Y2 and Y3 is hydrogen.

5. The macromolecule according to any one of claims 1-4, wherein q is an integer between 1 and 3.

6. The macromolecule according to any one of claims 1-5, wherein q is 1.

7. The macromolecule according to any one of claims 1-6, wherein R1 is hydrogen and R2 is selected from an optionally substituted C3-C6 bicycloalkylmethane, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted C3-C6 heterocycloalkyl, a C1-C6 hydroxy group, a C1-C6 ether, an optionally substituted -C1-C6 disulfide, an optionally substituted succinate, an optionally substituted C1-C6 alkylamine, a C1-C6 alkylacetamide or a C1-C6 alkylguanidine.

8. The macromolecule according to any one of claims 1-7, wherein R2 is an optionally substituted bicyclohexylmethane.

9. The macromolecule according to claim 8, wherein R2 is aminobicyclohexylmethane.

10. The macromolecule according to any one of claims 1-7, wherein R2 is an optionally substituted aryl.

11. The macromolecule according to claim 10, wherein R2 is a halotoluene.

12. The macromolecule according to claim 11, wherein R2 is 2-fluorotoluene.

13. The macromolecule according to any one of claims 1-7, wherein R2 is a C1-C6 hydroxy group.

14. The macromolecule according to claim 13, wherein R2 is a C3-C6 hydroxy group.

15. The macromolecule according to claim 14, wherein R2 is -(CH2)6OH.

16. The macromolecule according to any one of claims 1-7, wherein R2 is a C1-C6 ether.

17. The macromolecule according to claim 16, wherein R2 is -CH2OCH3, -CH2CH2OCH3, -CH2CH2OCH2CH3 or -CH2OCH2CH3.

18. The macromolecule according to claim 17, wherein R2 is -CH2CH2OCH3.

19. The macromolecule according to any one of claims 1-7, wherein R2 is a C1-C6 alkylacetamide.

20. The macromolecule according to claim 19, wherein R2 is -(CH2)2 acetamide (or ).

21. The macromolecule according to any one of claims 1-7, wherein R2 is an optionally substituted di-C1-C6 alkyldisulfide.

22. The macromolecule according to claim 21, wherein R2 is -CH2CH2-S-S-CH2CH2NH2.

23. The macromolecule according to any one of claims 1-7, wherein R2 is an optionally substituted succinate ester.

24. The macromolecule according to claim 23, wherein R2 is -(CH2) 1-6 NH(C=O)CH2CH2COOH.

25. The macromolecule according to claim 24, wherein R2 is - (CH2)6NH(C=O)CH2CH2COOH.

26. The macromolecule according to any one of claims 1-7, wherein R2 is an optionally substituted heteroaryl.

27. The macromolecule according to claim 26, wherein R2 is -(CH2) 1-6 imidazole.

28. The macromolecule according to claim 27, wherein R2 is -(CH2)3imidazole.

29. The macromolecule according to claim 26, wherein R2 is a disubstituted C2-C4 imidazole.

30. The macromolecule according to claim 29, wherein R2 is dipropylimidazole.

31. The macromolecule according to any one of claims 1-7, wherein R2 is an optionally substituted heterocycloalkyl.

32. The macromolecule according to claim 31, wherein R2 is -(CH2) 1-6 pyrrolidine.

33. The macromolecule according to claim 32, wherein R2 is -(CH2)2pyrrolidine.

34. The macromolecule according to any one of claims 1-7, wherein R2 is an optionally substituted C1-C6 alkylamine.

35. The macromolecule according to claim 34, wherein R2 is -(CH2) 1-3 dimethylamine.

36. The macromolecule according to claim 35, wherein R2 is -(CH2)2dimethylamine.

37. The macromolecule according to any one of claims 1-7, wherein R2 is a C1-C6 guanidine.

38. The macromolecule according to claim 37, wherein R2 is -(CH2)2guanidine.

39. The macromolecule according to any one of claims 1-6, wherein each of R1 and R2 is nitrogen.

40. The macromolecule according to claim 39, wherein R1 and R2 together form an optionally substituted heterocycle.

41. The macromolecule according to claim 40, wherein the optionally substituted heterocycle is a triazole.

42. The macromolecule according to claim 41, wherein the optionally substituted triazole contains a benzylamide.

43. The macromolecule according to claim 42, wherein the benzylamide is halogenated.

44. The macromolecule according to any one of claims 1-6, wherein R1 is an optionally substituted succinate ester, and R2 is an optionally substituted C3-C6 bicycloalkylmethane, an optionally substituted aryl, a C1-C6 thiol, or an optionally substituted succinate ester.

