IMMUNO-PCR AND NEXT GENERATION SEQUENCING FOR PRECISE ANTIGEN QUANTITATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Application No. 63/376,965, filed on September 23, 2022, the contents of which are herein incorporated by reference in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SLIN_014_01WO_SeqList_ST26.xml; Size: 3,514 bytes; and Date of Creation: September 21, 2023) are herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to polymer beads comprising (i) biomarkers and (ii) nucleic acids comprising: (a) a primer binding site; and (b) a unique molecular identifier; wherein the nucleic acids are bound to the biomarker; and wherein each nucleic acid on the polymer bead comprises a different UMI. The present disclosure also provides methods for quantitatively measuring biomarker concentration in a sample.

BACKGROUND

[0004] There is a need in the art for methods to quantify the number of antigens bound to a cell, bead, or surface. In medicine, quantifying the number of antigens on a cell surface may aid in the diagnosis of disease. For example, the amount of cell surface epithelial cell adhesion molecule (EpCAM) is associated with epithelial cancers and may be a useful tool for staging cancer. Mentink. Sci. Rep. 2023 Apr 13; 13(l):6051. Additionally, knowledge of the number of antigens on a cell, bead, or surface allows for optimal fluorophore selection in a flow cytometry experiment. For example, when brighter fluorophores are used to detect antigens that are present on a cell in low density and dimmer fluorophores are used to detect antigens present on a cell in higher density, fluorescent spill-over can be reduced.

[0005] Current flow cytometry based methods for determining the number of antigens bound to a cell, bead, or surface are limited by the requirement for a photosensitive fluorophore and by cell autofluorescence. SUMMARY

[0006] Provided herein are methods of quantitatively measuring biomarker concentration in a sample, comprising: (i) providing a sample comprising a first population of target biomarkers bound to a first population of nucleic acids, each of said nucleic acids comprising: (a) a primer binding site; and (b) a unique molecular identifier (UMI); wherein each nucleic acid in the first population of nucleic acids comprises a different UMI; (ii) amplifying the nucleic acids comprising the UMI; and (iii) sequencing the amplified nucleic acids; wherein the total number of different UMIs detected corresponds to the number of target biomarkers in the sample.

[0007] Provided herein are methods of quantitatively measuring biomarker concentration in a sample comprising a target biomarker, the method comprising: (i) contacting the sample with a first population of nucleic acids capable of selectively binding to the target biomarker; each of said nucleic acids comprising: (a) a primer binding site; and (b) a unique molecular identifier (UMI); wherein each nucleic acid in the first population of nucleic acids comprises a different UMI; (ii) separating unbound nucleic acids from the sample; (iii) amplifying the nucleic acids comprising the UMI after step (ii); and (iv) sequencing the amplified nucleic acids; wherein the total number of different UMIs detected corresponds to the number of target biomarkers in the sample.

[0008] Provided herein ae compositions comprising: (i) a plurality of target biomarkers; and (ii) a plurality of nucleic acids comprising: (a) a primer binding site; and (b) a unique molecular identifier (UMI); wherein the nucleic acids are bound to the biomarker; and wherein each nucleic acid in the population comprises a different UMI. In embodiments, the composition comprises a polymer bead. In embodiments, the polymer bead is a hydrogel bead.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying figures, which are incorporated herein and form a part of the specification, illustrate some, but not the only or exclusive, example embodiments and/or features. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

[0010] Figs. 1-3 illustrate the tuning of the optical properties of hydrogel beads to match the optical properties of a target cell. Fig. 1 shows that polymer beads can be modulated to match the forward scatter and side scatter of target cells, such as granulocytes, monocytes, and lymphocytes. Fig. 2 shows how side scatter (SSC) and forward scatter (FSC) optical properties can be independently modulated. Fig. 3 illustrates further polymer bead tuning via addition of selected biomarkers or labels. [0011] Fig. 4 shows a schematic of a polymer bead comprising a biomarker (labeled “Pl”) bound to a deoxyribonucleotide (labeled “DNA1,” “DNA2,” or “DNA3”) via an antibody linker (“Y”). Each deoxyribonucleotide contains a different unique molecular identifier (“UMI”).

[0012] Figs. 5-6 illustrates the quantitation method described in Example 3. Fig. 5 shows binding of a hydrogel bead comprising a biomarker (labeled “Pl”) to a deoxyribonucleotide (labeled “DNA1,” “DNA2,” or “DNA3”) via an antibody linker (“Y”). Each deoxyribonucleotide contains a different unique molecular identifier (“UMI”). Fig. 6 shows how the quantitation method of Example 3 allows for the identification of the number of biomarkers on the surface of the bead.

DETAILED DESCRIPTION

[0013] The present disclosure provides polymer beads (Section II) and methods of quantitatively measuring biomarker concentration in a sample (Section III). Advantageously, the methods provided herein allow for a determination of the absolute number of a target biomarker in a sample. The methods provided herein also allow for determination of the absolute number of antibodies bound to a sample, for example, a bead, a cell, or a population of cells. A determination of the absolute number of biomarkers in a sample is useful for determining disease state as the number of biomarkers in a sample is often correlated with disease.

I. Definitions

[0014] As used herein, the indefinite articles “a” and “an” and the definite article “the” are intended to include both the singular and the plural, unless the context in which they are used clearly indicates otherwise. “At least one” and “one or more” are used interchangeably to mean that the article may include one or more than one of the listed elements.

[0015] As used herein, the terms “polymer bead” and “polymer particle” may be used interchangeably.

[0016] “Substantially similar,” as used herein, denotes at least 40% similar, at least 50% similar, at least 60% similar, at least 70% similar, at least 80% similar, at least 90% similar, at least 95% similar, at least 96% similar, at least 97% similar, at least 98% similar or at least 99% similar.

[0017] Unless otherwise indicated, it is to be understood that all numbers expressing quantities, ratios, and numerical properties of ingredients, reaction conditions, and so forth, used in the specification and claims are contemplated to be able to be modified in all instances by the term “about”. For instance, throughout this application, the term “about” may be used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0018] As used herein the term “sequence identity” refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of residues, e.g. nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical residues which are shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e. the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. Comparison of sequences to determine percent identity can be accomplished by a number of well-known methods, including for example by using mathematical algorithms, such as, for example, those in the BLAST suite of sequence analysis programs. Unless noted otherwise, the term “sequence identity” in the claims refers to sequence identity as calculated by Clustal Omega® using default parameters.

[0019] The term “hydrogel” refers to a material comprising a macromolecular three- dimensional network that allows it to swell when in the presence of water (i.e., the “hydrated state"), to shrink in the absence of (or by reduction of the amount of) water (i.e., the “dehydrated state”), but not dissolve in water. As used herein, the term “hydrogel” refers to the material in either its hydrated or dehydrated state. The swelling, or absorption of water, is a consequence of the presence of hydrophilic functional groups attached to or dispersed within the macromolecular network. Crosslinks between adjacent macromolecules result in the aqueous insolubility of these hydrogels. The cross-links may be due to chemical (i.e., covalent) or physical (i.e., VanDer Waal forces, hydrogen-bonding, ionic forces, etc.) bonds. These chemical crosslinks may also be hydrolyzed under certain conditions, reversing the insolubility of the hydrogel. Multiple chemical crosslinking chemistries are described in the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools, lifetechnologies, com/content/sfs/brochures/ 1602163- Crosslinking-Reagents- Handbook.pdf, the disclosure of which is incorporated by reference in its entirety for all purposes.).

[0020] The term “binding” “bound” and similar variants refers to the association of two molecules via covalent or noncovalent interactions. Elements that are bound need not be in direct contact, but can instead be bound through one or more structures. For example, in embodiments, two elements are bound via a linker. For example, a nucleic acid may be bound to a target biomolecule through a linker that selectively binds to the target biomolecule.

[0021] The terms “amplicon” and “amplification product” as used herein generally refers to the product of an amplification reaction. An amplicon can be double-stranded or singlestranded, and can include the separated component strands obtained by denaturing a doublestranded amplification product. In some embodiments, an amplicon comprises a ligation product (for example but not limited to a ligated probe), the complement of at least part of a ligation product, or both. In certain embodiments, the amplicon of one amplification cycle can serve as a template in a subsequent amplification cycle.

[0022] The terms “annealing” and “hybridizing”, including without limitation variations of the root words hybridize and anneal, are used interchangeably and mean the nucleotide basepairing interaction of one nucleic acid with another nucleic acid that results in the formation of a duplex, triplex, or other higher-ordered structure. Conditions under which primers and probes anneal to complementary sequences are well known in the art, e.g., as described in Nucleic Acid Hybridization, A Practical Approach, Hames and Higgins, eds., IRL Press, Washington, D.C. (1985) and Wetmur and Davidson, Mol. Biol. 31 :349, 1968. Such conditions, however, can be routinely determined by persons of ordinary skill in the art, without undue experimentation. Preferably, annealing conditions are selected to allow the primers and/or probes to selectively hybridize with a complementary sequence in the corresponding target flanking sequence or amplicon, but not hybridize to any significant degree to different target nucleic acids or non-target sequences in the reaction composition at the second reaction temperature.

[0023] The term “selectively hybridize” and variations thereof means that, under appropriate stringency conditions, a given sequence (for example but not limited to a primer) anneals with a second sequence comprising a complementary string of nucleotides (for example but not limited to a target flanking sequence or a primer-binding site of an amplicon), but does not anneal to undesired sequences, such as non-target nucleic acids, probes, or other primers. Typically, as the reaction temperature increases toward the melting temperature of a particular double-stranded sequence, the relative amount of selective hybridization generally increases and mis-priming generally decreases. In this specification, a statement that one sequence hybridizes or selectively hybridizes with another sequence encompasses situations where the entirety of both of the sequences hybridize or selectively hybridize to one another, and situations where only a portion of one or both of the sequences hybridizes or selectively hybridizes to the entire other sequence or to a portion of the other sequence.

[0024] As used herein, the term “stringency” is used to define the temperature and solvent composition existing during hybridization and the subsequent processing steps at which a hybrid comprised of two complementary nucleotide sequences will form. Stringency also defines the amount of homology, the conditions necessary, and the stability of hybrids formed between two nucleotide sequences. As the stringency conditions increase, selective hybridization is favored and non-specific cross-hybridization is disfavored. Increased stringency conditions typically correspond to higher incubation temperatures, lower salt concentrations, and/or higher pH, relative to lower stringency conditions at which mis-priming, including without limitation, the mis-annealing of ligation probes and/or cleavage probes, is more likely to occur. Those in the art understand that appropriate stringency conditions to enable the selective hybridization of a primer or primer pair, a ligation probe pair, and/or a cleavage probe pair to a corresponding target flanking sequence and/or amplicon can be routinely determined using well known techniques and without undue experimentation (see, e.g., PCR: The Basics from background to bench, McPherson and Moller, Bios Scientific Publishers (2000; hereinafter “McPherson”)).

[0025] In this specification, a statement that one nucleic acid sequence is the same as or substantially the same as another nucleotide sequence encompasses situations where both of the nucleotide sequences are completely the same as or substantially the same as the other sequence, and situations where only a portion of one of the sequences is the same as or substantially the same as a portion of the entire other sequence. Likewise, a statement that one nucleic acid sequence is complementary to or substantially complementary to another nucleotide sequence encompasses situations where both of the nucleotide sequences are completely complementary or substantially complementary to one another, and situations where only a portion of one of the sequences is complementary to or substantially complementary to a portion of the entire other sequence.

[0026] The term “aptamer” as used herein refers to a DNA or RNA oligonucleotide that: 1) is typically identified originally using an in vitro selection process, for example but not limited to the “systematic evolution of ligands by exponential enrichment” (SELEX) process or a variation thereof, and 2) recognizes and binds to a binding partner, for example but not limited to an enzyme, biomarker, or antibody, in a highly specific, conformation-dependent manner.

[0027] The term “combinations thereof’ or “combination thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. [0028] As used herein, the terms “complementary” and “complementarity” are used in reference to at least two nucleic acids that are related by the base-pairing rules. For example but without limitation, the sequence “A-C-T” is complementary to the sequence “T-G-A ” Complementarity may be partial, in which case only some of the nucleotides are matched according to the base-pairing rules. Or, there may be complete or total complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between the nucleic acid strands. Complementarity need not be total for a stable duplex to form, i.e., stable duplexes may contain mismatched base pairs or unmatched bases. Those in the art can determine duplex stability empirically considering a number of variables including without limitation, the length of the nucleic acid, base composition and sequence of the nucleic acid, ionic strength, and incidence of mismatched base pairs. The stability of a nucleic acid duplex is typically measured by its melting temperature.

[0029] The term “corresponding” as used herein refers to at least one specific relationship between the elements to which the term relates. For illustration purposes but not as a limitation, at least one forward primer of a particular primer pair corresponds to at least one reverse primer of the same primer pair; at least one primer is designed to anneal with the flanking region of the corresponding target nucleic acid and/or the primer-binding portion of at least one corresponding amplicon; a first probe of a ligation probe set anneals to a target nucleic acid and/or an amplicon upstream of, and typically adjacent to, the ligation site and the corresponding second ligation probe anneals to the target nucleic acid and/or an amplicon downstream of, and typically adjacent to, the ligation site; and so forth.

[0030] The terms “denaturing” and “denaturation” as used herein refer to any process in which a double-stranded polynucleotide, including without limitation, a gDNA fragment comprising at least one target nucleic acid, a double-stranded amplicon, or a polynucleotide comprising at least one double-stranded segment, at a first temperature, is converted to two single-stranded polynucleotides or to a single-stranded or substantially single-stranded polynucleotide, as appropriate. Denaturing a double-stranded polynucleotide or a double-stranded segment includes without limitation, a variety of thermal and chemical techniques which render a double-stranded nucleic acid or a double-stranded segment single-stranded or substantially single-stranded, for example but not limited to, releasing the two individual single-stranded components of a double-stranded polynucleotide or a duplex comprising two oligonucleotides. Those in the art will appreciate that the denaturing technique employed is generally not limiting unless it substantially interferes with a subsequent annealing or enzymatic step of an amplification reaction or, in certain methods, the detection of a fluorescent signal.

[0031] The term “double-stranded,” as used herein refers to one or two nucleic acid strands that have hybridized along at least a portion of their lengths. Thus, in certain contexts, “doublestranded” can refer to a portion of a single oligonucleotide that can fold so that at least one segment of the first region of the oligonucleotide hybridizes to at least one segment of the third region of the same oligonucleotide, at least one segment of the fourth region of the oligonucleotide hybridizes with at least one segment of the sixth region of the oligonucleotide, or both, thereby forming one or more double-stranded segments and one or more singlestranded portions. Hence, a single nucleic acid strand can form hairpin or stem-loop conformations that have double-stranded and single-stranded segments (see, e.g., FIG. 1). Similarly, two complementary oligonucleotides can hybridize with each other to form a duplex (see, e.g., FIG. 2). Hence, “double-stranded” does not mean that a nucleic acid must be entirely double-stranded. Instead, a double-stranded nucleic acid can have one or more single-stranded segment and one or more double-stranded segment.

[0032] As used herein, the term “Tm” is used in reference to melting temperature. The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.

[0033] As used herein, the term “primer-binding site” refers to a region of a polynucleotide sequence, typically a target nucleic acid and/or an amplicon that can serve directly, or by virtue of its complement, as the template upon which a primer can anneal for any suitable primer extension reaction known in the art, for example but not limited to, PCR. It will be appreciated by those of skill in the art that when two primer-binding sites are present on a single polynucleotide, the orientation of the two primer-binding sites is generally different. For example, one primer of a primer pair is complementary to and can hybridize with to the first primer-binding site, while the corresponding primer of the primer pair is designed to hybridize with the complement of the second primer-binding site. Stated another way, in some embodiments the first primer-binding site can be in a sense orientation, and the second primerbinding site can be in an antisense orientation. A primer-binding site of an amplicon may, but need not comprise the same sequence as or at least some of the sequence of the target flanking sequence or its complement.

[0034] Those in the art understand that as a target nucleic acid and/or an amplification product is amplified by certain amplification means, the complement of the primer-binding site is synthesized in the complementary amplicon or the complementary strand of the amplicon. Thus, it is to be understood that the complement of a primer-binding site is expressly included within the intended meaning of the term primer-binding site, as used herein.

[0035] As used herein, the term “probe-binding site” refers to a region of a polynucleotide sequence, typically a target nucleic acid and/or an amplicon that can serve directly, or by virtue of its complement, as the template upon which probe can anneal. It will be appreciated by those of skill in the art that the probe-binding site for a ligation probe pair comprise an upstream probe-binding site and a downstream probe binding site and that these two sites are typically adjacent to each other. In certain embodiments, the upstream ligation probe-binding site and the downstream probe-binding site are not adjacent to each other and an amplifying step can comprises a gap-filling reaction. It will also be appreciated by those of skill in the art that the probe-binding site for a cleavage probe pair comprises an upstream probe-binding site that is adjacent to, and may but need not overlap at least part of the downstream cleavage probebinding site.

[0036] Those in the art understand that as a target nucleic acid and/or an amplification product is amplified by certain amplification means, the complement of the probe-binding site is synthesized in the complementary amplicon or the complementary strand of the amplicon. Thus, it is to be understood that the complement of a probe-binding site is expressly included within the intended meaning of the term probe-binding site, as used herein.

[0037] As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably and refer to single-stranded and double-stranded polymers of nucleotide monomers, including without limitation 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, or intemucleotide analogs, and associated counter ions, e.g., H ⁺ , NH4 ⁺ , trialkylammonium, Mg ²⁺ , Na ⁺ , and the like. A polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof and can include nucleotide analogs. The nucleotide monomer units may comprise any of the nucleotides described herein, including, but not limited to, nucleotides and/or nucleotide analogs. Polynucleotides typically range in size from a few monomeric units, e.g. 5-40 when they are sometimes referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units. Unless denoted otherwise, whenever a polynucleotide sequence is represented, it will be understood that the nucleotides are in 5' to 3' order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytosine, “G” denotes deoxyguanosine, “T” denotes thymidine, and “U” denotes deoxyuridine, unless otherwise noted.

[0038] The term “quencher” as used herein refers to a moiety that absorbs at least some of the intensity of a fluorescent emission. Quenchers can be categorized as fluorescent quenchers and dark quenchers (sometimes also referred to as non-fluorescent quenchers). A fluorescent quencher is a moiety, typically a fluorophore, that can absorb the fluorescent signal emitted from a source of fluorescence at a first wavelength, for example but not limited to, a nucleic acid dye associated with a double-stranded segment of nucleic acid, and after absorbing enough fluorescent energy, the fluorescent quencher can emit fluorescence at a second wavelength that is characteristic of the quencher, a process termed “fluorescent resonance energy transfer” or FRET. For example but not as a limitation, the FAM fluorophore associated with a TAMRA fluorescent quencher can be illuminated at 492 nm, the excitation peak for FAM, and emit fluorescence at 580 nm, the emission peak for TAMRA. A dark quencher, appropriately paired with a source of fluorescence, absorbs the fluorescent energy from the source, but does not itself fluoresce. Rather, the dark quencher dissipates the absorbed energy, typically as heat. In certain embodiments, a dark quencher comprises a chromophore that acts as an energy transfer acceptor from a fluorescent source, but does not emit a detectable fluorescent signal of its own. Non-limiting examples of dark or non-fluorescent quenchers include DABCYL (4-(4'- dimethylaminophenylazo) sulfonic acid); Black Hole Quenchers series quenchers, for example but not limited to BHQ-1, BHQ-2, and BHQ-3; Iowa Black; QSY series quenchers, for example but not limited to QSY-7; AbsoluteQuencher; Eclipse non-fluorescent quencher; nanocrystals for example but not limited to quantum dots; metals such as gold nanoparticles; and the like.

[0039] As used herein, the term “reaction vessel” generally refers to any container, chamber, device, or assembly, in which a reaction can occur in accordance with the present teachings. In some embodiments, a reaction vessel can be a microtube, for example but not limited to a 0.2 mL or a 0.5 mL reaction tube such as a MicroAmp® Optical tube (Applied Biosystems) or a micro-centrifuge tube, or other containers of the sort in common practice in molecular biology laboratories. In some embodiments, a reaction vessel comprises a well of a multi-well plate, a spot on a glass slide, or a channel or chamber of a microfluidics device, including without limitation an Applied Biosystems TaqMan Low Density Array. For example but not as a limitation, a plurality of reaction vessels can reside on the same support. In some embodiments, lab-on-a-chip like devices, available for example from Caliper and Fluidgm, can serve as reaction vessels in the disclosed methods. It will be recognized that a variety of reaction vessels are commercially available or can be designed for use in the context of the present teachings. [0040] The term “reporter group” is used in a broad sense herein and refers to any identifiable tag, label, or moiety.

[0041] The term “small RNA molecule” is used in a broad sense herein and refers to any nucleic acid sequence comprising ribonucleotides that are non-coding and typically have a length of: 150 nucleotides or less, 100 nucleotides or less, 75 nucleotides or less, 30 nucleotides or less, between 19 and 27 nucleotides, and between 21 and 23 nucleotides. A small RNA molecule can be single-stranded, double-stranded, or can comprise at least one single-stranded region and at least one double-stranded region, including without limitation, stem-loop or hairpin structures. Non-limiting examples of small RNA molecules include untranslated functional RNA, non-coding RNA (ncRNA), small non-messenger RNA (snmRNA), small interfering RNA (siRNA), tRNA, tiny non-coding RNA (tncRNA), small modulatory RNA (smRNA), snoRNA, stRNA, snRNA, microRNA (miRNA) including without limitation miRNA precursors such as primary miRNA (pri -miRNA) and precursor miRNA (pre-miRNA), and small interfering RNA (siRNA) (see, e.g., Eddy, Nature Reviews Genetics 2:919-29 (2001); Storz, Science 296:1260-63 (2002); Buckingham, Horizon Symposia: Understanding the RNAissance: l-3 (2003)). In certain embodiments, a target nucleic acid comprises a small RNA molecule.

[0042] The term “thermostable” when used in reference to an enzyme, indicates that the enzyme is functional or active (i.e., can perform catalysis) at an elevated temperature, for example but not limited to, at about 55° C. or higher. Thermostable enzymes that may be suitable for use in the current teachings are commercially available from various vendors, including without limitation, Applied Biosystems (Foster City, Calif.), Promega (Madison, Wis.), Stratagene (LaJolla, Calif.), and New England BioLabs (Beverly, Mass.). Those in the art will understand that thermostable enzymes can be isolated from a variety of thermophilic and/or hyperthermophilic organisms, for example but not limited to, certain species of eubacteria and archaea, including without limitation, certain viruses that infect such organisms and that such thermostable enzymes may be suitable for use in the disclosed complexes, methods, and kits. [0043] The terms “universal base” or “universal nucleotide” are generally used interchangeably herein and refer to a nucleotide analog that can substitute for more than one species of naturally-occurring nucleotide in a polynucleotide. Universal bases typically contain an aromatic ring moiety that may or may not contain nitrogen atoms and generally use aromatic ring stacking to stabilize a duplex. In certain embodiments, a universal base may be covalently attached to the C-l' carbon of a pentose sugar to make a universal nucleotide. In certain embodiments, a universal base does not hydrogen bond specifically with another nucleotide base. In certain embodiments, a nucleotide base may interact with adjacent nucleotide bases on the same nucleic acid strand by hydrophobic stacking. Non-limiting examples of universal nucleotides and universal bases include deoxy-7-azaindole triphosphate (d7AITP), deoxyisocarbostyril triphosphate (dICSTP), deoxypropynylisocarbostyril triphosphate (dPICSTP), deoxymethyl-7-azaindole triphosphate (dM7AITP), deoxylmPy triphosphate (dlmPyTP), deoxyPP triphosphate (dPPTP), deoxypropynyl-7-azaindole triphosphate (dP7AITP), 3-methyl isocarbostyril (MICS), 5-methyl isocarbyl (5MICS), imidazole-4- carboxamide, 3 -nitropyrrole, 5-nitroindole, hypoxanthine, inosine, deoxyinosine, 5- fluorodeoxyuridine, 4-nitrobenzimidazole, and certain PNA-bases, including without limitation certain pseudocomplementary PNA (pcPNA) bases. Descriptions of universal bases can be found in, among other places, Loakes, Nucl. Acids Res. 29:2437-47 (2001); Berger et al., Nucl. Acids Res. 28:2911-14 (2000); Loakes et al., J. Mol. Biol. 270:426-35 (1997); Verma and Eckstein, Ann. Rev. Biochem. 67:99-134 (1998); Published PCT Application No. US02/33619, and Patron and Pervin, U.S. Pat. No. 6,433,134.

[0044] The term “DNA polymerase” is used in a broad sense herein and refers to any polypeptide that can catalyze the 5 '-3 'extension of a hybridized primer by the addition of deoxyribonucleotides and/or certain nucleotide analogs in a template-dependent manner. For example but not limited to, the sequential addition of deoxyribonucleotides to the 3 '-end of a primer that is annealed to a nucleic acid template during a primer extension reaction. Nonlimiting examples of DNA polymerases include RNA-dependent DNA polymerases, including without limitation reverse transcriptases, and DNA-dependent DNA polymerases. It is to be appreciated that certain DNA polymerases (for example but not limited to certain eubacterial Type A DNA polymerases and Taq DNA polymerase) may further comprise a structurespecific nuclease activity and that when an amplification reaction comprises an invasive cleavage reaction, for example but not limited to, FEN-LCR or PCR-FEN (see, e.g., Bi et al., U.S. Pat. No. 6,511,810; and Neville et al., BioTechniques 32:S34-43 (2002)), wherein the cleaving enzyme comprises a DNA polymerase, such polymerase is referred to herein as a cleaving enzyme in the invasive cleavage context and the corresponding enzymatic activity comprises structure-specific oligonucleotide cleavage. In certain embodiments, a DNA polymerase provides both a polymerization activity and a structure-specific cleaving activity. The term “RNA polymerase” refers to a DNA-dependent RNA polymerase or an RNA- dependent polymerase (sometimes referred to as an RNA replicase), and includes any polypeptide that can catalyze the 5 '-3' addition of ribonucleotides in a template-dependent manner. In certain embodiments, an RNA polymerase binds to a promoter sequence and catalyzes transcription. Non-limiting examples of RNA polymerases include the RNA polymerases from the bacteriophages T3, T7, SP6, f2, MS2, and Qp.

[0045] The term “primer” refers to a polynucleotide, generally an oligonucleotide comprising a “target” binding portion that is typically about 12 to about 35 nucleotides long, that is designed to selectively hybridize with a target nucleic acid flanking sequence or to a corresponding primer-binding site of an amplification product under appropriate stringency conditions; and serve as the initiation point for the synthesis of a nucleotide sequence that is complementary to the corresponding polynucleotide template from its 3 '-end.

[0046] The terms “forward” and “reverse” when used in reference to the primers of a primer pair indicate the relative orientation of the primers on a polynucleotide sequence. For illustration purposes but not as a limitation, consider a single-stranded polynucleotide drawn in a horizontal, left to right orientation with its 5 '-end on the left. The “reverse” primer is designed to anneal with the downstream primer-binding site at or near the “3 '-end” of this illustrative polynucleotide in a 5' to 3' orientation, right to left. The corresponding “forward primer is designed to anneal with the complement of the upstream primer-binding site at or near the “5 '-end” of the polynucleotide in a 5' to 3' “forward” orientation, left to right. Thus, the reverse primer comprises a sequence that is complementary to the reverse or downstream primer-binding site of the polynucleotide and the forward primer comprises a sequence that is the same as or substantially the same as the forward or upstream primer-binding site. It is to be understood that the terms “3-end” and “5'-end” as used in this paragraph are illustrative only and do not necessarily refer literally to the respective ends of the polynucleotide. Rather, the only limitation is that the reverse primer of this exemplary primer pair anneals with a reverse primer-binding site that is downstream of the forward primer-binding site that comprises the same sequence or substantially the same sequence as the “target” binding portion of the corresponding forward primer. As will be recognized by those of skill in the art, these terms are not intended to be limiting, but rather to provide illustrative orientation in a given embodiment. [0047] A “primer pair” of the current teachings comprises a forward primer and a corresponding reverse primer. The forward primer comprises a first target-specific portion that comprises a sequence that is the same as or substantially the same as the nucleotide sequence of the first or upstream target flanking sequence, and that is designed to selectively hybridize with the complement of the upstream target flanking sequence that is present in, among other places, the reverse amplification product. The reverse primer of the primer pair comprises a second target-specific portion that comprises a sequence that is complementary to or substantially complementary to, and that is designed to selectively hybridize with, the second or downstream target region flanking sequence that is present in among other places, the forward amplification product. In certain embodiments, a forward primer, a reverse primer, or a forward primer and a reverse primer of a primer pair further comprises a reporter-probe binding site, a universal primer-binding site, and/or a reporter group, for example but not limited to a fluorescent reporter group. In some embodiments, a sequencing primer comprises a fluorescent reporter group. In certain embodiments, a forward primer and the corresponding reverse primer of a primer pair have different melting temperatures to permit temperaturebased asymmetric PCR.

[0048] A universal primer or primer set may be employed according to certain embodiments of the current teachings. In certain embodiments, a universal primer or a universal primer set hybridizes with and can be used to amplify two or more different target nucleic acid species and/or two or more different species of desired amplicon.

[0049] The term “probe” refers to a polynucleotide that comprises a portion that is designed to hybridize in a sequence-specific manner with a complementary probe-binding site on a particular nucleic acid sequence, for example but not limited to a target nucleic acid or an amplification product. In certain embodiments, corresponding probes of a ligation probe set are ligated together to form a ligated probe. In some embodiments, corresponding probes of a cleavage probe set anneal with a template strand to form a nucleic acid cleavage structure, which can be cleaved by an appropriate cleaving enzyme under suitable conditions to form a hybridization structure comprising the template strand, the upstream cleavage probe, and a hybridized fragment of the second cleavage probe. In certain embodiments, the annealed upstream cleavage probe and the hybridized fragment of the downstream cleavage probe are ligated together to form a ligated probe. In certain embodiments, a probe comprises a reporter group, for example but not limited to, a reporter probe. In some embodiments, a probe comprises a primer-binding site. [0050] The term “reporter probe” refers to a sequence of nucleotides and/or nucleotide analogs, that anneals with a target nucleic acid and/or an amplicon, and when detected, including but not limited to a change in intensity or of emitted wavelength, is used to identify and/or quantify the corresponding target nucleic acid in an end-point or real-time detection technique, for example but not limited to a Q-PCR technique. Most reporter probes can be categorized based on their mode of action, for example but not limited to: nuclease probes, including without limitation TaqMan® probes (see, e.g., Livak, Genetic Analysis: Biomolecular Engineering 14: 143-149 (1999); Yeung et al., BioTechniques 36:266-75 (2004)); extension probes such as scorpion primers, Lux™ primers, Amplifluors, and the like; hybridization probes such as molecular beacons, Eclipse probes, light-up probes, pairs of singly-labeled reporter probes, hybridization probe pairs, and the like; or combinations thereof. In certain embodiments, reporter probes comprise a PNA, an LNA, a universal base, or combinations thereof, and can include stem-loop and stem-less reporter probe configurations. Certain reporter probes are singly-labeled, while other reporter probes are doubly-labeled. Dual probe systems that comprise FRET between adjacently hybridized probes are within the intended scope of the term reporter probe (see, e.g., Zhang et al., Hepatology 36:723-28 (2003)).

[0051] The term “target nucleic acid” or “target” refers to the nucleic acid sequence that is specifically amplified and/or detected using the compositions, methods, and kits of the present teachings (in contrast to a secondary amplification product, which is the result of a spurious side-reaction, typically due to mis-priming). In certain embodiments, a target nucleic acid serves as a template in a primer extension reaction. In some embodiments, a target nucleic acid serves as a ligation template. In some embodiments, a target nucleic acid serves as a template strand in a nucleic acid cleavage structure. In certain embodiments, the target nucleic acid comprises DNA and is present in genomic DNA (gDNA) or mitochondrial DNA (mtDNA). In certain embodiments, the target nucleic acid comprises RNA, for example but not limited to, ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), or an RNA molecule such as a miRNA precursor, including without limitation, a pri-miRNA, a pre- miRNA, or a pri-miRNA and a pre-miRNA. In some embodiments, the target nucleic acid comprises a small RNA molecule, including without limitation, a miRNA, a siRNA, a stRNA, a snoRNA, or other ncRNA. The target nucleic acid need not constitute the entirety of a nucleic acid molecule. For example but not as a limitation, a large nucleic acid, for example a gDNA fragment, can comprise a multiplicity of different target nucleic acids. Typically, a target nucleic acid has at least one defined end. In many nucleic acid amplification reactions the target has two defined ends. [0052] The terms “amplifying” and “amplification” are used in a broad sense and refer to any technique known in the art in which a target nucleic acid, an amplicon, at least part of a target nucleic acid, or at least part of an amplicon, is reproduced or copied (including the synthesis of a complementary strand or the formation of a ligation probe), typically in a templatedependent manner, including a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Some amplifying techniques are performed isothermally; some amplification techniques are performed using temperature cycling; some amplification techniques comprise at least one isothermal amplifying step and at least one amplifying step comprising thermocycling. Some non-limiting examples of amplification techniques include primer extension, including without limitation PCR, RT-PCR, asynchronous PCR (A-PCR), asymmetric PCR, quantitative or Q-PCR; ligase chain reaction (LCR), ligase detection reaction (LDR), including without limitation gap-filling and gap oligonucleotide versions of each (see, e.g., Cao, Chapter 1.3 in DNA Amplification: Current Techniques and Applications, Demidov and Broude, eds., Horizon Bioscience (2004; hereinafter “Demidov and Broude”); Abravaya et al., Nucl. Acids Res. 23:675-82 (1995); Lizardi et al., Nat. Genetics 19:225-32 (1998); and Segev, U.S. Pat. No. 6,004,826); rolling circle amplification (RCA), sometimes referred to as rolling circle replication (RCR); strand displacement amplification (SDA) and multiple displacement amplification (MDA); nucleic acid strand-based amplification (NASBA), sometimes referred to as transcription-mediated amplification (TMA) or self-sustained replication (3 SR); SPIA™ and RiboSPIA™ amplification (see, e.g., Kurn, U.S. Pat. No. 6,251,639 and U.S. Patent Application Publication No. US 2003/0017591A1); and helicase-dependent amplification (HDA; see, e.g., Vincent et al., EMBO Reports 5:795-800 (2004)), and including without limitation multiplex versions and/or combinations thereof, for example but not limited to, OLA/PCR, PCR/LDR, PCR/LCR, also known as combined chain reaction (CCR). Descriptions of certain amplification techniques can be found in, among other places, Molecular Cloning, A Laboratory Manual, Sambrook and Russell, eds., Cold Spring Harbor Press, 3d ed. (2001; hereinafter “Sambrook and Russell”); Sambrook et al.; Ausubel et al.; PCR Primer; McPherson; Rapley; Lizardi et al., Nat. Genetics 19:225-32 (1998); Wiedmann et al., S51-64, in PCR Methods and Applications, Cold Spring Harbor Laboratory Press (1994); Cao, Trends in Biotechnol. 22:38-44 (2004); and Wenz and Schroth, U.S. Patent Application Publication No. US 2003/0190646A1.

[0053] The term “antibody” refers to an immunoglobulin molecule, including, but not limited to IgA, IgG, IgD, IgE, IgM, and combinations thereof. In embodiments, the antibody is a monoclonal antibody. In embodiments the antibody is a polyclonal antibody. In embodiment, the antibody is a human antibody.

[0054] The term “antibody fragment” refers to a protein comprising at least one complementarity determining region (CDR) of an antibody (i.e., a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2, or a light chain CDR3). Non-limiting examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins (“scFv”), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (“dsFv”), single-domain antibodies (sdAb, nanobody), heavychain only antibodies (e.g., camelid VHH, camelid nanobody, shark Ig NAR), a variable heavy domain, and a variable light domain.