45. The macromolecule according to claim 44, wherein R1 is a succinate ester, and R2 is an optionally substituted C3-C6 bicycloalkylmethane, an optionally substituted aryl, a C1-C6 thiol, or an optionally substituted succinate ester.

46. The macromolecule according to claim 45, wherein R2 is dicyclohexylmethane succinate.

47. The macromolecule according to claim 45, wherein R2 is an optionally substituted aryl.

48. The macromolecule according to claim 47, wherein R2 is 2-fluorotoluene.

49. The macromolecule according to claim 45, wherein R2 is a C1-C6 thiol.

50. The macromolecule according to claim 49, wherein R2 is -(CH2)2SH.

51. The macromolecule according to claim 45, wherein R2 is an optionally substituted succinate ester.

52. The macromolecule according to claim 51, wherein R2 is -(CH2) 1-12 NH(C=O)CH2CH2COOH.

53. The macromolecule according to claim 52, wherein R2 is -(CH2) 1-3 NH(C=O)CH2CH2COOH.

54. The macromolecule according to claim 53, wherein R2 is - (CH2)2NH(C=O)CH2CH2COOH.

55. The macromolecule according to claim 51, wherein R2 is -(CH2) 10-12 NH(C=O)CH2CH2COOH.

56. The macromolecule according to claim 55, wherein R2 is - (CH2) 12 NH(C=O)CH2CH2COOH。 57. The macromolecule according to claim 44, wherein R1 is a C2-C 12 alkenyl succinate, and R2 is a substituted succinate.

58. The macromolecule according to claim 57, wherein R1 is a C8 alkenyl succinate and R2 is a C8 alkenyl ethylamino succinate.

59. The macromolecule according to any one of claims 1 - 6, wherein R1 is a C1 - C6 alkyl sulfone and R2 is an optionally substituted C1 - C6 alkylamine.

60. The macromolecule according to claim 59, wherein R1 is -(CH2)3SOOOH, and R2 is -(CH2) 1-6 N(CH3)2(CH2CH2CH2SOOOH) or -(CH2) 1-6 N(CH2CH2CH2SOOOH)2.

61. The macromolecule according to claim 60, wherein R2 is - (CH2)2N(CH3)2(CH2CH2CH2SOOOH).

62. The macromolecule according to claim 61, wherein R2 is - (CH2)2N(CH2CH2CH2SOOOH)2.

63. The macromolecule according to any one of claims 1 - 6, wherein R1 is a phthalate and R2 is a C1 - C6 aminophthalate.

64. The macromolecule according to claim 63, wherein R2 is a C2 - C6 aminophthalate.

65. The macromolecule according to claim 64, wherein R2 is a C2 aminophthalate.

66. The macromolecule according to claim 64, wherein R2 is a C6 aminophthalate.

67. A macromolecule comprising a repeating unit of formula (II): wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1 - C6 alkyl; R4 is hydrogen or a C1 - C6 thiol; and R5 is a succinate, a C1 - C6 thiol, an optionally substituted aryl, or an optionally substituted C1 - C6 disulfide.

68. A macromolecule comprising a repeating unit of formula (III - A): wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1 - C6 alkyl; X is O or NH; and q is an integer between 1 and 6.