[0055] The term "next generation sequencing,” also referred to as “NGS,” refers to a variety of high-throughput sequencing technologies for sequencing DNA or RNA that parallelize the sequencing process, producing thousands or millions of sequences at once. NGS is generally conducted with the following steps: First, DNA sequencing libraries are generated by clonal amplification by PCR in vitro; second, the DNA is sequenced by synthesis, such that the DNA sequence is determined by the addition of nucleotides to the complementary strand rather through chain-termination chemistry; third, the spatially segregated, amplified DNA templates are sequenced simultaneously in a massively parallel fashion without the requirement for a physical separation step. NGS parallelization of sequencing reactions generates hundreds of megabases to gigabases of nucleotide sequence reads in a single instrument run. Unlike conventional sequencing techniques, such as Sanger sequencing, which simply report the average genotype of an aggregate collection of molecules, NGS technologies digitally tabulate the sequence of many individual DNA fragments, such that low frequency variants (i.e., variants present at less than about 10%, 5% or 1% frequency in a heterogeneous population of nucleic acid molecules) can be detected. For this reason, NGS technologies are often referred to as "ultradeep sequencing." The term "massively parallel" can also be used to refer to the simultaneous generation of sequence information from many different template molecules by NGS .

[0056] NGS strategies can include several methodologies, including, but not limited to: (i) microelectrophoretic methods; (ii) sequencing by hybridization; (iii) real-time observation of single molecules, and (iv) cyclic-array sequencing. Cyclic-array sequencing refers to technologies in which a sequence of a dense array of DNA is obtained by iterative cycles of template extension and imaging-based data collection. Commercially available cyclic- array sequencing technologies include, but are not limited to 454 sequencing, for example, used in 454 Genome Sequencers (Roche Applied Science; Basel), Solexa technology, for example, used in the Illumina Genome Analyzer (San Diego, CA), the SOLiD platform (Applied Biosystems; Foster City, CA), the Polonator (Dover/Harvard) and HeliScope Single Molecule Sequencer technology (Helicos; Cambridge, MA). Although these platforms are quite diverse in sequencing biochemistry as well as in how the array is generated, their work flows are conceptually similar. Other next generation sequencing methods include single molecule real time sequencing (Pacific Bio) and Ion semiconductor sequencing (Ion Torrent sequencing). See, Shendure J and Ji H. (2008) Next Generation DNA Sequencing. Nature Biotech. 26(10): 1135- 1145 for a more detailed discussion of

NGS sequencing technologies.

[0057] As used herein, the term “biomarker” refers to any element of interest in a biological sample, such as a protein, or nucleic acid.

II. Polymer Beads

[0058] In embodiments, provided herein is a polymer bead, or polymer particle, comprising: (i) a target biomarker; and (ii) nucleic acids comprising: (a) a primer binding site; and (b) a unique molecular identifier (UMI); wherein the nucleic acids are bound to the biomarker and wherein each nucleic acid comprises a different UMI.

II-A. Beads/Particles

[0059] The polymer beads, or polymer particles, of the disclosure may be of any shape, including but not limited to spherical, non-spherical, elongated, cube, cuboid, cones and cylinders. In some embodiments, a hydrogel bead of the disclosure has material modulus properties (e.g., elasticity) more closely resembling that of a target cell as compared to a polystyrene bead of the same diameter. The polymer beads of the disclosure may also mimic extracellular vesicles, viruses, virus-like particles, spheroids, organoids, or any other biological target of interest.

[0060] Polymer beads can be functionalized, allowing them to mimic optical properties of labeled biological particles. Functionalization can be mediated by a compound comprising a free amine group, e.g. allylamine, which can be incorporated into a polymer bead during the formation process. The polymer beads of the present invention may be functionalized with any biomarker, polypeptide, peptide, protein, epitope, or antigen known in the art. [0061] Polymer beads, in one embodiment, are functionalized with one or more cell surface markers (see, e.g., Tables 1, 2, and 3), or fragments thereof, for example, extracellular portions thereof in the case of transmembrane proteins, for example, by attaching the one or more cell surface markers, extracellular portions or ligand binding regions thereof to the particle via a free amine, free carboxyl and/or free hydroxyl group present on the surface of the polymer bead. Functionalization of a polymer bead with a dye or cell surface molecule can also occur through a linker, for example a streptavidin/biotin conjugate.

[0062] Particles of the disclosure may comprise a hydrogel or hydrophobic polymer. A hydrogel is a material comprising a macromolecular three-dimensional network that allows it to swell when in the presence of water, to shrink in the absence of (or by reduction of the amount of) water but not dissolve in water.

[0063] In embodiments, the polymer beads may have a diameter of less than about 1 pm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, or less than 1000 pm in diameter. In some embodiments, the polymer beads may have a diameter of more than about 1pm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, or greater than 1000 pm in diameter. In one embodiment, the polymer beads may have a diameter in the range of 5 pm to 100 pm. [0064] In another aspect, a polymer bead can comprise non-polystyrene-based material such as PLGA. In other aspects, the polymer bead is generated using polystyrene and latex.

[0065] The hydrogels provided herein, in the form of beads/particles, are synthesized by polymerizing one or more of the monomers provided herein. The synthesis is carried out to form individual hydrogel particles. The monomeric material (monomer) in one embodiment is polymerized to form a homopolymer. However, in another embodiment copolymers of different monomeric units (z.e., co-monomers) are synthesized and used in the methods provided herein. The monomer or co-monomers used in the methods and compositions described herein, in one embodiment, is a bifunctional monomer or includes a bifunctional monomer (where co-monomers are employed). In one embodiment, the polymer/hydrogel is synthesized in the presence of a crosslinker. In a further embodiment, embodiment, the polymer/hydrogel is synthesized in the presence of a polymerization initiator. In embodiments, any form of polymerization known to those skilled in the art, can be employed to form polymers. In embodiments, polymerization is catalyzed by ultraviolet light-induced radical formation and reaction progression.

[0066] In embodiments, polymer beads are formed by precipitation polymerization, as described in Elbert (2011), Acta Biomater. 7, pp. 31-56. This reference is incorporated by reference herein in its entirety for all purposes. Precipitation polymerization is a technique that takes advantage of the differences in the solubility of monomer and polymer to produce microparticles. Specifically, it is known that larger polymer chains generally have lower solubility than smaller ones. Accordingly, above a specific molecular weight, phase separation may be favored. Precipitation polymerization initially begins as solution polymerizations in a single phase, homogenous system. Shortly after the start of the polymerization, in one embodiment, a relatively high concentration of polymer chains is present, favoring phase separation by nucleation. As polymerization proceeds, the concentration of polymer chains is low and existing particles capture the chains before nucleation of new particles can occur. Thus, nucleation of particles occurs only for a brief period of time shortly after the start of the reaction, which in one embodiment, results in a narrow size distribution of particles. In embodiments, polymer beads may be formed by one or more methods selected from the group consisting of: lithographic particle formation (Helgeson et al. (2011). Curr. Opin. Colloid. Interface Sci. 16, pp. 106-117, incorporated by reference herein in its entirety for all purposes) membrane emulsification (e.g., by the micosieve emulsification technology techniques described by Nanomi B.V. (Netherlands)), microchannel emulsification (Sugiura et al. (2002). Languimir 18, pp. 5708-5712, incorporated by reference herein in its entirety), or bulk emulsification (SNF Floerger, available at snf.com.au/downloads/Emulsion_Handbook_E.pdf, incorporated by reference herein in its entirety).

[0067] In embodiments, polymer beads are formed within a microfluidic device having two oil channels that focus on a central stream of aqueous monomer solution. In embodiments, droplets form at the interface of the two channels and central stream to break off droplets in water-in-oil emulsion. In embodiments, after droplets are formed, they are stabilized prior to polymerization. In embodiments, droplets are stabilized by adding a surfactant to the oil phase. In embodiments, droplets are not stabilized prior to polymerization. In embodiments, polymerization of the monomer is triggered by adding an accelerator (e.g., N,N,N' ,N'tetramethylethylenediamine) to one or both of the oil channels after initial droplets are formed.

[0068] In embodiments, acrylate is the monomer that is polymerized. In embodiments, acrylamide is the monomer that is polymerized.

[0069] The amount of monomer can be varied by the user of the invention, for example to obtain a particular optical property that is substantially similar to that of a target cell. In one embodiment, the monomeric component(s) (z.e., monomer, co-monomer, bifunctional monomer, or a combination thereof, for example, bis/acrylamide in various crosslinking ratios, allyl amine or other co-monomers which provide chemical functionality for secondary labeling/conjugation or alginate is present at about 10 percent by weight to about 95 percent weight of the hydrogel. In a further embodiment, the monomeric component(s) is present at about 15 percent by weight to about 90 percent weight of the hydrogel, or about 20 percent by weight to about 90 percent weight of the hydrogel.

[0070] Examples of various monomers and cross-linking chemistries available for use with the present invention are provided in the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools, lifetechnologies. com/content/sfs/brochures/1602163-Crosslinking-Reagents- Handbook.pdf, the disclosure of which is incorporated by reference in its entirety for all purposes. For example, hydrazine (e.g., with an NHS ester compound) or EDC coupling reactions (e.g., with a maleimide compound) can be used to construct the hydrogels of the invention.

[0071] In one embodiment, a monomer for use with the polymers provided herein is lactic acid, glycolic acid, acrylic acid, 1 -hydroxy ethyl methacrylate, ethyl methacrylate, 2-hydroxy ethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, a derivatized version thereof, or a combination thereof.

[0072] In one embodiment, one or more of the following monomers is used herein to form a polymer of the present invention: 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, polyethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate or a combination thereof.

[0073] In another embodiment, one or more of the following monomers is used herein to form a tunable polymer: phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3- dibromopropyl acrylate, 2,3 -dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4- chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N- benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N- diphenylmethyl acrylamide N-(4-methylphenyl)methyl acrylamide, N-l -naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N- (4-hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N, N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)methyl methacrylamide, N-l -naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2- phenylethyl)methacrylamide, N-triphenylmethyl methacrylamide, N-(4- hydroxyphenyl)methacrylamide, N,N-methylphenyl methacrylamide, N,N'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine. In embodiments, a monomer is selected from any one of: 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate. In embodiments, a monomer is selected from any one of lactic acid, glycolic acid, acrylic acid, 1 -hydroxy ethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, a derivatized version thereof, or a combination thereof.

[0074] Additional polymer monomers are described in U.S. Patent No. 6,657,030, which is incorporated by reference in its entirety herein for all purposes.

[0075] In embodiments, the polymer monomer is a monosaccharide, disaccharide, or polysaccharide. In embodiments, the monosaccharide, disaccharide, or polysaccharide is functionalized. In one embodiment, an acrylamide or acrylate functionalized monosaccharide, disaccharide or polysaccharide is used as a polymerizable polymer monomer. In one embodiment, a structural polysaccharide is used as a polymerizable polymer monomer. In a further embodiment, the structural polysaccharide is an arabinoxylan, cellulose, chitin or a pectin. In another embodiment, alginic acid (alginate) is used as a polymerizable polymer monomer. In yet another embodiment, a glycosaminoglycan (GAG) is used as a polymerizable monomer in the polymers provided herein. In a further embodiment, the GAG is chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate or hyaluronic acid (also referred to in the art as hyaluron or hyaluronate) is used as a polymerizable polymer monomer. The additional range of compatible biomonomers and their reactive chemistries are known be individuals skilled in the art and follow general chemical reactivity principles.

[0076] Biocompatible monomers for use with the polymers described herein include in one embodiment, ethyleglycol dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA), methylmethacrylte (MMA), methacryloxymethyltrimethylsilane (TMS-MA), N-vinyl-2- pyrrolidon (N-VP), styrene, or a combination thereof. In embodiments, the polysaccharide monomer is any one of agar, agarose, alginic acid, alguronic acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta-glucan, callose, capsullan, carrageenan polysaccharides (e.g., kappa, iota or lambda class), cellodextrin, cellulin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminoogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof.

[0077] In embodiments, the polymer monomer comprises a peptide or protein. In embodiments, the protein is a structural protein, or a domain thereof. In embodiments, the structural protein is silk, elastin, titin or collagen, or a domain thereof. In embodiments, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycan). In embodiments, the structural protein is collagen. In embodiments, the collagen is collagen type I, collagen type II or collagen type III or a combination thereof. In another embodiment, the polymer monomer comprises a proteoglycan. In a further embodiment, the proteoglycan is decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.

[0078] In another embodiment, an acrylate-functionalized structural protein polymer monomer is used as a component of the polymer provided herein (e.g., an acrylate functionalized protein or protein domain, for example, silk, elastin, titin, collagen, proteoglycan, or a functionalized domain thereof). In a further embodiment, the acrylate functionalized structural protein polymer monomer comprises a proteoglycan, e.g., decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.

[0079] In embodiments, PEG monomers and oligopeptides that mimic extracellular matrix proteins are used in the polymers provided herein. In embodiments, the PEG monomer is vinyl sulfone-functionalized multiarm PEG. In embodiments, the monomer is an integrin binding peptide. In embodiments, the monomer is a bis-cysteine matrix metalloproteinase peptide. Biscysteine matrix metalloproteinase peptides are described in the following reference which is incorporated by reference herein in its entirety for all purposes: Lutolf et al. (2003). Proc. Natl. Acad. Sci. U.S.A. 100, 5413-5418. In embodiments, polymers are formed by a Michael-type addition reaction between the di-thiolated oligopeptides and vinyl sulfone groups on the PEG. [0080] In embodiments, the polymer monomer is a bioactive domains in a natural proteins. In embodiments, the polymer monomer is a cell-adhesive integrin binding domain, a controlled release affinity binding domain, or a transglutaminase cross-linking domain can be used in the polymers provided herein. Details for producing such polymers are described in the following references which are incorporated by reference herein in its entirety for all purposes: Martino et al. (2009). Biomaterials 30, 1089; Martino et al. (2011). Sci. Trans. Med. 3, 100ra89; Hu and Messersmith (2003). J. Am. Chem. Soc. 125, 14298.

[0081] In one embodiment, recombinant DNA methods are used to create proteins that form polymers in response to changes in pH or temperature. In embodiments, the protein comprises a coiled-coil domain. The following reference describes a coiled-coil protein that forms a polymer and is incorporated by reference herein in is entirety for all purposes: Petka et al. (1998). Science 281, pp. 389-392.

[0082] In embodiments, the polymer is a naturally occurring polymer. Naturally occurring polymers useful in this disclosure include various polysaccharides available from natural sources such as plants, algae, fungi, yeasts, marine invertebrates and arthropods. Non-limiting examples include agarose, dextrans, chitin, cellulose-based compounds, starch, derivatized starch, and the like. These generally will have repeating glucose units as a major portion of the polysaccharide backbone. Cross-linking chemistries for such polysaccharides are known in the art, see for example Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology,” (available at tools. lifetechnologies. com/content/sfs/brochures/1602163-Crosslinking-Reagents- Handbook.pdf).

[0083] In embodiments, the polymer monomer is hyaluronan. In embodiments, hyaluronan is functionalized, for example with acrylate or acrylamide. Hyaluronan is a high molecular weight GAG composed of disaccharide repeating units of N-acetylglucosamine and glucuronic acid linked together through alternating P-1,4 and P-1,3 glycosidic bonds. In the human body, hyaluronate is found in several soft connective tissues, including skin, umbilical cord, synovial fluid, and vitreous humor. Accordingly, in one embodiment, where one or more optical properties of a skin cell, umbilical cord cell or vitreous humor cell is desired to be mimicked, in one embodiment, hyaluronan is used as a polymer monomer. Methods for fabricating polymer beads/particles are described in Xu et al. (2012). Soft Matter. 8, pp. 3280-3294, the disclosure of which is incorporated herein in its entirety for all purposes. As described therein, hyaluronan can be derivatized with various reactive handles depending on the desired crosslinking chemistry and other monomers used to form a polymer bead/particle.

[0084] In embodiments, the polymer monomer is chitosan. Chitosan is a linear polysaccharide composed of randomly distributed P-(l-4)-linked D-glucosamine (deacetylated unit) and N- acetyl-D-glucosamine (acetylated unit).

[0085] In embodiments, the polymer comprises greater than about 30%, greater than about 40%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% water by weight. In embodiments, the polymer has a water content of from about 10 percent by weight to about 95 percent by weight, or from about 20 percent by weight to about 95 percent by weight, or from about 30 percent by weight to about 95 percent by weight, or from about 40 percent by weight to about 95 percent by weight, or from about 50 percent by weight to about 95 percent by weight, or from about 60 percent by weight to about 95 percent by weight, or from about 70 percent by weight to about 95 percent by weight, or from about 80 percent by weight to about 95 percent by weight. In embodiments, the polymer retains the same shape in the dehydrated and hydrated conditions. For example, if the polymer has an approximately spherical shape in the dehydrated condition, it will be approximately spherical in the hydrated condition.

[0086] In embodiments, the amount of monomer can be varied, for example to obtain a particular optical property or morphological property that is substantially similar to that of a target cell. In embodiments, the monomer is present at about 10 percent by weight to about 95 percent weight of the polymer. In embodiments, the monomer is present at about 10 %, about 11 %, about 12 %, about 13 %, about 14 %, about 15 %, about 16 %, about 17 %, about 18 %, about 19 %, about 20 %, about 21 %, about 22 %, about 23 %, about 24 %, about 25 %, about 26 %, about 27 %, about 28 %, about 29 %, about 30 %, about 31 %, about 32 %, about 33 %, about 34 %, about 35 %, about 36 %, about 37 %, about 38 %, about 39 %, about 40 %, about 41 %, about 42 %, about 43 %, about 44 %, about 45 %, about 46 %, about 47 %, about 48 %, about 49 %, about 50 %, about 51 %, about 52 %, about 53 %, about 54 %, about 55 %, about 56 %, about 57 %, about 58 %, about 59 %, about 60 %, about 61 %, about 62 %, about 63 %, about 64 %, about 65 %, about 66 %, about 67 %, about 68 %, about 69 %, about 70 %, about 71 %, about 72 %, about 73 %, about 74 %, about 75 %, about 76 %, about 77 %, about 78 %, about 79 %, about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 %, about 92 %, about 93 %, about 94 %, about 95 % by weight of the polymer, including all values and subranges in between inclusive of endpoints.

[0087] In some embodiments, the polymer beads, or polymer particles, are engineered to degrade to provide such biomolecules to a cell in culture. Degradation can include, without limitation, dissolution (i.e., dissolving) or lysis. The polymer can be engineered to have multiple layers, as shown in FIG. 1, with different rates of degradation for at least two of the layers. The polymer, whether in its entirety or various layers thereof, can be degraded chemically (e.g., reagents, detergents, bursting, or the like), mechanically (e.g., vibration, acoustic, freeze-thaw, bursting, or the like), or both chemically and mechanically.

[0088] The rate of degradation of the entire polymer particles, individual layers of the polymer particles, or groups or subpopulations of a polymer population can be fast (i.e., less than 24 hours) or slow (i.e., 24 hours or more). For example, a first layer of a polymer particle can degrade in less than 24 hours and a second layer of the same polymer particle can degrade in 48 hours. As yet another example, a first subpopulation of polymer particles can degrade in less than 1 hour, a second subpopulation of polymer particles can degrade in 24 hours, and a third subpopulation of polymer particles can degrade in one week. The first, second, and third subpopulations form a population of polymer particles.

[0089] In some embodiments, a population of polymer particles can include groups or subpopulations of polymer particles having different rates of degradation.

[0090] In some embodiments, the polymer particle can be engineered to have pore sizes which correlate to various rates of degradation. The pore sizes can range from 0.1 nm to 1 pm. For example, a first polymer particle can have a first pore size, such that the first polymer particle has a first rate of degradation; and, a second polymer can have a second pore size, such that the second polymer particle has a second rate of degradation with the first and second rates of degradation not being equal (e.g., first rate is faster than the second rate; or, the first rate is slower than the second rate).

[0091] In some embodiments, the polymer particle can be engineered to have a rate of degradation based on a plurality of factors, including, without limitation, pore size, chemical composition (i.e., chemical bonds, monomers, co-monomer), layer composition, the like, and combinations thereof. [0092] In some embodiments, an individual polymer particle or a plurality thereof comprises a biodegradable polymer as a polymer monomer. In one embodiment, the biodegradable polymer is a poly(esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), poly(lactic-co-glycolic) acid (PLGA) and their copolymers. In some embodiments, the biodegradable polymer is a carbohydrate or a protein, or a combination thereof. For example, in one embodiment, a monosaccharide, disaccharide or polysaccharide, (e.g., glucose, sucrose, or maltodextrin) peptide, protein (or domain thereof) is used as a polymer monomer. Other biodegradable polymers include poly(hydroxyalkanoate)s of the PHB-PHV class, additional poly(ester)s, and natural polymers, for example, modified poly(saccharide)s, e.g., starch, cellulose, and chitosan. In other embodiments, the biocompatible polymer is an adhesion protein, cellulose, a carbohydrate, a starch (e.g., maltodextrin, 2-hydroxyethyl starch, alginic acid), a dextran, a lignin, a polyaminoacid, an amino acid, or chitin. Such biodegradable polymers are available commercially, for example, from Sigma Aldrich (St. Louis, Mo.).

[0093] The protein in some embodiments comprises only natural amino acids. However, the present disclosure is not limited thereto. For example, self-assembling artificial proteins and proteins with non-natural amino acids (e.g., those incorporated into non-ribosomal peptides or synthetically introduced via synthetic approaches, see for example, Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587, the disclosure of which is incorporated by reference in its entirety for all purposes), or protein domains thereof, can also be used as polymer monomers. The range of non-natural (unnatural) amino acids that can be incorporated into such compositions is well known to those skilled in the art (Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587; incorporated by reference in its entirety for all purposes). The biodegradable polymer in one embodiment, is used as a co-monomer, i.e., in a mixture of monomers. The biodegradable polymer in one embodiment is a bifunctional monomer.

[0094] The biomonomer, in some embodiments, is functionalized with acrylamide or acrylate. For example, in one embodiment, the polymerizable acrylamide functionalized biomolecule is an acrylamide or acrylate functionalized protein (for example, an acrylamide functionalized collagen or functionalized collagen domain), an acrylamide or acrylate functionalized peptide, or an acrylamide or acrylate functionalized monosaccharide, disaccharide or polysaccharide.

[0095] Any monosaccharide, disaccharide or polysaccharide (functionalized or otherwise) can be used as a polymer monomer. In some embodiments, an acrylamide or acrylate functionalized monosaccharide, disaccharide or polysaccharide is used as a polymerizable polymer monomer. In some embodiments, a structural polysaccharide is used as a polymerizable polymer monomer. In further embodiments, the structural polysaccharide is an arabinoxylan, cellulose, chitin or a pectin. In other embodiments, alginic acid (alginate) is used as a polymerizable polymer monomer. In yet other embodiments, a glycosaminoglycan (GAG) is used as a polymerizable monomer in the polymers provided herein. In further embodiments, the GAG is chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate or hyaluronic acid (also referred to in the art as hyaluronan or hyaluronate) is used as a polymerizable polymer monomer. The additional range of compatible biomonomers and their reactive chemistries are known be individuals skilled in the art and follow general chemical reactivity principles.

[0096] The passive optical properties of the polymer beads may be modulated to mimic the passive optical properties of a target cell, or otherwise modulated to achieve experimental goals. Exemplary target cells are included in the non-exhaustive listing in Table 1. In embodiments, the target cell is a lymphocyte, a monocyte, or a granulocyte. In embodiments, the target cell is a prokaryotic cell. In embodiments, the target cell is a eukaryotic cell. In embodiments, the target cell is a white blood cell. In embodiments, the target cell is a platelet. In embodiments, the target cell is a red blood cell. In embodiments, the target cell is an immune cell. In embodiments, the immune cell is a T cell, a B cell, an NK cell, a lymphocyte, a monocyte, a granulocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a macrophage, or a dendritic cell.

[0097] In embodiments, each polymer bead comprises less than 10%, 20%, 30%, or 40% polystyrene by hydrated or dehydrated volume. Depending on their composition, the polymer beads hydrated and dehydrated volume may be identical.

[0098] In embodiments, the polymer beador polymer particle is functionalized to mimic one or more optical properties of a target cell or labeled target cell. In embodiments, the polymer or polymer particle comprises one or more high-refractive index molecules. In embodiments, the hydrogel or polymer particle comprises a plurality of high-refractive index molecules. In embodiments, the high-refractive index molecule enables for mimicking of the S SC of a target cell. In embodiments, the high-refractive index molecule is selected from one or more of colloidal silica, alkyl acrylate, alkyl methacrylate or a combination thereof. In embodiments, the high-refractive index molecule is alkyl acrylate, alkyl methacrylate, or both. In embodiments, alkyl acrylates or alkyl methacrylates contain 1 to 18, 1 to 8, or 2 to 8, carbon atoms in the alkyl group. In embodiments, the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tertbutyl, 2-ethylhexyl, heptyl or octyl. In embodiments, the alkyl group is branched. In embodiments, the alkyl group is linear. [0099] In some embodiments, the polymer beads are designed to have distinguishable optical properties. For example, in some embodiments, a composition comprises a first and a second population of polymer beads, each population of polymer beads having been engineered to have distinct optical properties, allowing for deconvolution in the cytometer.

[0100] The three primary modes of deconvolution for flow cytometry are the two passive optical properties of a polymer bead (FSC, corresponding to the refractive index, or RI, and SSC) and biomarkers present on the surface of a given cell type. Therefore, compositions that allow polymer beads, or polymer beads, of the disclosure to mimic specific cell types with respect to these three modes are useful for providing synthetic, robust calibrants for flow cytometry.

[0101] In one embodiment, the RI of a disclosed polymer bead is greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.1 0, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0102] In another embodiment, the RI of a disclosed polymer bead is about 1.10 to about 3.0, or about 1.15 to about 3.0, or about 1.20 to about 3.0, or about 1.25 to about 3.0, or about 1.30 to about 3.0, or about 1.35 to about 3.0, or about 1.4 to about 3.0, or about 1.45 to about 3.0, or about 1.50 to about 3.0, or about 1.6 to about 3.0, or about 1.7 to about 3.0, or about 1.8 to about 3.0, or about 1.9 to about 3.0, or about 2.0 to about 3.0.

[0103] In some embodiments, the RI of a disclosed polymer bead is less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.

[0104] In embodiments, the dimensions (e.g., diameter, width, thickness) of a polymer bead of the present disclosure are substantially similar to a target cell. In embodiments, a polymer bead has a diameter of less than about 1 pm, about 2 pm, about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 120 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 600 pm, about 800 pm, or less than about 1000 pm in diameter. In some embodiments, a polymer particle has a diameter of more than about 1 pm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, or greater than 1000 pm in diameter. In some embodiments, a polymer particle has a diameter in the range of 2.5 pm to 100 pm. In some embodiments, a polymer particle has a diameter of from about 2.5 pm to about 25 pm, from about 3 pm to about 20 pm, from about 3.5 pm to about 15 pm, from about 4 pm to about 12 pm, from about 5 pm to about 10 pm, from about 6 pm to about 9 pm, from about 7 pm to about 8 pm, or from about 10 pm to about 20 pm. In embodiments, the polymer bead has a diameter of less than about 100 pm. In embodiments, the polymer bead has a diameter of less than about 10 pm. In embodiments, the polymer bead has a diameter of more than about 10 pm. In In embodiments, the polymer bead has a diameter from about 10 pm to about 20 pm. In embodiments, a polymer bead has a diameter of less than about 1 pm. In embodiments, a polymer bead has a diameter of more than about 1 pm. In embodiments, the diameter of a polymer bead is measured using dynamic light scattering.

[0105] In embodiments, a polymer bead has a width of less than about 1 pm, about 2 pm, about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 120 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 600 pm, about 800 pm, or less than about 1000 pm. In some embodiments, a polymer particle has a width of more than about 1 pm, about 2 pm, about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 120 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 600 pm, about 800 pm, or about 1000 pm. In some embodiments, a polymer particle has a width in the range of 2.5 pm to 100 pm. In some embodiments, a polymer particle has a width of from about 2.5 pm to about 25 pm, from about 3 pm to about 20 pm, from about 3.5 pm to about 15 pm, from about 4 pm to about 12 pm, from about 5 pm to about 10 pm, from about 6 pm to about 9 pm, or from about 7 pm to about 8 pm. [0106] In embodiments, a polymer particle has a thickness of less than about 1 pm, about 2 pm, about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 120 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 600 pm, about 800 pm, or less than about 1000 pm. In some embodiments, a polymer particle has a thickness of more than about 1 pm, about 2 pm, about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 120 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 600 pm, about 800 pm, or less than about 1000 pm, including all ranges and subranges therebetween. In some embodiments, a polymer particle has a thickness in the range of 2.5 pm to 100 pm. In some embodiments, a polymer particle has a thickness of from about 2.5 pm to about 25 pm, from about 3 pm to about 20 pm, from about 3.5 pm to about 15 pm, from about 4 pm to about 12 pm, from about 5 pm to about 10 pm, from about 6 pm to about 9 pm, or from about 7 pm to about 8 pm.

[0107] In embodiments, the polymer bead exhibits a mean fluorescence intensity (MFI) when labeled with the pre-apoptotic signal that is at least as high as the MFI of a target cell labeled with the same pre-apoptotic signal. In embodiments, the MFI of the polymer bead and the MFI of the target cell are within 10 % to within 50 %. In embodiments, the MFI of the polymer bead and the MFI of the target cell are within 10 %, within 11 %, within 12 %, within 13 %, within 14 %, within 15 %, within 16 %, within 17 %, within 18 %, within 19 %, within 20 %, within

21 %, within 22 %, within 23 %, within 24 %, within 25 %, within 26 %, within 27 %, within

28 %, within 29 %, within 30 %, within 31 %, within 32 %, within 33 %, within 34 %, within 35 %, within 36 %, within 37 %, within 38 %, within 39 %, within 40 %, within 41 %, within

42 %, within 43 %, within 44 %, within 45 %, within 46 %, within 47 %, within 48 %, within

49 %, or within 50 %, including all values and subranges in between inclusive of endpoints.

[0108] In embodiments, the MFI of the polymer bead and the MFI of the target cell is within 50%, 40%, 30%, 20%, or 10%.

[0109] The SSC of a disclosed polymer bead is most meaningfully measured in comparison to that of target cell. In some embodiments, a disclosed polymer bead has an SSC within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% that of a target cell, as measured by a cytometric device. [0110] The SSC of a polymer bead in one embodiment, is modulated by incorporating a high- refractive index molecule (or plurality thereof) in the polymer bead. In one embodiment, a high-refractive index molecule is provided in a polymer bead, and in a further embodiment, the high-refractive index molecule is colloidal silica, alkyl acrylate, alkyl methacrylate or a combination thereof. Thus, in some embodiments, a polymer bead of the disclosure comprises alkyl acrylate and/or alkyl methacrylate. Concentration of monomer in one embodiment is adjusted to further adjust the refractive index of the polymer bead.

[0111] Alkyl acrylates or alkyl methacrylates can contain 1 to 18, 1 to 8, or 2 to 8, carbon atoms in the alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tertbutyl, 2-ethylhexyl, heptyl or octyl groups. The alkyl group may be branched or linear.

[0112] High-refractive index molecules can also include vinylarenes such as styrene and methylstyrene, optionally substituted on the aromatic ring with an alkyl group, such as methyl, ethyl or tert-butyl, or with a halogen, such as chlorostyrene.

[0113] In some embodiments, FSC is modulated by adjusting the percentage of monomer present in the composition thereby altering the water content present during polymer bead formation. In one embodiment, where a monomer and co-monomer are employed, the ratio of monomer and co-monomer is adjusted to change the polymer bead ‘s forward scatter properties.

[0114] For example, the ratio of monomer and co-monomer can be used to adjust the polymer bead’s elasticity (i.e., Young’s Modulus) to be substantially similar to the elasticity of the target cell. The ratio of the monomer and co-monomer can change the Young’s Modulus for the polymer bead can range from 0.2 kiloPascals (kPa) to 400 kPa, based on the elasticity of the target cell. The elasticity of the polymer bead (e.g., softness or firmness) can affect the function of the target cell with which the polymer bead interacts.

[0115] The FSC of a disclosed polymer bead is most meaningfully measured in comparison to that of target cell. In some embodiments, a disclosed polymer bead has an FSC within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% that of a target cell, as measured by a cytometric device.

[0116] FSC is related to particle volume, and thus can be modulated by altering particle diameter, as described herein. Generally, it has been observed that large objects refract more light than smaller objects leading to high forward scatter signals (and vice versa). Accordingly, particle diameter in one embodiment is altered to modulate FSC properties of a polymer bead. For example, polymer bead diameter is increased in one embodiment is altered by harnessing larger microfluidic channels during particle formation. [0117] SSC can be engineered by encapsulating nanoparticles within polymer bead to mimic organelles in a target cell. In some embodiments, a polymer bead of the disclosure comprises one or more types of nanoparticles selected from the group consisting of: polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS) nanoparticles, and silica nanoparticles. Without wishing to be bound by theory, the ability to selectively tune both forward and side scatter of a polymer bead, as described herein, allows for a robust platform to mimic a vast array of cell types.

[0118] Polymers and other polymer beads are known in the art and are described in US Patent Nos. 9,915,598 and 10,753,846, incorporated herein by reference in their entirety.

[0119] In embodiments, a polymer bead described herein may be lysed. A lysable synthetic bead of the present invention allows a user to measure both the intact bead as well as the lysed bead. In embodiments, the polymer bead is lysed with a lysis buffer. In embodiments, the lysis buffer is ammonium chloride. Hematological lysis buffers often use ammonium chloride, including the IX RBC Lysis Buffer and 10X RBC Lysis Buffer from Thermo Fisher Scientific. Hematological lysis buffers used on clinical blood samples are designed to lyse non-nucleated red blood cells and preserve white blood cells in order to perform white blood cell counts and the quantitative measurement of hemoglobin. Other lysis buffers may be designed to dissolve the engineered particle including strong reducing agents such as dithiothreitol (DTT) or betamercaptoethanol (BME). Additional non-limiting examples include divalent ions such as ethylenediaminetetraacetic acid (EDTA) or citrate.

[0120] In some embodiments the polymer beads of the present disclosure contain magnetic particles that make the polymer bead responsive to magnetic fields. In some embodiments, the magnetic particles are magnets. In some embodiments the magnetic particles comprise ferromagnetic materials. In some embodiments, the magnetic particles make it possible to separate out the polymer beads from solution, or from unbound nucleic acids. They can also be used to immobilize the polymer beads during washes/transfers, mixing.

II-B. Biomarkers

[0121] In embodiments, the polymer beads described herein comprise one or more biomarkers. In embodiments, the one or more biomarkers are proteins. In embodiments, the proteins are typically found inside of cells. In embodiments, the proteins are cell surface markers, an epitope binding region of a cell surface marker, or a combination thereof. [0122] In embodiments, the polymer beads comprise multiple copies of a single biomarker. In embodiments, the polymer beads comprise one or more biomarkers selected from any one of: CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-gamma, CD20, TCR gamma/delta, TNF alpha, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CDl lc, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, TCR V alpha 7.2, CD 11b, CD21, CD24, IL-4, Biotin, CCR10, CD31, CD44, CD 138, CD294, NKp46, TCR V delta 2, TIGIT, CDlc, CD2, CD7, CD8a, CD15, CD32, CD103, CD107a, CD141, CD158, CD159c, IL-13, IL-21, KLRG1, TIM-3, CCR5, CD5, CD33, CD45.2, CD80, CD159a (NKG2a), CD244, CD272, CD278, CD337, Granzyme B, Ig Lambda Light Chain, IgA, IL- 17A, Streptavidin, TCR V delta 1, CDld, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD 137, CD 163, CD 193, CTLA-4, CX3CR1, Fc epsilon R1 alpha, IL-22, Lag-3, MIP-1 beta, Perforin, TCR V gamma 9, CDla, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD 172a, CD 186, CD226, CD303, CLEC12A, CXCR4, Helios, Ig Kappa Light Chain, IgE, IgGl, IgG3, IL-5, IL-8, IL-21 R, KIR3dl05, KLRC1/2, Ly-6C, Ly-6G, MHC Class II (I-A/I- E), MHC II, TCR alpha/beta, TCR beta, TCR V alpha 24, Akt (pS473), ALDH1 Al, Annexin V, Bcl-2, c-Met, CCR7, cdl6/32, cd41a, CD3 epsilon, CD8b, CDl lb/c, CD16/CD32, CD23, CD29, CD43, CD45.1, CD48, CD49b, CD49d, CD66, CD68, CD71, CD85k, CD93, CD99, CD106, CD122, CD133, CD134, CD146, CD150, CD158b, CD158bl/b2, j, CD158e, CD166, CD169, CD184, CD200, CD200 R, CD235a, CD267, CD268, CD273, CD274, CD317, CD324, CD326, CD328, CD336, CD357, CD366, DDR2, eFluor 780 Fix Viability, EGF Receptor, EGFR (pY845), EOMES, EphA2, ERK1/2 (pT202/pY204), F4/80, FCRL5, Flt-3, FVS575V, FVS700, Granzyme A, HER2/ErbB2, Hesl, Hoechst (33342), ICAM-1, IFN-alpha, IgAl, IgAl/IgA2, IgA2, IgG2, IgG4, IL-1 RAcP, IL-6, IL-10, IL-12, IL-17, Integrin alpha 4 beta 7, Isotype Ctrl, KLRC1, KLRC2, Live/Dead Fix Aqua, Ly-6A/Ly-6E, Ly-6G/Ly-6C, Mannose Receptor, MDR1, Met (pY1234/pY1235), MMP-9, NGF Receptor p75, ORAI1, ORAI2, ORAI3, p53, P2RY12, PARP, cleaved, RT1B, S6 (pS235/pS236), STIM1, STIM2, TCR delta, TCR delta/gamma, TCR V alpha 24 J alpha 18, TCR V beta 11, TCR V gamma 1.1, TCR V gamma 2, TER-119, TIMP-3, TRAF3, TSLP Receptor, VDAC1, Vimentin, XCR1, and YAP 1.