69. A macromolecule comprising repeating units of a first component and a second component, wherein the first component comprises the structure of component (A), and the second component comprises the structure of component (B’): wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1 - C6 alkyl; each of X1, X2, and X3 is independently selected from hydrogen or a C1 - C6 alkyl; A is R1 is hydrogen, an optionally substituted succinate, a C1 - C6 alkyl sulfone, a phthalate, R2 is C1-C 12 amine, C1-C6 hydroxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted C3-C6 heterocycloalkyl, two or more fused 3-6 membered rings; optionally substituted C3-C6 bicycloalkylmethane, C1-C6 alkylguanidine, C1-C6 ether, optionally substituted -C1-C6 disulfide, C1-C6 thiol, optionally substituted succinate, optionally substituted C1-C6 alkylamine, C1-C6 alkylacetamide, C1-C6 aminophthalate, boric acid, C1-C6 thiol, C1-C 11 optionally substituted cycloalkyl or monosaccharide; or R1 and R2 together with the nitrogen to which they are attached form an optionally substituted heterocycle; or R1 and R2 together with the nitrogen to which they are attached form an azide group; R4 is hydrogen or a C1 - C6 thiol; R5 is a succinate, an optionally substituted aryl, or an optionally substituted - C1 - C6 disulfide; B is q is an integer between 1 and 6; and p is an integer between 1 and 20.

70. A macromolecule comprising repeating units of a first component and cross - linked repeating units, wherein the first component comprises the structure of component (A’): wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or a C1 - C6 alkyl; A’ is G’ or W’ includes Q’; Q’ is a peptide.

71. A macromolecule comprising repeating units of a first component and a second component, wherein the first component comprises the structure of component (A’), and the second component comprises the structure of (B’): wherein each of Y1, Y2, and Y3 is independently selected from hydrogen or C1-C6 alkyl; each of X1, X2, and X3 is independently selected from hydrogen or C1-C6 alkyl; A' is G’ or W’ comprises Q’; Q’ is a peptide; B is q is an integer between 1 and 6; and p is an integer between 1 and 20.

72. A system comprising: a. a surface; b. a macromolecule as claimed in any one of claims 70 or 71 coupled to the surface, wherein the peptide comprises a binding site; c. a protein that interacts with the peptide at the binding site.

73. A surface comprising a moiety of formula (IV’): wherein Z is a linking moiety comprising a straight chain having 2 to 20 atoms selected from carbon, oxygen, and nitrogen, and optional substituents on the carbon and any nitrogen atoms in the straight chain; R1’ is hydrogen or succinate; and R2’ is C1-C6 alkyl-G’; G’ comprises Q’; Q’ is a peptide; wherein the peptide does not contain cysteine.

74. A surface comprising a moiety of formula (IV): wherein Z is a linking moiety comprising a straight chain having 2 to 20 atoms selected from carbon, oxygen, and nitrogen, and optional substituents on the carbon and any nitrogen atoms in the straight chain; R1 is hydrogen, an optionally substituted succinate, an optionally substituted glutarate, an optionally substituted adipate, an optionally substituted pimelate, an optionally substituted suberate, an optionally substituted azelate, or an optionally substituted sebacate; and R2 is an optionally substituted -C1-C6 disulfide or C1-C6 thiol.

75. The surface as claimed in claim 74, wherein Z is C1-C6 alkyl.

76. The surface as claimed in claim 74 or 75, wherein Z is C3 alkyl.

77. The surface as claimed in claim 74, wherein Z is C1-C8 alkyl substituted with a hydroxyl group.

78. The surface as claimed in claim 77, wherein Z is:

79. The surface as claimed in any one of claims 74-78, wherein R1 is hydrogen and R2 is a substituted -C1-C6 alkyl disulfide.

80. The surface as claimed in any one of claims 74-78, wherein R2 is a substituted di-C1-C6 alkyl disulfide.

81. The surface as claimed in claim 80, wherein R2 is -CH2CH2-S-S-CH2CH2NH2.

82. The surface as claimed in any one of claims 74-78, wherein R1 is succinate and R2 is C1-C6 thiol.

83. The surface as claimed in claim 82, wherein R2 is -(CH2)2SH.

84. A surface comprising a macromolecule as claimed in any one of claims 1-71, wherein the macromolecule is immobilized on a modified surface.

85. The surface as claimed in claims 73-84, wherein the macromolecule is covalently coupled to the surface.

86. The surface according to any one of claims 73 - 84, wherein the macromolecule is electrostatically coupled to the surface.

87. The surface according to any one of claims 73 - 84, wherein the macromolecule is coupled to the surface by a polymerization event.

88. The surface according to claim 87, wherein the polymerization event comprises reacting with vinyl groups on the surface.

89. The surface according to any one of claims 73 - 84, wherein the surface is a bead or a particle.

90. The surface according to claim 89, wherein the surface is a particle.

91. The surface according to claim 90, wherein the particle is a nanoparticle or a microparticle.

92. The surface according to claim 90, wherein the particle has a diameter of from about 200 nanometers (nm) to about 400 nm.

93. The surface according to any one of claims 90 - 92, wherein the particle is a superparamagnetic iron oxide particle.

94. The surface according to any one of claims 90 - 92, wherein the particle comprises an iron oxide material.

95. The surface according to any one of claims 90 - 92, wherein the particle has an iron oxide core.

96. The surface according to any one of claims 90 - 92, wherein the particle has an iron oxide crystal embedded in a polystyrene core.

97. The surface according to any one of claims 90 - 92, wherein the particle comprises an iron oxide core with a silica shell coating.

98. The surface according to any one of claims 73 - 97, wherein the surface is selected from Table 3 or Table 4.

99. The surface according to claim 74, wherein the surface comprises a structure 100. The surface according to claim 74, wherein the surface comprises a structure 101. The surface according to claim 69, wherein the surface comprises a structure wherein represents the connection point of the unit of component (A) or component (B).

102. The surface according to claim 69, wherein the surface comprises a structure wherein denotes the connection point of the unit of component (A) or component (B).

103. The surface according to claim 69, wherein the surface comprises a structure wherein denotes the connection point of the unit of component (A) or component (B).

104. The surface according to claim 69, wherein the surface comprises a structure wherein denotes the connection point of the unit of component (A) or component (B).

105. The surface according to claim 69, wherein the surface comprises a structure wherein represents the connection point of the unit constituting component (A) or component (B).

106. The surface according to claim 69, wherein the surface comprises a structure wherein represents a connection point of a unit of component (A) or component (B).

107. The surface according to claim 69, wherein the surface comprises a structure wherein represents a connection point of a unit of component (A) or component (B).

108. The surface according to claim 69, wherein the surface comprises a structure wherein denotes the connection point of the unit of component (A) or component (B).

109. The surface according to claim 69, wherein the surface comprises a structure Wherein represents a connection point of a unit of component (A) or component (B).

110. The surface according to claim 69, wherein the surface comprises a structure wherein denotes the connection point of the unit of component (A) or component (B).

111. A method, comprising: a. contacting a biological sample with the surface according to any one of claims 73 - 110; b. releasing a plurality of biomolecules or portions thereof from the surface; and c. identifying at least the plurality of biomolecules or portions thereof from the surface.

112. The method according to claim 111, wherein the surface comprises at least two unique surfaces.

113. The method according to claim 112, wherein the at least two specific surfaces comprise structures wherein represents a connection point of units of component (A) or component (B).

114. The method according to claim 112, wherein the at least two distinctive surfaces comprise structures wherein represents a connection point of a unit of component (A) or component (B).

115. The method according to claim 112, wherein the at least two specific surfaces comprise structures wherein represents a connection point of a unit of component (A) or component (B).

116. The method according to claim 112, wherein the at least two distinctive surfaces comprise structures wherein represents a connection point of a unit of component (A) or component (B).

117. The method according to claim 112, wherein the at least two specific surfaces comprise structures wherein represents a connection point of a unit of component (A) or component (B).

118. The method according to claim 112, wherein the at least two specific surfaces comprise structures wherein and independently represent connection points of units of component (A) or component (B).

119. The method according to any one of claims 111 - 118, wherein the biomolecule comprises a protein.

120. The method according to claim 119, wherein the protein comprises a targeting protein.

121. The method according to claim 119, wherein the protein comprises a vacuolar lumen, a lysosomal lumen, a spliceosome tri-snRNP complex, a U4 / U6 xU5 tri-snRNP complex, a secretory granule lumen, an intracellular organelle lumen, a membrane raft, a spliceosome snRNP complex, a sperm proteasome complex, or a Golgi lumen protein.