[0123] In embodiments, the biomarker is any biomarker of Tables 1-3.

II-C. Nucleic Acids

[0124] In embodiments, the polymer beads comprise nucleic acids comprising: (a) a primer binding site; and (b) a unique molecular identifier (UMI).

[0125] In embodiments, a polymer bead comprises a plurality of target molecules, wherein each target molecule is bound to a nucleic acid, wherein the UMI of each nucleic acid is different.

[0126] In embodiments, a nucleic acid that is bound to the polymer beads is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,

390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,

580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760,

770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,

960, 970, 980, 990, or 1000 nucleotides long, including all ranges and subranges therebetween. [0127] In embodiments, a nucleic acid that is bound to the polymer beads may have from about 6 to about 50, from about 7 to about 50, from about 8 to about 50, from about 9 to about 50, from about 10 to about 50, from about 11 to about 50, from about 12 to about 50, from about 13 to about 50, from about 14 to about 50, from about 15 to about 50, from about 16 to about 50, from about 17 to about 50, from about 18 to about 50, from about 19 to about 50, from about 20 to about 50, from about 21 to about 50, from about 22 to about 50, from about 23 to about 50, from about 24 to about 50, from about 25 to about 50, from about 26 to about 50, from about 27 to about 50, from about 28 to about 50, from about 29 to about 50, from about 30 to about 50, from about 31 to about 50, from about 32 to about 50, from about 33 to about 50, from about 34 to about 50, from about 35 to about 50, from about 36 to about 50, from about 37 to about 50, from about 38 to about 50, from about 39 to about 50, from about 40 to about 50, from about 5 to about 45, from about 6 to about 45, from about 7 to about 45, from about 8 to about 45, from about 9 to about 45, from about 10 to about 45, from about 11 to about 45, from about 12 to about 45, from about 13 to about 45, from about 14 to about 45, from about 15 to about 45, from about 16 to about 45, from about 17 to about 45, from about 18 to about 45, from about 19 to about 45, from about 20 to about 45, from about 21 to about 45, from about 22 to about 45, from about 23 to about 45, from about 24 to about 45, from about 25 to about 45, from about 26 to about 45, from about 27 to about 45, from about 28 to about 45, from about 29 to about 45, from about 30 to about 45, from about 31 to about 45, from about 32 to about 45, from about 33 to about 45, from about 34 to about 45, from about 35 to about 45, from about 36 to about 45, from about 37 to about 45, from about 38 to about 45, from about 39 to about 45, from about 40 to about 45, from about 5 to about 40, from about 6 to about 40, from about 7 to about 40, from about 8 to about 40, from about 9 to about 40, from about 10 to about 40, from about 11 to about 40, from about 12 to about 40, from about 13 to about 40, from about 14 to about 40, from about 15 to about 40, from about 16 to about 40, from about 17 to about 40, from about 18 to about 40, from about 19 to about 40, from about 20 to about 40, from about 21 to about 40, from about 22 to about 40, from about 23 to about 40, from about 24 to about 40, from about 25 to about 40, from about 26 to about 40, from about 27 to about 40, from about 28 to about 40, from about 29 to about 40, from about 30 to about 40, from about 5 to about 35, from about 6 to about 35, from about 7 to about 35, from about 8 to about 35, from about 9 to about 35, from about 10 to about 35, from about 11 to about 35, from about 12 to about 35, from about 13 to about 35, from about 14 to about 35, from about 15 to about 35, from about 16 to about 35, from about 17 to about 35, from about 18 to about 35, from about 19 to about 35, from about 20 to about 35, from about 21 to about 35, from about 22 to about 35, from about 23 to about 35, from about 24 to about 35, from about 25 to about 35, from about 26 to about 35, from about 27 to about 35, from about 28 to about 35, from about 29 to about 35, from about 30 to about 35, from about 5 to about 30, from about 6 to about 30, from about 7 to about 30, from about 8 to about 30, from about 9 to about 30, from about 10 to about 30, from about 11 to about 30, from about 12 to about 30, from about 13 to about 30, from about 14 to about 30, from about 15 to about 30, from about 16 to about 30, from about 17 to about 30, from about 18 to about 30, from about 19 to about 30, from about 20 to about 30, from about 21 to about 30, from about 22 to about 30, from about 23 to about 30, from about 24 to about 30, from about 25 to about 30, from about 5 to about 25, from about 6 to about 25, from about 7 to about 25, from about 8 to about 25, from about 9 to about 25, from about 10 to about 25, from about 11 to about 25, from about 12 to about 25, from about 13 to about 25, from about 14 to about 25, from about 15 to about 25, from about 16 to about 25, from about 17 to about 25, from about 18 to about 25, from about 19 to about 25, from about 20 to about 25, from about 5 to about 20, from about 6 to about 20, from about 7 to about 20, from about 8 to about 20, from about 9 to about 20, from about 10 to about 20, from about 11 to about 20, from about 12 to about 20, from about 13 to about 20, from about 14 to about 20, from about 15 to about 20, from about 1 to about 15, from about 2 to about 15, from about 3 to about 15, from about 4 to about 15, from about 5 to about 15, from about 6 to about 15, from about 7 to about 15, from about 8 to about 15, from about 9 to about 15, or from about 10 to about 15 nucleotides.

[0128] In embodiments, the UMI of the nucleic acid that is bound to the polymer beads is 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides long, including all ranges and subranges therebetween.

[0129] In embodiments, the UMI of the nucleic acid that is bound to the polymer beads may have from about 6 to about 50, from about 7 to about 50, from about 8 to about 50, from about 9 to about 50, from about 10 to about 50, from about 11 to about 50, from about 12 to about 50, from about 13 to about 50, from about 14 to about 50, from about 15 to about 50, from about 16 to about 50, from about 17 to about 50, from about 18 to about 50, from about 19 to about 50, from about 20 to about 50, from about 21 to about 50, from about 22 to about 50, from about 23 to about 50, from about 24 to about 50, from about 25 to about 50, from about 26 to about 50, from about 27 to about 50, from about 28 to about 50, from about 29 to about 50, from about 30 to about 50, from about 31 to about 50, from about 32 to about 50, from about 33 to about 50, from about 34 to about 50, from about 35 to about 50, from about 36 to about 50, from about 37 to about 50, from about 38 to about 50, from about 39 to about 50, from about 40 to about 50, from about 5 to about 45, from about 6 to about 45, from about 7 to about 45, from about 8 to about 45, from about 9 to about 45, from about 10 to about 45, from about 11 to about 45, from about 12 to about 45, from about 13 to about 45, from about 14 to about 45, from about 15 to about 45, from about 16 to about 45, from about 17 to about 45, from about 18 to about 45, from about 19 to about 45, from about 20 to about 45, from about 21 to about 45, from about 22 to about 45, from about 23 to about 45, from about 24 to about 45, from about 25 to about 45, from about 26 to about 45, from about 27 to about 45, from about 28 to about 45, from about 29 to about 45, from about 30 to about 45, from about 31 to about 45, from about 32 to about 45, from about 33 to about 45, from about 34 to about 45, from about 35 to about 45, from about 36 to about 45, from about 37 to about 45, from about 38 to about 45, from about 39 to about 45, from about 40 to about 45, from about 5 to about 40, from about 6 to about 40, from about 7 to about 40, from about 8 to about 40, from about 9 to about 40, from about 10 to about 40, from about 11 to about 40, from about 12 to about 40, from about 13 to about 40, from about 14 to about 40, from about 15 to about 40, from about 16 to about 40, from about 17 to about 40, from about 18 to about 40, from about 19 to about 40, from about 20 to about 40, from about 21 to about 40, from about 22 to about 40, from about 23 to about 40, from about 24 to about 40, from about 25 to about 40, from about 26 to about 40, from about 27 to about 40, from about 28 to about 40, from about 29 to about 40, from about 30 to about 40, from about 5 to about 35, from about 6 to about 35, from about 7 to about 35, from about 8 to about 35, from about 9 to about 35, from about 10 to about 35, from about 11 to about 35, from about 12 to about 35, from about 13 to about 35, from about 14 to about 35, from about 15 to about 35, from about 16 to about 35, from about 17 to about 35, from about 18 to about 35, from about 19 to about 35, from about 20 to about 35, from about 21 to about 35, from about 22 to about 35, from about 23 to about 35, from about 24 to about 35, from about 25 to about 35, from about 26 to about 35, from about 27 to about 35, from about 28 to about 35, from about 29 to about 35, from about 30 to about 35, from about 5 to about 30, from about 6 to about 30, from about 7 to about 30, from about 8 to about 30, from about 9 to about 30, from about 10 to about 30, from about 11 to about 30, from about 12 to about 30, from about 13 to about 30, from about 14 to about 30, from about 15 to about 30, from about 16 to about 30, from about 17 to about 30, from about 18 to about 30, from about 19 to about 30, from about 20 to about 30, from about 21 to about 30, from about 22 to about 30, from about 23 to about 30, from about 24 to about 30, from about 25 to about 30, from about 5 to about 25, from about 6 to about 25, from about 7 to about 25, from about 8 to about 25, from about 9 to about 25, from about 10 to about 25, from about 11 to about 25, from about 12 to about 25, from about 13 to about 25, from about 14 to about 25, from about 15 to about 25, from about 16 to about 25, from about 17 to about 25, from about 18 to about 25, from about 19 to about 25, from about 20 to about 25, from about 5 to about 20, from about 6 to about 20, from about 7 to about 20, from about 8 to about 20, from about 9 to about 20, from about 10 to about 20, from about 11 to about 20, from about 12 to about 20, from about 13 to about 20, from about 14 to about 20, from about 15 to about 20, from about 1 to about 15, from about 2 to about 15, from about 3 to about 15, from about 4 to about 15, from about 5 to about 15, from about 6 to about 15, from about 7 to about 15, from about 8 to about 15, from about 9 to about 15, or from about 10 to about 15 nucleotides.

[0130] In some embodiments, the primer site is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,

260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,

450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630,

640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,

830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides long, including all ranges and subranges therebetween.

[0131] In embodiments, the primer binding site comprises from 1 to about 50 nucleotides. In embodiments, the primer binding site comprises from about 6 to about 50, from about 7 to about 50, from about 8 to about 50, from about 9 to about 50, from about 10 to about 50, from about 11 to about 50, from about 12 to about 50, from about 13 to about 50, from about 14 to about 50, from about 15 to about 50, from about 16 to about 50, from about 17 to about 50, from about 18 to about 50, from about 19 to about 50, from about 20 to about 50, from about 21 to about 50, from about 22 to about 50, from about 23 to about 50, from about 24 to about 50, from about 25 to about 50, from about 26 to about 50, from about 27 to about 50, from about 28 to about 50, from about 29 to about 50, from about 30 to about 50, from about 31 to about 50, from about 32 to about 50, from about 33 to about 50, from about 34 to about 50, from about 35 to about 50, from about 36 to about 50, from about 37 to about 50, from about 38 to about 50, from about 39 to about 50, from about 40 to about 50, from about 5 to about 45, from about 6 to about 45, from about 7 to about 45, from about 8 to about 45, from about 9 to about 45, from about 10 to about 45, from about 11 to about 45, from about 12 to about 45, from about 13 to about 45, from about 14 to about 45, from about 15 to about 45, from about 16 to about 45, from about 17 to about 45, from about 18 to about 45, from about 19 to about 45, from about 20 to about 45, from about 21 to about 45, from about 22 to about 45, from about 23 to about 45, from about 24 to about 45, from about 25 to about 45, from about 26 to about 45, from about 27 to about 45, from about 28 to about 45, from about 29 to about 45, from about 30 to about 45, from about 31 to about 45, from about 32 to about 45, from about 33 to about 45, from about 34 to about 45, from about 35 to about 45, from about 36 to about 45, from about 37 to about 45, from about 38 to about 45, from about 39 to about 45, from about 40 to about 45, from about 5 to about 40, from about 6 to about 40, from about 7 to about 40, from about 8 to about 40, from about 9 to about 40, from about 10 to about 40, from about 11 to about 40, from about 12 to about 40, from about 13 to about 40, from about 14 to about 40, from about 15 to about 40, from about 16 to about 40, from about 17 to about 40, from about 18 to about 40, from about 19 to about 40, from about 20 to about 40, from about 21 to about 40, from about 22 to about 40, from about 23 to about 40, from about 24 to about 40, from about 25 to about 40, from about 26 to about 40, from about 27 to about 40, from about 28 to about 40, from about 29 to about 40, from about 30 to about 40, from about 5 to about 35, from about 6 to about 35, from about 7 to about 35, from about 8 to about 35, from about 9 to about 35, from about 10 to about 35, from about 11 to about 35, from about 12 to about 35, from about 13 to about 35, from about 14 to about 35, from about 15 to about 35, from about 16 to about 35, from about 17 to about 35, from about 18 to about 35, from about 19 to about 35, from about 20 to about 35, from about 21 to about 35, from about 22 to about 35, from about 23 to about 35, from about 24 to about 35, from about 25 to about 35, from about 26 to about 35, from about 27 to about 35, from about 28 to about 35, from about 29 to about 35, from about 30 to about 35, from about 5 to about 30, from about 6 to about 30, from about 7 to about 30, from about 8 to about 30, from about 9 to about 30, from about 10 to about 30, from about 11 to about 30, from about 12 to about 30, from about 13 to about 30, from about 14 to about 30, from about 15 to about 30, from about 16 to about 30, from about 17 to about 30, from about 18 to about 30, from about 19 to about 30, from about 20 to about 30, from about 21 to about 30, from about 22 to about 30, from about 23 to about 30, from about 24 to about 30, from about 25 to about 30, from about 5 to about 25, from about 6 to about 25, from about 7 to about 25, from about 8 to about 25, from about 9 to about 25, from about 10 to about 25, from about 11 to about 25, from about 12 to about 25, from about 13 to about 25, from about 14 to about 25, from about 15 to about 25, from about 16 to about 25, from about 17 to about 25, from about 18 to about 25, from about 19 to about 25, from about 20 to about 25, from about 5 to about 20, from about 6 to about 20, from about 7 to about 20, from about 8 to about 20, from about 9 to about 20, from about 10 to about 20, from about 11 to about 20, from about 12 to about 20, from about 13 to about 20, from about 14 to about 20, from about 15 to about 20, from about 1 to about 15, from about 2 to about 15, from about 3 to about 15, from about 4 to about 15, from about 5 to about 15, from about 6 to about 15, from about 7 to about 15, from about 8 to about 15, from about 9 to about 15, or from about 10 to about 15 nucleotides. In embodiments, the primer binding site comprises from 18 to 22 nucleotides.

[0132] In embodiments, the nucleic acid further comprises an adapter. In embodiments, the adapter comprises a nucleic acid that is complementary to a nucleic acid bound to a solid surface.