122. A method for preparing a surface having repeating units comprising a first component and a second component, the method comprising: a. Providing a monomer mixture comprising a first monomer and a second monomer in a solvent, wherein the first monomer comprises a vinyl group and wherein the second monomer comprises an epoxy group; b. Contacting the surface with the monomer mixture to produce a reaction mixture; c. Initiating radical polymerization to produce macromolecules immobilized on the surface; d. Contacting the macromolecules immobilized on the surface with an amine to produce aminated macromolecules; and e. Optionally, contacting the aminated macromolecules with a compound comprising a succinate ester, a phthalate ester, or a propane sulfone.

123. A method for preparing a surface having repeating units comprising a first component and a second component, the method comprising: (a) Providing a monomer mixture comprising a first monomer and a second monomer in a solvent, wherein the first monomer comprises a vinyl group and the second monomer comprises an epoxy group; (b) Contacting the surface with the monomer mixture to produce a reaction mixture; (c) Initiating radical polymerization to produce macromolecules immobilized on the surface; (d) Contacting the macromolecules immobilized on the surface with an azide salt to produce macromolecules containing azide; and (e) Optionally, contacting the macromolecules containing azide with a molecule containing an alkyne to form macromolecules containing a triazole.

124. A kit for identifying biomolecules in a biological sample, the kit comprising one or more surfaces according to any one of claims 73-110.

125. A composition for identifying biomolecules in a biological sample, the composition comprising one or more surfaces according to any one of claims 73-110 and a biological sample in contact with the surface.

126. A system for identifying biomolecules in a biological sample, the composition comprising: i. A surface according to any one of claims 73-110; ii. A suspension solution; iii. A biological sample containing a certain concentration of protein; and iv. An automated system comprising a network of units having differentiated functions in differentiating the states of complex biological samples using a plurality of surfaces having different physicochemical properties, and wherein the automated system is programmed to perform a series of steps.

127. A method for identifying a protein in a sample, the method comprising: a. Incubating one or more surfaces according to any one of claims 73-110 with a biological sample containing a biomolecule to form a unique biomolecular corona; b. Isolate at least a portion of the biomolecules in the unique biomolecular corona; and c. Determine the unique biomolecular corona.

128. The method of claim 127, wherein the determination is capable of identifying 1 to 20,000 protein groups.

129. The method of any one of claims 127-128, wherein the determination is capable of identifying 1000 to 10,000 protein groups.

130. The method of any one of claims 127-129, wherein the determination is capable of identifying 1,000 to 5,000 protein groups.

131. The method of any one of claims 127-130, wherein the determination is capable of identifying 1,200 to 2,200 protein groups.

132. The method of any one of claims 127-131, wherein the protein group comprises a peptide sequence having a minimum length of 7 amino acid residues.

133. The method of any one of claims 127-132, wherein the determination is capable of identifying 1,000 to 10,000 proteins.

134. The method of any one of claims 127-133, wherein the determination is capable of identifying 1,800 to 5,000 proteins.

135. The method of any one of claims 127-134, wherein the sample comprises a plurality of samples.

136. The method of any one of claims 127-135, wherein the plurality of samples comprises at least two or more spatially isolated samples.

137. The method of any one of claims 127-136, wherein the incubation comprises contacting the at least two or more spatially isolated samples with the one or more surfaces simultaneously.

138. The method of any one of claims 127-137, wherein isolation comprises magnetically isolating the one or more surfaces simultaneously from unbound proteins in the at least two or more spatially isolated samples of the plurality of samples.

139. The method of any one of claims 127-138, wherein the determination comprises determining a plurality of unique biomolecular coronas to simultaneously identify proteins in the at least two or more spatially isolated samples.

140. The method of any one of claims 127-139, further comprising repeating, wherein when repeated, the incubation, isolation, and determination result in a percentile normalized coefficient of variation (QNCV) of 20% or less, as determined by comparing peptide mass spectrometry features of at least three complete determination repeats from each of the one or more surfaces.

141. The method according to any one of claims 127 - 140, wherein, When repeated, the incubation, isolation, and determination result in a percentile normalized coefficient of variation (QNCV) of 10% or less, as determined by comparing peptide mass spectrometry features of at least three complete determination repeats from each of the one or more surfaces.

142. The method of any one of claims 127-141, wherein the determination is capable of identifying proteins in a dynamic range of at least 7, at least 8, at least 9, or at least 10.

143. The method according to any one of claims 127 - 142, further comprising washing the one or more surfaces at least once or at least twice after magnetically isolating the one or more surfaces from the unbound protein.

144. The method according to any one of claims 127 - 143, wherein after the assay, the method further comprises lysing the proteins in the plurality of distinct biomolecular coronas.

145. The method according to any one of claims 127 - 144, further comprising digesting the proteins in the plurality of distinct biomolecular coronas to generate digested peptides.

146. The method according to any one of claims 127 - 145, further comprising purifying the digested peptides.

147. The method according to any one of claims 127 - 146, wherein the assay comprises identifying the proteins in the sample using mass spectrometry.

148. The method according to any one of claims 127 - 147, wherein the assay is performed in about 2 to about 4 hours.

149. The method according to any one of claims 127 - 148, wherein the assay is performed in about 1 to about 20 hours.

150. The method according to any one of claims 127 - 149, wherein the assay is performed in about 2 to about 10 hours.

151. The method according to any one of claims 127 - 150, wherein the assay is performed in about 4 to about 6 hours.

152. The method according to any one of claims 127 - 151, wherein the isolation takes no more than about 30 minutes, no more than about 15 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 2 minutes.

153. The method according to any one of claims 127 - 152, wherein the plurality of samples comprises at least 10 spatially isolated samples, at least 50 spatially isolated samples, at least 100 spatially isolated samples, at least 150 spatially isolated samples, at least 200 spatially isolated samples, at least 250 spatially isolated samples, or at least 300 spatially isolated samples.

154. The method according to any one of claims 127 - 153, wherein the plurality of samples comprises at least 96 samples.

155. The method according to any one of claims 127 - 154, wherein the one or more surfaces comprise at least 2 distinct surfaces, at least 3 distinct surfaces, at least 4 distinct surfaces, at least 5 distinct surfaces, at least 6 distinct surfaces, at least 7 distinct surfaces, at least 8 distinct surfaces, at least 9 distinct surfaces, at least 10 distinct surfaces, at least 11 distinct surfaces, at least 12 distinct surfaces, at least 13 distinct surfaces, at least 14 distinct surfaces, at least 15 distinct surfaces, at least 20 distinct surfaces, at least 25 surfaces, or at least 30 distinct surfaces.

156. The method according to any one of claims 127 - 155, wherein the one or more surfaces comprise at least 10 distinct surfaces.

157. The method according to any one of claims 127 - 156, wherein the at least two spatially separated samples differ in at least one physicochemical property.

158. The method according to any one of claims 127 - 157, wherein the one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface share at least one physicochemical property and differ in at least one physicochemical property such that the first unique surface and the second unique surface are different.

159. The method according to any one of claims 127 - 158, wherein the one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface share at least two physicochemical properties and differ in at least two physicochemical properties such that the first unique surface and the second unique surface are different.

160. The method according to any one of claims 127 - 159, wherein the one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface share at least one physicochemical property and differ in at least two physicochemical properties such that the first unique surface and the second unique surface are different.

161. The method according to any one of claims 127 - 160, wherein the one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface share at least two physicochemical properties and differ in at least one physicochemical property such that the first unique surface and the second unique surface are different.

162. The method according to any one of claims 127 - 161, wherein the physicochemical property comprises size, charge, core material, shell material, porosity, or surface hydrophobicity.

163. The method according to any one of claims 127 - 162, wherein the size is a diameter or a radius measured by dynamic light scattering, SEM, TEM, or any combination thereof.

164. The method according to any one of claims 127 - 163, wherein the one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface comprise a carboxylate material, wherein the first unique particle is a microparticle, and wherein the second unique surface is a nanoparticle.