[0133] In embodiments, the nucleic acid further comprises a barcode. [0134] In embodiments, the nucleic acid further comprises a nucleotide comprising an azide. The nucleotide comprising an azide may be 8-Azido-adenosine-5'-monophosphate, 8-Azido- adenosine-5'-diphosphate, 8-Azido-adenosine-5'-triphosphate, 2'-Azido-2'-deoxyadenosine-5'- triphosphate; 3'-Azido-3'-deoxyadenosine-5'-triphosphate, 3'-Azido-2',3'-dideoxyadenosine- 5'-triphosphate, 3'-O-Azidomethyl-2'-deoxyadenosine-5'-triphosphate, y-(2-Azidoethyl)- adenosine-5'-triphosphate, y-(6-Azidohexyl)-adenosine-5'-triphosphate, y-[(6-Azidohexyl)- imido]-adenosine-5'-triphosphate, N6-(6-Azido)hexyl-adenosine-5'-triphosphate, N6-(6- Azido)hexyl-2'deoxy-adenosine-5'-triphosphate, N6-(6-Azido)hexyl-3'-deoxyadenosine-5'- triphosphate, 8-N3-ATP[y]biotinpentylamine, adenosine-5'-monophosphate, 8-Azido- adenosine-5'-monophosphate, 2'-Azido-2'-deoxyadenosine-5'-monophosphate; 3'-Azido-3'- deoxyadenosine-5'-monophosphate, 3'-Azido-2',3'-dideoxyadenosine-5'-monophosphate, 3'- O-Azidomethyl-2'-deoxyadenosine-5'-monophosphate, y-(2-Azidoethyl)-adenosine-5'- monophosphate, y-(6-Azidohexyl)-adenosine-5'-monophosphate, y-[(6-Azidohexyl)-imido]- adenosine-5'-monophosphate, N6-(6-Azido)hexyl-adenosine-5'-monophosphate, N6-(6- Azido)hexyl-2'deoxy-adenosine-5'-monophosphate, N6-(6-Azido)hexyl-3'-deoxyadenosine- 5'-monophosphate, 3'-Azido-3'-deoxyguanosine-5'-triphosphate, 3'-Azido-3'-deoxyguanosine- 5'-monophosphate, 3'-O-Azidomethyl-2'-deoxyguanosine-5'-triphosphate, 3'-O-Azidomethyl- 2'-deoxyguanosine-5'-monophosphate, 3'-Azido-2',3'-dideoxyguanosine-5'-triphosphate, 3'- Azido-2',3'-dideoxyguanosine-5'-monophosphate, 3'-Azido-3'-deoxyuridine-5'-triphosphate, 3'-Azido-3'-deoxyuridine-5'-monophosphate, 3'-Azido-2',3'-dideoxyuridine-5'-triphosphate, 3'-Azido-2',3'-dideoxyuridine-5'-monophosphate, 5-Azido-PEG4-uridine-5'-triphosphate, 5- Azido-PEG4-uridine-5'-monophosphate, 5-(3-Azidopropyl)-uridine-5'-triphosphate, 5-(3- Azidopropyl)-uridine-5'-monophosphate, 5-Azidomethyl-uridine-5'-triphosphate, 5- Azidomethyl-uridine-5'-monophosphate, 5-Azidomethyl-2'-deoxyuridine-5'-triphosphate, 5- Azidomethyl-2'-deoxyuridine-5'-monophosphate, 5-(15-Azido-4,7,10,13-tetraoxa- pentadecanoyl-aminoallyl)-2'-deoxyuridine-5'-triphosphate, 5-(15-Azido-4,7,10,13-tetraoxa- pentadecanoyl-aminoallyl)-2'-deoxyuridine-5'-monophosphate, 5-Azido-PEG4-cytidine-5'- triphosphate, 5-Azido-PEG4-cytidine-5'-monophosphate, 5-Azido-PEG4-2'-deoxycytidine-5'- triphosphate, 5-Azido-PEG4-2'-deoxycytidine-5'-monophosphate, 3'-Azido-3'-deoxycytidine- 5'-triphosphate, 3'-Azido-3'-deoxycytidine-5'-monophosphate, 3'-Azido-2',3'-dideoxycytosine- 5'-triphosphate, 3'-Azido-2',3'-dideoxycytosine-5'-monophosphate, 3'-O-Azidomethyl-2'- deoxycytidine-5'-triphosphate, 3'-O-Azidomethyl-2'-deoxycytidine-5'-monophosphate, Cytidine-5'-phosphate-3'-(15-azido-4,7,10,13-tetraoxa-pentad ecanoyl-6- aminohexyl)phosphate, 3'-Azido-2',3'-dideoxythymidine-5'-monophosphate, 3'-Azido-2',3'- dideoxythymidine-5'-triphosphate, and 3'-O-Azidomethyl-2'-deoxythymidine-5'-triphosphate. [0135] In embodiments, the nucleic acid comprises the sequence of any one of SEQ ID NOS: 1-2 or a sequence with at least 80 %, at least 81 % at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identity to any one of SEQ ID NOS: 1-2.

[0136] In embodiments, the polymer beads are bound to the nucleic acids via a linker that binds specifically to a biomarker described herein. In embodiments, the linker is an antibody or an antigen binding fragment thereof. In embodiments the linker is an aptamer.

III. Methods of Quantitatively Measuring Biomarker Concentration in a Sample [0137] The methods described herein allow for the absolute quantitation of biomarkers in a sample. Provided herein are methods of quantitatively measuring biomarker concentration in a sample, comprising: (i) providing a sample comprising a target biomarker, wherein the target biomarker is bound to a population of nucleic acids, said nucleic acids comprising: (a) a primer binding site; and (b) a unique molecular identifier (UMI); wherein each nucleic acid comprises a different UMI; (ii) amplifying the nucleic acids comprising the UMI; and (iii) sequencing the amplified nucleic acids; wherein the total number of different UMIs detected corresponds to the number of target biomarkers in the sample. Provided herein are methods comprising quantitatively measuring biomarker concentration in a sample comprising a target biomarker, the method comprising: (i) contacting the sample with a population of nucleic acids capable of selectively binding to the target biomarker; said nucleic acids comprising: (a) a primer binding site; and (b) a unique molecular identifier (UMI); wherein each nucleic acid comprises a different UMI; (ii) separating unbound nucleic acid from the sample; (iii) amplifying the nucleic acid comprising UMI after step (ii) (i.e amplifying the subset of nucleic acids that were bound to the target biomarker); and (iv) sequencing the amplified nucleic acids; wherein the total number of different UMIs detected corresponds to the number of target biomarkers in the sample.

III-A. Samples

[0138] In embodiments, the methods comprise quantitatively measuring the biomarker concentration in a sample. In embodiments, the sample is a polymer bead, cell, or surface. The polymer bead may be any bead described herein, for example, any polymer bead described in Section II-A of this application.

[0139] In embodiments, the sample is a single cell. In embodiments, the sample is a population of cells. In embodiments, the sample is a cell membrane. In embodiments, the sample comprises a lymphocyte, a monocyte, a granulocyte, or a combination thereof. In embodiments, the target cell is a prokaryotic cell. In embodiments, the target cell is a eukaryotic cell. In embodiments, the target cell is a white blood cell. In embodiments, the cell is a platelet. In embodiments, the target cell is a red blood cell. In embodiments, the cell is an immune cell. In embodiments, the immune cell is a T cell, a B cell, an NK cell, a lymphocyte, a monocyte, a granulocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a macrophage, or a dendritic cell. In embodiments, the cell is a peripheral blood mononuclear. In embodiments, the sample comprises a cell membrane from any one of the cells described herein.

[0140] In embodiments, the sample is a surface. In embodiments, the surface is a microplate.

[0141] In embodiments, the sample comprises a target biomarker. The target biomarker may be any biomarker described herein, for example, any biomarker described in Section II-B of this application.

III-B. Nucleic Acids

[0142] In embodiments, the methods comprise contacting a sample with a population of nucleic acids capable of selectively binding to the target biomarker. Each nucleic acid in the population comprise (a) a primer binding site; and (b) a unique molecular identifier (UMI); wherein each nucleic acid comprises a different UMI. Suitable nucleic acids for the methods described herein are described in Section II-C of this application.

III-C. Linkers

[0143] In embodiments, the nucleic acid is bound to the sample via a linker. In embodiments, the linker selectively binds to the target biomarker. In embodiments, the linker is an antibody or an antigen-binding fragment thereof. Non-limiting examples of antigen-binding antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins (“scFv”), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (“dsFv”), singledomain antibodies (sdAb, nanobody), heavy-chain only antibodies (e.g., camelid VHH, camelid nanobody, shark Ig NAR), a variable heavy domain, and a variable light domain. In some embodiments, the antibody or antigen-binding fragment thereof is monoclonal. In some embodiments, the antibody or antigen-binding fragment thereof is polyclonal.

[0144] In embodiments, a nucleic acid described herein is covalently attached to the linker. In embodiments, a nucleic acid described herein is non-covalently attached to the linker. In embodiments, the nucleic acid is covalently attached to an antibody linker using an oYo-Link® (described at alphathera.com) or GLYCLICK® methods (described at genovis.com/smartenzymes/antibody-conjugation/glyclick/). The following publication describes the oYo-Link® method and is incorporated by reference herein in its entirety for all purposes: Niu et al. Methods Mol Bio. 2023;2593: 113=136.

[0145] In embodiments, the nucleic acid is attached to the Fc region of an antibody or antigen fragment. In embodiments, the nucleic acid comprises an azide or alkyne group. In embodiments, a linker comprises an azide or an alkyne group. In embodiments, the nucleic acid is attached to the linker via a triazole. The triazole may be formed by reacting a nucleic acid comprising an azide with a linker comprising an alkyne. Alternatively, the triazole may be formed by reacting a nucleic acid comprising an alkyne with a linker comprising an azide.

[0146] U.S. Publication No. 20210381040 describes alternative methods for attaching nucleic acids to linkers and is incorporated by reference herein in its entirety.

[0147] In embodiments, the linker is an aptamer. In embodiments, the linker is a T cell receptor (TCR) or a fragment thereof. In embodiments, the linker is the ligand of a biomarker described herein. For example, the ligand of the biomarker PD1 is PD-L1.

III-D. Separating Unbound Nucleic Acid from Samples

[0148] In embodiments, the methods described herein comprise separating nucleic acid that is not bound to the biomarker. In embodiments, separating nucleic acid that is not bound to the biomarker comprises washing the sample with a buffer. In embodiments, the buffers comprises sodium acetate, saline, glycine-HCL, cacodylate buffer, Tris-HCl, 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N- morpholino)propanesulfonic acid (MOPS), citrate, phosphate buffer, tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), and tris(hydroxymethyl)aminomethane (Tris). In some embodiments, the buffer comprises one or more of arginine, histidine, urea, pluronic acid, and octoxynol-9 (Triton-X-100™).

[0149] In embodiments, the pH of the buffer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, including all ranges and subranges therebetween. [0150] In embodiments, the pH of the buffer is from about 1 to about 14, from about 2 to about 14, from about 3 to about 14, from about 4 to about 14, from about 5 to about 14, from about 6 to about 14, from about 7 to about 14, from about 8 to about 14, from about 9 to about 14, from about 10 to about 14, from about 11 to about 14, from about 12 to about 14, from about 1 to about 13, from about 2 to about 13, from about 3 to about 13, from about 4 to about 13, from about 5 to about 13, from about 6 to about 13, from about 7 to about 13, from about 8 to about 13, from about 9 to about 13, from about 10 to about 13, from about 11 to about 13, from about 1 to about 12, from about 2 to about 12, from about 3 to about 12, from about 4 to about 12, from about 5 to about 12, from about 6 to about 12, from about 7 to about 12, from about 8 to about 12, from about 9 to about 12, from about 10 to about 12, from about 1 to about 11, from about 2 to about 11, from about 3 to about 11, from about 4 to about 11, from about 5 to about 11, from about 6 to about 11, from about 7 to about 11, from about 8 to about 11, from about 9 to about 11, from about 1 to about 10, from about 2 to about 10, from about 3 to about 10, from about 4 to about 10, from about 5 to about 10, from about 6 to about 10, from about 7 to about 10, from about 8 to about 10, from about 1 to about 9, from about 2 to about 9, from about 3 to about 9, from about 4 to about 9, from about 5 to about 9, from about 6 to about 9, from about 7 to about 9, from about 1 to about 8, from about 2 to about 8, from about 3 to about 8, from about 4 to about 8, from about 5 to about 8, from about 6 to about 8, from about 1 to about 7, from about 2 to about 7, from about 3 to about 7, from about 4 to about 7, from about 5 to about 7, from about 1 to about 6, from about 2 to about 6, from about 3 to about 6, from about 4 to about 6, from about 5 to about 6, from about 1 to about 5, from about 2 to about 5, from about 3 to about 5, from about 4 to about 5, from about 1 to about 4, from about 2 to about 4, from about 3 to about 4, from about 1 to about 3, from about 2 to about 3, or from about 1 to about 2.

[0151] In embodiments, separating nucleic acid that is not bound to the biomarker comprises (i) contacting the sample with a magnetic bead that binds to the unbound nucleic acid; and (ii) exposing the sample to a magnetic field.

[0152] In some embodiment, the polymer particles of the present disclosure are magnetic, such that the separating step can involve applying a magnetic field to the sample and taking out any unmagnetized portion of the sample (e.g., pipetting it out or pouring it out). In embodiments the separating step can be inserting a magnetic probe into the sample to separate out polymer beads into a another container. Non-limiting suitable examples of magnetic beads that bind to DNA are OMEGA BIO-TEK®’s Mag-Bind® magnetic beads (SKU: M1378-01), Thermo Fischer Scientific®’ s DYNABEADS®. III-E. Amplifying Nucleic Acids

[0153] In embodiments, amplification techniques comprise at least one cycle of amplification, for example, but not limited to, the steps of denaturing a double-stranded nucleic acid to separate the component strands; hybridizing a primer to a target flanking sequence or a primerbinding site of an amplicon (or complements of either, as appropriate); and synthesizing a strand of nucleotides in a template-dependent manner using a DNA polymerase. In some embodiments the amplification step is polymerase chain reaction amplification. In certain embodiments, a cycle of amplification comprises the steps of denaturing a double-stranded nucleic acid to separate the component strands; hybridizing a first ligation probe and a corresponding second ligation probe to (1) the target nucleic acid or the complement of the target nucleic acid or (2) an amplicon; and ligating the adjacently hybridized probes with a ligase to form a ligated probe (an exemplary amplicon). In certain embodiments, a cycle of amplification comprises the steps of denaturing a double-stranded nucleic acid to separate the component strands; hybridizing an upstream cleavage probe and a corresponding downstream cleavage probe to (1) the target nucleic acid or the complement of the target nucleic acid or (2) an amplicon, to form a nucleic acid cleavage structure; cleaving the cleavage structure to release the flap and form a hybridization structure comprising the upstream cleavage probe annealed adjacent to the hybridized fragment of the downstream cleavage probe; and optionally ligating the adjacently hybridized probes with a ligase to form a ligated probe. The cycle may or may not be repeated. In certain embodiments, a cycle of amplification comprises a multiplicity of amplification cycles, for example but not limited to 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles or more than 45 cycles of amplification.

[0154] In some embodiments, amplifying comprises thermocycling using an instrument, for example but not limited to, a GeneAmp® PCR System 9700, 9600, 2700, or 2400 thermocycler (all from Applied Biosystems), a PTC Tempo Thermal Cycler, Cl 000 Touch Thermal Cycler, SI 000™ Thermal Cycler, and T100 Thermal Cycler (all from Bio-Rad Laboratories), SureCycler 8800 Thermal Cycler (Agilent Technologies), ProFlex, Veriti® Thermal Cycler, QuantStudio 5 (all from Thermo Fisher Scientific), PCRmax Alpha Cycler 4 (PCRMax Limited), Prime Thermal Cycler (Techne), Mastercycler x50 (Eppendorf North America), and AlllnOneCycler (Bioneer). In certain embodiments, single-stranded amplicons are generated in an amplification reaction, for example but not limited to asymmetric PCR or A-PCR.

[0155] Devices have been developed that can perform a thermal cycling reaction and detection with reaction compositions containing a nucleic acid dye, emit a light beam of a specified wavelength, read the intensity of the fluorescent signal emitted from the nucleic acid dye molecules associated with double-stranded nucleic acids, and display the intensity of fluorescence after each cycle. Devices comprising a thermal cycler, light beam emitter, and a fluorescent signal detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907; 6,015,674; and 6,174,670, and include, but are not limited to the ABI Prism® 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.), the ABI GeneAmp® 5700 Sequence Detection System (Applied Biosystems, Foster City, Calif.), LightCycler 480, LightCycler 1.5, andLightCycler 2.0 (all from Roche), Mx4000, Mx3000P, and Mx3005P (all from Statagene), SmartCycler (Cepheid), Rotor-Gene 6000 (Corbett), Mastercycle ep realplex (Eppendorf North America), MiniOpticon, MyiQ, Opticon2, Chromo4, iQ5 (all from Bio-Rad Laboratories).

[0156] In some embodiments, digital polymerase chain reaction (dPCR) or droplet dPCR (ddPCR) can be utilized. dPCR is a refinement of conventional PCR and can be used to directly quantify and clonally amplify nucleic acids, e.g., DNA, cDNA or RNA. Conventional PCR is generally used for measuring nucleic acid amounts and is carried out by a single reaction per sample. Utilizing dPCR methodology, a single reaction is also carried out on a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows for a more reliable collection and sensitive measurement of nucleic acid amounts.

[0157] In dPCR, a sample is partitioned so that individual nucleic acid molecules within the sample are localized and concentrated within many separate regions. The capture or isolation of individual nucleic acid molecules can be performed in micro well plates, capillaries, the dispersed phase of an emulsion, and arrays of miniaturized chambers, as well as on nucleic acid binding surfaces. The partitioning of the sample allows one to estimate the number of different molecules by assuming that the molecule population follows the Poisson distribution. As a result, each partitioned sample will contain “0” or “1” molecules, or a negative or positive reaction, respectively. After PCR amplification, nucleic acids can be quantified by counting the regions that contain PCR end-product, positive reactions. In conventional PCR, the number of PCR amplification cycles is proportional to the starting copy number. dPCR, however, is not dependent on the number of amplification cycles to determine the initial sample amount, eliminating the reliance on uncertain exponential data to quantify target nucleic acids and therefore provides absolute quantification.

[0158] The dPCR utilized herein may include, but is not limited to, a Digital LightCycler (Roche Sequencing), QX 100™ and QX600™ Droplet Digital™ PCR System (Bio-Rad Laboratories), BioMark HD dPCR system (Fluidigm), OpenArray® and QuantStudio® 12K Flex dPCR systems (Life Techol ogies), and RainDrop™ Instrument (RainDance Technologies).

[0159] Those in the art will appreciate that the disclosed enzyme inhibitors, complexes, methods, and kits can be applied in a variety of different contexts in which an enzyme-mediated amplification reaction is performed that may be subject to mis-annealing of primers and/or probes and the subsequent formation of undesired secondary amplicons. Any enzyme-mediated amplification technique that can benefit from the use of an enzyme inhibitor comprising a quencher to at least decrease background fluorescence is within the intended scope of the current teachings.

[0160] An amplified or sequenced target nucleic acid can be detected by any suitable technique known in the art that comprises measuring, quantitating, and/or observing directly or indirectly, a quenchable emission, including without limitation, fluorescence, chemiluminescence, bioluminescence, phosphorescence, and so forth, for example but not limited to, laser-induced fluorescence and electrochemiluminescence. According to some embodiments of the disclosed methods, detecting can comprise any suitable real-time or end-point detection technique. Some non-limiting examples of suitable detection techniques include melting curve analysis, Q-PCR or other real-time technique comprising a nucleic acid dye, and in some embodiments, at least one reporter probe; and electrophoresis techniques, including without limitation gel electrophoresis. Those in the art will appreciate that various quencher moieties are available that collectively cover a broad range of detectable emissions and that by pairing a quencher with an appropriate absorption spectra with an emission source, at least some of the emission from that source can be reduced.

[0161] In some embodiments, the methods of the current teachings comprise Q-PCR. The term “quantitative PCR”, or “Q-PCR”, also known as real-time PCR, refers to a variety of methods used to quantify PCR amplification products, either specifically, non-specifically, or both (see, e.g., Raeymakers, Mol. Biotechnol. 15: 115-22 (2000); Joyce, Quantitative RT-PCR, in Methods in Mol Biol., vol. 193, O'Connell, ed., Humana Press; Pierson et al., Nucl. Acids Res. 3 l(14):e73 (2003)). Such methods typically are categorized as kinetics-based systems, that generally determine or compare the amplification factor, such as determining the threshold cycle (Ct), or as co-amplification methods, that generally compare the amount of product generated from simultaneous amplification of target and standard templates. Q-PCR techniques typically comprise reporter probes, a nucleic acid dye, or both. For example but not limited to TaqMan® probes (Applied Biosystems), i-probes, molecular beacons, Eclipse probes, scorpion primers, Lux™ primers, FRET primers, ethidium bromide, and unsymmetrical cyanine dyes, for example but not limited to, SYBR® Green I (Molecular Probes), YO-PRO-1, Hoechst 33258, BOXTO (TATAA Biocenter, Goteborg, Sweden) and PicoGreen® (Molecular Probes).

III-F. Sequencing Amplified Nucleic Acids

[0162] In some embodiments, the methods herein are performed before or in conjunction with a sequencing reaction. The term “sequencing” is used in a broad sense herein and refers to any technique known in the art that allows the order of at least some consecutive nucleotides in at least part of a polynucleotide, for example but not limited to a target nucleic acid or an amplicon, to be identified. Some non-limiting examples of sequencing techniques include Sanger's dideoxy terminator method and the chemical cleavage method of Maxam and Gilbert, including variations of those methods; sequencing by hybridization; sequencing by synthesis; and restriction mapping. Some sequencing methods comprise electrophoreses, including capillary electrophoresis and gel electrophoresis; sequencing by hybridization including microarray hybridization; mass spectrometry; and single molecule detection. In some embodiments, sequencing comprises direct sequencing, duplex sequencing, cycle sequencing, single base extension sequencing (SBE), solid-phase sequencing, or combinations thereof.

[0163] In some embodiments, sequencing comprises detecting the sequencing product using an instrument, for example but not limited to an ABI PRISM® 377 DNA Sequencer, an ABI PRISM® 310, 3100, 3100-Avant, 3730, or 3730x1 Genetic Analyzer, an ABI PRISM® 3700 DNA Analyzer (all from Applied Biosystems), a mass spectrometer, MiSeq, HiSeq 3000/4000, NextSeq 1000/2000, NovaSeq - SI, S2, S4, SP, NovaSeq X - 1.5B, 10B, 25B, and NovaSeq X Plus - 1.5, 10B, 25B (all from Illumina), PacBio RS, PacBio Revio, and PacBio Sequel (all from PacBio), PromethlON 2, GridlON, and MinlON (all from Oxford Nanopore), PGM 314 Chip, Proton I Chip, and SS/SS XL 520 Chip (all from Ion Torrent), GS FLX 1 PTP (Roche454), AVITI (Element Biosciences), DNBSEQ-E25, DNBSEQ-G99, and DNBSEQ-T7 (all form Complete Genomics), and G4-F2 and G4-F3 (both from Singular Genomics).

[0164] In some embodiments, sequencing comprises incorporating a dNTP, including a dATP, a dCTP, a dGTP, a dTTP, a dUTP, a diTP, or combinations thereof and including dideoxyribonucleotide analogs of dNTPs, into an amplification product.

[0165] Those in the art will appreciate that the sequencing method employed is not typically a limitation of the present methods. Rather any sequencing technique that provides the order of at least some consecutive nucleotides of at least part of the corresponding amplicon or target nucleic acid can typically be used with the current methods. In some embodiments, unincorporated primers and/or dNTPs are removed prior to a sequencing step by enzymatic degradation, including without limitation exonuclease I and shrimp alkaline phosphatase digestion, for example but not limited to the ExoSAP-IT® reagent (USB Corp., Cleveland, Ohio). In some embodiments, unincorporated primers, dNTPs, and/or ddNTPs are removed by gel or column purification, sedimentation, filtration, beads, magnetic separation, or hybridization-based pull out, as appropriate (see, e.g., ABI PRISM® Duplex™ 384 Well F/R Sequence Capture Kit, Applied Biosystems P/N 4308082).

[0166] In certain embodiments, a reaction composition comprising an amplification product, or at least part of such a reaction composition, is subjected to a sequencing reaction without an intervening purification step (see, e.g., Baskin et al., U.S. Patent Application Publication No. US 2002/0137047 Al). Descriptions of sequencing techniques can be found in, among other places, McPherson, particularly in Chapter 5; Sambrook and Russell; Ausubel et al.; Siuzdak, The Expanding Role of Mass Spectrometry in Biotechnology, MCC Press, 2003, particularly in Chapter 7; and Rapley, particularly in Part VI.

[0167] The following documents described next generation sequencing and are incorporated by reference herein in their entireties for all purposes: U.S. Patent No. 9957564; U.S. Publication No. 2021/0254154; Qin. Cancer Biol Med. 2019 Feb; 16(1): 4-10; and International Publication No. 2011/139371.

NUMBERED EMBODIMENTS OF THE INVENTION

[0168] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

[0169] Embodiment Al. A method of quantitatively measuring biomarker concentration in a sample, comprising (i) providing a sample comprising a first population of target biomarkers bound to a first population of nucleic acids, each of said nucleic acids comprising (a) a primer binding site, and (b) a unique molecular identifier (UMI), wherein each nucleic acid in the first population of nucleic acids comprises a different UMI, (ii) amplifying the nucleic acids comprising the UMI, and (iii) sequencing the amplified nucleic acids, wherein the total number of different UMIs detected corresponds to the number of target biomarkers in the sample.

[0170] Embodiment A2. A method of quantitatively measuring biomarker concentration in a sample comprising a target biomarker, the method comprising (i) contacting the sample with a first population of nucleic acids capable of selectively binding to the target biomarker, each of said nucleic acids comprising (a) a primer binding site, and (b) a unique molecular identifier (UMI), wherein each nucleic acid in the first population of nucleic acids comprises a different UMI, (ii) separating unbound nucleic acids from the sample, (iii) amplifying the nucleic acids comprising the UMI after step (ii), and (iv) sequencing the amplified nucleic acids, wherein the total number of different UMIs detected corresponds to the number of target biomarkers in the sample.

[0171] Embodiment A3. The method of any one of embodiments A1-A2, wherein the sample comprises a cell, a bead, or a surface.

[0172] Embodiment A4. The method of any one of embodiments A1-A3, wherein the sample comprises one or more fluorophores.

[0173] Embodiment A5. The method embodiment A4, wherein the fluorophore is selected from of the group consisting of: peridinin chlorophyll protein-cyanine 5.5 dye (PerCP-Cy5.5); phycoerythrin-cyanine7 (PE Cy7); allophycocyanin-cyanine 7 (APC-Cy7); fluorescein isothiocyanate (FITC); phycoerythrin (PE); allophyscocyanin (APC); 6-carboxy-4', 5'- dichloro-2', 7'-dimethoxyfluorescein succinimidylester; 5-( and-6)-carboxyeosin; 5- carboxyfluorescein; 6 carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S- carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether ,-alanine-carboxamide, or succinimidyl ester; 5-carboxy fluorescein succinimidyl ester; 6-carboxyfluorescein succinimidyl ester; 5-( and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl) amino fluorescein; 2', 7'-difluoro fluorescein; eosin-5-isothiocyanate; erythrosin5- isothiocyanate;6-( fluorescein-5-carboxamido) hexanoic acid or succinimidyl ester; 6- (fluorescein-5-( and-6)-carboxamido) hexanoic acid or succinimidylester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen® 488 carboxylic acid, or succinimidyl ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimidyl ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimidylester or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimidyl ester; RhodamineGreen™ carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidylester; Rhodamine Green™-X succinimidyl ester or hydrochloride; RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidylester; bis-(4- carboxypiperidinyl) sulfonerhodamine or di(succinimidylester); 5-( and- 6)carboxynaphtho fluorescein, 5 -( and-6)carboxynaphthofluorescein succinimidyl ester;5- carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine6Ghydrochloride, 5- carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-( and- 6)-carboxyrhodamine6G succinimidyl ester; 5-carboxy-2',4',5',7'- tetrabromosulfonefluorescein succinimidyl esteror bis-( diisopropylethylammonium) salt; 5- carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)- carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimidyl ester; 6- carboxytetramethylrhodaminesuccinimidyl ester; 5-(and -6)-carboxytetramethylrhodamine succinimidyl ester;6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester; 6- carboxy-X-rhodamine succinimidyl ester; 5-( and-6)-carboxy-X-rhodamine succinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; NANOGOLD® mono(sulfosuccinimidyl ester); QSY® 21carboxylic acid or succinimidyl ester; QSY® 7 carboxylic acid or succinimidyl ester; Rhodamine RedTM-X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido) hexanoic acid; succinimidyl ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; X-rhodamine-5-(and-6) isothiocyanate, BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630/650-X STPester; BODIPY® 650/665-X STP ester; 6-dibromo-4, 4-difluoro-5, 7 -dimethyl-4-bora-3 a, 4a-diaza-s-indacene-3 -propionic acid succinimidyl ester; 4,4-difluoro- 4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid; 4,4- difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-pentanoicacid; 4,4-difluoro-5,7- dimethyl-4-bora3a,4a-diaza-s-indacene- 3-pentanoicacid succinimidyl ester; 4,4-difluoro-5,7- dimefhyl-4-bora-3 a, 4a-diaza-s- indacene-3propionicacid; 4, 4-difluoro-5, 7 -dimethyl-4-bora- 3 a, 4adiaza-s-indacene-3- propionicacid succinimidyl ester; 4, 4-difluoro-5, 7 -dimefhyl-4-bora- 3a,4a-diaza-s-indacene- 3propionic acid; sulfosuccinimidyl ester or sodium salt; 6-(( 4,4- difluoro-5, 7 -dimethyl-4- bora-3a,4a-diaza-s-indacene-3propionyl)amino)hexanoicacid; 6-( ( 4,4-difluoro-5, 7 dimethyl- 4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester; N-(4, 4-difluoro 5, 7 -dimethyl-4-bora-3 a, 4a-diaza-s-indacene-3-propionyl) cysteic acid, succinimidyl ester or triethylammonium salt; 6-4,4-difluoro-l,3- dimethyl-5-( 4- methoxyphenyl)-4-bora3a, 4a4, 4-difluoro-5, 7-diphenyl-4-bora-3a,4a-diaza-sindacene-3- propionicacid; 4, 4-difluoro-5, 7 -diphenyl-4-bora3 a, 4a-diaza-s-indacene-3- propionicacid succinimidyl ester; 4, 4-difluoro-5-phenyl-4-bora-3 a, 4a-diaza-s-indacene-3- propionic acid; succinimidyl ester; 6-(( 4, 4-difluoro-5-phenyl-4 bora-3 a, 4a-diaza-s-indacene-3- propionyl)amino) hexanoicacid or succinimidyl ester; 4,4-difluoro-5-(4-phenyl- l,3butadienyl)-4-bora-3 a, 4a-diaza-s-indacene-3-propionicacid succinimidyl ester; 4, 4- difluoro-5-(2- pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6- (((4,4- difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3- yl)styryloxy)acetyl)aminohexanoicacid or succinimidyl ester; 4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid; 4, 4-difluoro-5 -styryl-4-bora-3 a, 4a-diaza-sindacene- 3-propionic acid; succinimidyl ester; 4,4-difluoro-l,3,5,7-tetramethyl-4-bora-3a,4adiaza-s- indacene-8-propionicacid; 4,4-difluoro-l,3,5,7-tetramethyl-4bora-3a,4a-diaza-sindacene - 8- propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-sindacene- 3- propionic acid succinimidyl ester; 6-( ( ( 4-( 4, 4-difluoro-5 -(2-thienyl)-4-bora-3 a, 4adiazas- indacene-3-yl)phenoxy)acetyl)amino )hexanoic acid or succinimidyl ester; and 6-(((4,4- difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3- yl)styryloxy)acetyl) aminohexanoic acid or succinimidyl ester, Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid; Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532 carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 64 7 carboxylic acid; Alexa Fluor® 660 carboxylic acid; Alexa Fluor® 680 carboxylic acid, Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHSester; and Cy7 NHS ester.

[0174] Embodiment A6. The method of any one of embodiments A1-A5, wherein the sample comprises a bead.

[0175] Embodiment A7. The method of embodiment A6, wherein the bead is a polymer bead. [0176] Embodiment A7.1. The method of embodiment A7, wherein the polymer bead has an average diameter ranging from about 1 pm to about 20 pm. [0177] Embodiment A7.2. The method of embodiment A7, wherein the polymer bead has an average diameter ranging from about 5 pm to about 40 pm.

[0178] Embodiment A7.3. The method of embodiment A7, wherein the polymer bead has an average diameter ranging from about 5 pm to about 10 pm.

[0179] Embodiment A8. The method of embodiment A7, wherein the polymer bead comprises less than 10%, 20%, 30%, or 40% polystyrene by hydrated volume.

[0180] Embodiment A9. The method of embodiment A7, wherein the polymer beads comprise less than 10%, 20%, 30%, or 40% polystyrene by dehydrated volume.

[0181] Embodiment A10. The method of any one of embodiments A7-A9, wherein the polymer beads are hydrogel beads.

[0182] Embodiment Al l. The method of embodiment A10, wherein the hydrogel comprises a monomer.

[0183] Embodiment A12. The method of embodiment Al l, wherein the monomer is hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxypoly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2- phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6- tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2, 3 -dibromopropyl acrylate, 2,3- dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4- methoxybenzylacrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2- benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenyl acrylamide, Nphenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N- dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide N-(4- methylphenyl)methyl acrylamide, N-l -naphthyl acrylamide, N-4-nitrophenyl acrylamide, N- (2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4- hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)m ethyl methacrylamide, N-l -naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl)methacrylamide, N- triphenylmethyl methacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenyl methacrylamide, N'-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, or a combination thereof.

[0184] Embodiment A12.1. The method of embodiment Al l, wherein the monomer is a dissolvable monomer or a biodegradable monomer.

[0185] Embodiment A12.2. The method of embodiment A12.1, wherein the biodegradable monomer is a monosaccharide, disaccharide, polysaccharide, peptide, protein, or protein domain.

[0186] Embodiment A12.3. The method of embodiment A12.1, wherein the biodegradable monomer is a protein or protein domain comprising at least one non-natural amino acid.

[0187] Embodiment A12.4. The method of embodiment A12.1, wherein the biodegradable monomer is a structural polysaccharide.

[0188] Embodiment A12.5. The method of embodiment A12.1, wherein the biodegradable monomer is agar, agarose, alginic acid, alguronic acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta-glucan, callose, capsullan, carrageenan polysaccharide, cellodextrin, cellulin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminoogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectc acid, pectin, pentastarch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof.

[0189] Embodiment A12.6. The method of embodiment A12.1, wherein the biodegradable monomer is chitosan or hyaluronan.

[0190] Embodiment A12.7. The method of embodiment A12.2, wherein the protein is a structural protein, a domain thereof, or a combination thereof.

[0191] Embodiment A12.8. The method of embodiment A12.2, wherein the protein is a proteoglycan, a domain thereof, or a combination thereof.

[0192] Embodiment A12.9. The method of embodiment A12.2, wherein the protein is an extracellular matrix component. [0193] Embodiment A12.10. The method of embodiment A12.2, wherein the protein is collagen, elastin or a proteoglycan.

[0194] Embodiment A12. l l. The method of embodiment A12.10, wherein the collagen is collagen type I, collagen type II, collagen type III, a domain thereof or a combination thereof. [0195] Embodiment A12.12. The method of embodiment A12.1, wherein the dissolvable monomer is dissolvable by one or more of chemical means and physical means.

[0196] Embodiment A13. The method of any one of embodiments A7-A12.12, wherein the polymer beads comprise a known optical property.

[0197] Embodiment A13.1. The method of any one of embodiments A7-A13, wherein each polymer bead exhibits at least one optical property having a quantitative profile that is substantially similar to a quantitative profile of a corresponding optical property of a target cell.

[0198] Embodiment A14. The method of embodiment A13 or A13.1, wherein the at least one optical property comprises side scatter (SSC).

[0199] Embodiment A14.1. The method of embodiment A14, wherein the polymer bead comprises scatter-modulating additives.

[0200] Embodiment A14.2. The method of embodiment A14.1, wherein the scattermodulating additives comprises one or more of a nanoparticle, a colloidal silica, an encapsulated material, and a chemical side-group.

[0201] Embodiment A15. The method of embodiment A14, wherein the scatter comprises forward scatter (FSC).

[0202] Embodiment Al 5.1. The method of embodiment Al 5, wherein the forward scatter is defined by a refractive index (RI) of each of the polymer beads.

[0203] Embodiment A15.2. The method of embodiment A15.1, wherein the sample comprises polymer beads with an RI of greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.1 0, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0204] Embodiment A15.3. The method of embodiment A15.1, wherein the sample comprises polymer beads with an RI of about 1.10 to about 3.0, or about 1.15 to about 3.0, or about 1.20 to about 3.0, or about 1.25 to about 3.0, or about 1.30 to about 3.0, or about 1.35 to about 3.0, or about 1.4 to about 3.0, or about 1.45 to about 3.0, or about 1.50 to about 3.0, or about 1.6 to about 3.0, or about 1.7 to about 3.0, or about 1.8 to about 3.0, or about 1.9 to about 3.0, or about 2.0 to about 3.0.

[0205] Embodiment A15.4. The method of embodiment A15.1, wherein the sample comprises polymer beads with an RI of less than about 1.1 0, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.

[0206] Embodiment A16. The method of any one of embodiments A13-A15.4, wherein the optical property comprises forward scatter and side scatter

[0207] Embodiment A17. The method of any one of embodiments A1-A5, wherein the sample comprises a cell.

[0208] Embodiment A18. The method of embodiment A17, wherein the cell is selected any one of natural killer cells, B cells, or T cells.

[0209] Embodiment A19. The method of any one of embodiments A1-A5, wherein the sample comprises peripheral blood mononuclear cells.

[0210] Embodiment A20. The method of any one of embodiments Al -Al 9, wherein the target biomarker is attached to a solid surface.

[0211] Embodiment A21. The method of embodiment A20, wherein the solid surface is a microplate .