165. The method according to any one of claims 127 - 164, wherein the one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface have a surface charge of 0 mV to - 50 mV, wherein the first unique surface has a diameter of less than 200 nm, and wherein the second unique surface has a diameter of greater than 200 nm.

166. The method according to any one of claims 127 - 165, wherein the one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface have a diameter of 100 to 400 nm, wherein the first unique surface has a positive surface charge, and wherein the second unique surface has a neutral surface charge.

167. The method according to any one of claims 127 - 166, wherein the one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface are nanoparticles, wherein the first unique surface has a surface charge less than -20 mV, and the second unique surface has a surface charge greater than -20 mV.

168. The method according to any one of claims 127 - 167, wherein the one or more surfaces comprise a first unique surface and a second unique surface, wherein the first unique surface and the second unique surface are microparticles, wherein the first unique surface has a negative surface charge, and wherein the second unique surface has a positive surface charge.

169. The method according to any one of claims 127 - 168, wherein the one or more surfaces comprise a subset of negatively charged nanoparticles, wherein each particle in the subset is distinct in at least one surface chemical group.

170. The method according to any one of claims 127 - 169, wherein the one or more surfaces comprise a first unique surface, a second particle, and a third unique surface, wherein the first unique surface, the second unique surface, and the third unique surface comprise an iron oxide core, a polymer shell, and have a diameter less than about 500 nm, and wherein the first unique surface comprises a negative charge, the second unique surface comprises a positive charge, and the third unique surface comprises a neutral charge, wherein the diameter is the average diameter as measured by dynamic light scattering.

171. The method according to any one of claims 127 - 170, wherein at least one unique surface of the one or more surfaces is a nanoparticle.

172. The method according to any one of claims 127 - 171, wherein at least one unique surface of the one or more surfaces is a microparticle.

173. The method according to any one of claims 127 - 172, wherein at least one unique surface of the one or more surfaces is a superparamagnetic iron oxide particle.

174. The method according to any one of claims 127 - 173, wherein each particle of the one or more surfaces comprises an iron oxide material.

175. The method according to any one of claims 127 - 174, wherein at least one unique surface of the one or more surfaces has an iron oxide core.

176. The method according to any one of claims 127 - 175, wherein at least one unique surface of the one or more surfaces has an iron oxide crystal embedded in a polystyrene core.

177. The method according to any one of claims 127 - 176, wherein each unique surface of the one or more surfaces is a superparamagnetic iron oxide particle.

178. The method according to any one of claims 127 - 177, wherein each unique surface of the one or more surfaces comprises an iron oxide core.

179. The method according to any one of claims 127 - 178, wherein each unique surface of the one or more surfaces has iron oxide crystals embedded in a polystyrene core.

180. The method according to any one of claims 127 - 179, wherein at least one unique surface of the one or more surfaces comprises a carboxylated polymer, an aminated polymer, a zwitterionic polymer, or any combination thereof.

181. The method according to any one of claims 127 - 180, wherein at least one surface of the one or more surfaces comprises an iron oxide core having a silica shell coating.

182. The method according to any one of claims 127 - 181, wherein at least one unique surface of the one or more surfaces comprises a negative surface charge.

183. The method according to any one of claims 127 - 182, wherein at least one unique surface of the one or more surfaces comprises a positive surface charge.

184. The method according to any one of claims 127 - 183, wherein at least one unique surface of the one or more surfaces comprises a neutral surface charge.

185. A surface for adsorbing biomolecules from a biological sample, wherein the surface is functionalized with carboxylic acid and thiol, and wherein the functionalization promotes the adsorption of biomolecules upon contact with the biological sample.

186. A method, comprising: (a) Contacting a biological sample with the surface according to claim 185 such that biomolecules are adsorbed onto the surface, wherein the surface is functionalized with carboxylic acid and thiol; (b) Separating the biological sample from the surface; and (c) Analyzing the biomolecules adsorbed to the surface.

187. A kit for identifying biomolecules in a biological sample, the kit comprising the surface according to claim 185.

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