[0212] Embodiment A22. The method of any one of embodiments A1-A21, wherein the target biomarker is selected from the group consisting of: CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-gamma, CD20, TCR gamma/delta, TNF alpha, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CDl lc, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, TCR V alpha 7.2, CDl lb, CD21, CD24, IL-4, Biotin, CCR10, CD31, CD44, CD 138, CD294, NKp46, TCR V delta 2, TIGIT, CDlc, CD2, CD7, CD8a, CD15, CD32, CD103, CD107a, CD141, CD158, CD159c, IL-13, IL-21, KLRG1, TIM- 3, CCR5, CD5, CD33, CD45.2, CD80, CD159a (NKG2a), CD244, CD272, CD278, CD337, Granzyme B, Ig Lambda Light Chain, IgA, IL- 17 A, Streptavidin, TCR V delta 1, CD Id, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD 137, CD 163, CD 193, CTLA-4, CX3CR1, Fc epsilon R1 alpha, IL-22, Lag-3, MIP-1 beta, Perforin, TCR V gamma 9, CDla, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Ig Kappa Light Chain, IgE, IgGl, IgG3, IL-5, IL-8, IL-21 R, KIR3dlO5, KLRC1/2, Ly-6C, Ly-6G, MHC Class II (I-A/I-E), MHC II, TCR alpha/beta, TCR beta, TCR V alpha 24, Akt (pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, cdl6/32, cd41a, CD3 epsilon, CD8b, CDl lb/c, CD16/CD32, CD23, CD29, CD43, CD45.1, CD48, CD49b, CD49d, CD66, CD68, CD71, CD85k, CD93, CD99, CD106, CD122, CD133, CD134, CD146, CD150, CD158b, CD158bl/b2, j, CD158e, CD166, CD169, CD184, CD200, CD200 R, CD235a, CD267, CD268, CD273, CD274, CD317, CD324, CD326, CD328, CD336, CD357, CD366, DDR2, eFluor 780 Fix Viability, EGF Receptor, EGFR (pY845), EOMES, EphA2, ERK1/2 (pT202/pY204), F4/80, FCRL5, Flt-3, FVS575V, FVS700, Granzyme A, HER2/ErbB2, Hesl, Hoechst (33342), ICAM-1, IFN-alpha, IgAl, IgAl/IgA2, IgA2, IgG2, IgG4, IL-1 RAcP, IL-6, IL- 10, IL- 12, IL- 17, Integrin alpha 4 beta 7, Isotype Ctrl, KLRC1, KLRC2, Live/Dead Fix Aqua, Ly-6A/Ly-6E, Ly-6G/Ly-6C, Mannose Receptor, MDR1, Met (pY1234/pY1235), MMP-9, NGF Receptor p75, ORAI1, ORAI2, ORAI3, p53, P2RY12, PARP, cleaved, RT1B, S6 (pS235/pS236), STIM1, STIM2, TCR delta, TCR delta/gamma, TCR V alpha 24 J alpha 18, TCR V beta 11, TCR V gamma 1.1, TCR V gamma 2, TER- 119, TIMP-3, TRAF3, TSLP Receptor, VDAC1, Vimentin, XCR1, and YAP1.

[0213] Embodiment A23. The method of any one of embodiments A1-A21, wherein the target biomarker is selected from any one of Tables 1-3.

[0214] Embodiment A24. The method of any one of embodiments A1-A23, wherein the nucleic acids in the first population of nucleic acids comprise deoxyribonucleic acids (DNA). [0215] Embodiment A25. The method of any one of embodiments A1-A23, wherein the nucleic acids in the first population of nucleic acids comprise ribonucleic acids (RNA).

[0216] Embodiment A26. The method of any one of embodiments A1-A25, wherein the nucleic acids in the first population of nucleic acids comprises from 1 to about 50, from 2 to about 50, from 3 to about 50, from 4 to about 50, from about 5 to about 50, from about 6 to about 50, from about 7 to about 50, from about 8 to about 50, from about 9 to about 50, from about 10 to about 50, from about 11 to about 50, from about 12 to about 50, from about 13 to about 50, from about 14 to about 50, from about 15 to about 50, from about 16 to about 50, from about 17 to about 50, from about 18 to about 50, from about 19 to about 50, from about 20 to about 50, from about 21 to about 50, from about 22 to about 50, from about 23 to about 50, from about 24 to about 50, from about 25 to about 50, from about 26 to about 50, from about 27 to about 50, from about 28 to about 50, from about 29 to about 50, from about 30 to about 50, from about 31 to about 50, from about 32 to about 50, from about 33 to about 50, from about 34 to about 50, from about 35 to about 50, from about 36 to about 50, from about 37 to about 50, from about 38 to about 50, from about 39 to about 50, from about 40 to about 50, from about 5 to about 45, from about 6 to about 45, from about 7 to about 45, from about 8 to about 45, from about 9 to about 45, from about 10 to about 45, from about 11 to about 45, from about 12 to about 45, from about 13 to about 45, from about 14 to about 45, from about 15 to about 45, from about 16 to about 45, from about 17 to about 45, from about 18 to about 45, from about 19 to about 45, from about 20 to about 45, from about 21 to about 45, from about 22 to about 45, from about 23 to about 45, from about 24 to about 45, from about 25 to about 45, from about 26 to about 45, from about 27 to about 45, from about 28 to about 45, from about 29 to about 45, from about 30 to about 45, from about 31 to about 45, from about 32 to about 45, from about 33 to about 45, from about 34 to about 45, from about 35 to about 45, from about 36 to about 45, from about 37 to about 45, from about 38 to about 45, from about 39 to about 45, from about 40 to about 45, from about 5 to about 40, from about 6 to about 40, from about 7 to about 40, from about 8 to about 40, from about 9 to about 40, from about 10 to about 40, from about 11 to about 40, from about 12 to about 40, from about 13 to about 40, from about 14 to about 40, from about 15 to about 40, from about 16 to about 40, from about 17 to about 40, from about 18 to about 40, from about 19 to about 40, from about 20 to about 40, from about 21 to about 40, from about 22 to about 40, from about 23 to about 40, from about 24 to about 40, from about 25 to about 40, from about 26 to about 40, from about 27 to about 40, from about 28 to about 40, from about 29 to about 40, from about 30 to about 40, from about 5 to about 35, from about 6 to about 35, from about 7 to about 35, from about 8 to about 35, from about 9 to about 35, from about 10 to about 35, from about 11 to about 35, from about 12 to about 35, from about 13 to about 35, from about 14 to about 35, from about 15 to about 35, from about 16 to about 35, from about 17 to about 35, from about 18 to about 35, from about 19 to about 35, from about 20 to about 35, from about 21 to about 35, from about 22 to about 35, from about 23 to about 35, from about 24 to about 35, from about 25 to about 35, from about 26 to about 35, from about 27 to about 35, from about 28 to about 35, from about 29 to about 35, from about 30 to about 35, from about 5 to about 30, from about 6 to about 30, from about 7 to about 30, from about 8 to about 30, from about 9 to about 30, from about 10 to about 30, from about 11 to about 30, from about 12 to about 30, from about 13 to about 30, from about 14 to about 30, from about 15 to about 30, from about 16 to about 30, from about 17 to about 30, from about 18 to about 30, from about 19 to about 30, from about 20 to about 30, from about 21 to about 30, from about 22 to about 30, from about

23 to about 30, from about 24 to about 30, from about 25 to about 30, from about 5 to about 25, from about 6 to about 25, from about 7 to about 25, from about 8 to about 25, from about 9 to about 25, from about 10 to about 25, from about 11 to about 25, from about 12 to about 25, from about 13 to about 25, from about 14 to about 25, from about 15 to about 25, from about 16 to about 25, from about 17 to about 25, from about 18 to about 25, from about 19 to about 25, from about 20 to about 25, from about 5 to about 20, from about 6 to about 20, from about 7 to about 20, from about 8 to about 20, from about 9 to about 20, from about 10 to about 20, from about 11 to about 20, from about 12 to about 20, from about 13 to about 20, from about 14 to about 20, from about 15 to about 20, from about 1 to about 15, from about 2 to about 15, from about 3 to about 15, from about 4 to about 15, from about 5 to about 15, from about 6 to about 15, from about 7 to about 15, from about 8 to about 15, from about 9 to about 15, or from about 10 to about 15 nucleotides.

[0217] Embodiment 27. The method of any one of embodiments 1-26, wherein the UMI comprises from 1 to about 50, from 2 to about 50, from 3 to about 50, from 4 to about 50, from about 5 to about 50, from about 6 to about 50, from about 7 to about 50, from about 8 to about 50, from about 9 to about 50, from about 10 to about 50, from about 11 to about 50, from about 12 to about 50, from about 13 to about 50, from about 14 to about 50, from about 15 to about 50, from about 16 to about 50, from about 17 to about 50, from about 18 to about 50, from about 19 to about 50, from about 20 to about 50, from about 21 to about 50, from about 22 to about 50, from about 23 to about 50, from about 24 to about 50, from about 25 to about 50, from about 26 to about 50, from about 27 to about 50, from about 28 to about 50, from about 29 to about 50, from about 30 to about 50, from about 31 to about 50, from about 32 to about 50, from about 33 to about 50, from about 34 to about 50, from about 35 to about 50, from about 36 to about 50, from about 37 to about 50, from about 38 to about 50, from about 39 to about 50, from about 40 to about 50, from about 5 to about 45, from about 6 to about 45, from about 7 to about 45, from about 8 to about 45, from about 9 to about 45, from about 10 to about 45, from about 11 to about 45, from about 12 to about 45, from about 13 to about 45, from about 14 to about 45, from about 15 to about 45, from about 16 to about 45, from about 17 to about 45, from about 18 to about 45, from about 19 to about 45, from about 20 to about 45, from about 21 to about 45, from about 22 to about 45, from about 23 to about 45, from about

24 to about 45, from about 25 to about 45, from about 26 to about 45, from about 27 to about 45, from about 28 to about 45, from about 29 to about 45, from about 30 to about 45, from about 31 to about 45, from about 32 to about 45, from about 33 to about 45, from about 34 to about 45, from about 35 to about 45, from about 36 to about 45, from about 37 to about 45, from about 38 to about 45, from about 39 to about 45, from about 40 to about 45, from about 5 to about 40, from about 6 to about 40, from about 7 to about 40, from about 8 to about 40, from about 9 to about 40, from about 10 to about 40, from about 11 to about 40, from about 12 to about 40, from about 13 to about 40, from about 14 to about 40, from about 15 to about 40, from about 16 to about 40, from about 17 to about 40, from about 18 to about 40, from about 19 to about 40, from about 20 to about 40, from about 21 to about 40, from about 22 to about 40, from about 23 to about 40, from about 24 to about 40, from about 25 to about 40, from about 26 to about 40, from about 27 to about 40, from about 28 to about 40, from about 29 to about 40, from about 30 to about 40, from about 5 to about 35, from about 6 to about 35, from about 7 to about 35, from about 8 to about 35, from about 9 to about 35, from about 10 to about 35, from about 11 to about 35, from about 12 to about 35, from about 13 to about 35, from about 14 to about 35, from about 15 to about 35, from about 16 to about 35, from about 17 to about 35, from about 18 to about 35, from about 19 to about 35, from about 20 to about 35, from about 21 to about 35, from about 22 to about 35, from about 23 to about 35, from about 24 to about 35, from about 25 to about 35, from about 26 to about 35, from about 27 to about 35, from about 28 to about 35, from about 29 to about 35, from about 30 to about 35, from about 5 to about 30, from about 6 to about 30, from about 7 to about 30, from about 8 to about 30, from about 9 to about 30, from about 10 to about 30, from about 11 to about 30, from about 12 to about 30, from about 13 to about 30, from about 14 to about 30, from about 15 to about 30, from about 16 to about 30, from about 17 to about 30, from about 18 to about 30, from about 19 to about 30, from about 20 to about 30, from about 21 to about 30, from about 22 to about 30, from about 23 to about 30, from about 24 to about 30, from about 25 to about 30, from about 5 to about 25, from about 6 to about 25, from about 7 to about 25, from about 8 to about 25, from about 9 to about 25, from about 10 to about 25, from about 11 to about 25, from about 12 to about 25, from about 13 to about 25, from about 14 to about 25, from about 15 to about 25, from about 16 to about 25, from about 17 to about 25, from about 18 to about 25, from about 19 to about 25, from about 20 to about 25, from about 5 to about 20, from about 6 to about 20, from about 7 to about 20, from about 8 to about 20, from about 9 to about 20, from about 10 to about 20, from about 11 to about 20, from about 12 to about 20, from about 13 to about 20, from about 14 to about 20, from about 15 to about 20, from about 1 to about 15, from about 2 to about 15, from about 3 to about 15, from about 4 to about 15, from about 5 to about 15, from about 6 to about 15, from about 7 to about 15, from about 8 to about 15, from about 9 to about 15, or from about 10 to about 15 nucleotides. [0218] Embodiment A28. The method of any one of embodiments A1-A27, wherein the population of nucleic acids binds to the target biomarker through a linker, wherein the linker selectively binds to the population of nucleic acids.

[0219] Embodiment A29. The method of embodiment A28, wherein the linker is an aptamer or an antibody or antigen binding fragment thereof.

[0220] Embodiment A29.1 The method of embodiment A28, wherein the nucleic acids in the first population of nucleic acids are antibody-nucleic acid conjugates.

[0221] Embodiment A30. The method of any one of embodiments A1-A29, wherein amplifying the nucleic acids comprising the UMI comprises (i) contacting the nucleic acids with a primer that binds to the primer binding site; and (ii) subjecting the nucleic acids to polymerase chain reaction.

[0222] Embodiment A31. The method of any one of embodiments A1-A30, wherein sequencing comprises high throughput generation sequencing.

[0223] Embodiment A32. The method of any one of embodiments A2-A31, wherein separating unbound nucleic acid from the sample comprises washing the sample with a buffer.

[0224] Embodiment A33. The method of any one of embodiments A2-A31, wherein separating unbound nucleic acid from the sample comprises (i) removing the target biomarker bound to the nucleic acid from the sample.

[0225] Embodiment A33.1. The method of embodiment A33, wherein the target biomarker is attached to a magnetic particle, and the removing step comprises exposing the sample to a magnetic field.

[0226] Embodiment A34. The method of any one of embodiments A1-A33.1, further comprising isolating the bound nucleic acid from the sample.

[0227] Embodiment A35. The method of embodiment A34, wherein the isolating comprises eluting the bound nucleic acid from the sample.

[0228] Embodiment A36. The method of embodiment A34, wherein the isolating comprises dissolving substrates connected to the target biomarker, thereby releasing the bound nucleic acid from the sample.

[0229] Embodiment Bl . A composition comprising (i) a plurality of target biomarkers, and (ii) a plurality of nucleic acids comprising (a) a primer binding site and (b) a unique molecular identifier (UMI), wherein the nucleic acids are bound to the biomarker and wherein each nucleic acid in the population comprises a different UMI.

[0230] Embodiment Bl.l. The composition of embodiment Bl, wherein the composition comprises a polymer bead. [0231] Embodiment Bl. The composition of embodiment B 1.1, wherein the plurality of target biomarkers are bound to the polymer bead.

[0232] Embodiment B2. The composition of embodiment Bl.l or Bl.2, wherein the polymer bead comprises less than 10%, 20%, 30%, or 40% polystyrene by hydrated volume.

[0233] Embodiment B3. The composition of embodiment Bl.l or Bl.2, wherein the polymer beads comprise less than 10%, 20%, 30%, or 40% polystyrene by dehydrated volume.

[0234] Embodiment B4. The composition of any one of embodiments B1.1-B3, wherein the polymer beads are hydrogel beads.

[0235] Embodiment B5. The composition of embodiment B4, wherein the hydrogel comprises a monomer.

[0236] Embodiment B6. The composition of embodiment B5, wherein the monomer is selected from the group consisting of hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxy ethoxy ethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, polyethylene glycol) methacrylate, methoxypoly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6- tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2, 3 -dibromopropyl acrylate, 2,3- dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4- methoxybenzylacrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2- benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenyl acrylamide, Nphenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N- dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide N-(4- methylphenyl)methyl acrylamide, N-l -naphthyl acrylamide, N-4-nitrophenyl acrylamide, N- (2-phenylethyl)acrylamide, N-triphenylmethyl acrylamide, N-(4- hydroxyphenyl)acrylamide, N,N-methylphenyl acrylamide, N, N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl)m ethyl methacrylamide, N-l -naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl)methacrylamide, N- triphenylmethyl methacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenyl methacrylamide, N,N' -phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, and a combination thereof.

[0237] Embodiment B6.1. The composition of embodiment B5, wherein the monomer is a dissolvable monomer or a biodegradable monomer.

[0238] Embodiment B6.2. The composition of embodiment B6.1, wherein the monomer is a biodegradable monomer and the biodegradable monomer is a monosaccharide, disaccharide, polysaccharide, peptide, protein, or protein domain.

[0239] Embodiment B6.3. The composition of embodiment B6.1, wherein the monomer is a biodegradable monomer and the biodegradable monomer is a protein or protein domain comprising at least one non-natural amino acid.

[0240] Embodiment B6.4. The composition of embodiment B6.1, wherein the monomer is a biodegradable monomer and the biodegradable monomer is a structural polysaccharide.

[0241] Embodiment B6.5. The composition of embodiment B6.1, wherein the monomer is a biodegradable monomer and the biodegradable monomer is agar, agarose, alginic acid, alguronic acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta-glucan, callose, capsullan, carrageenan polysaccharide, cellodextrin, cellulin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminoogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof.

[0242] Embodiment B6.6. The composition of embodiment B6.1, wherein the monomer is a biodegradable monomer and the biodegradable monomer is chitosan or hyaluronan.

[0243] Embodiment B6.7. The composition of embodiment B6.2, wherein the protein is a structural protein, a domain thereof, or a combination thereof.

[0244] Embodiment B6.8. The composition of embodiment B6.2, wherein the protein is a proteoglycan, a domain thereof, or a combination thereof.

[0245] Embodiment B6.9. The composition of embodiment B6.2, wherein the protein is an extracellular matrix component. [0246] Embodiment B6.10. The composition of embodiment B6.2, wherein the protein is collagen, elastin or a proteoglycan.

[0247] Embodiment B6. l l. The composition of embodiment B6.10, wherein the collagen is collagen type I, collagen type II, collagen type III, a domain thereof or a combination thereof. [0248] Embodiment B6.12. The composition of embodiment B6.1, wherein the dissolvable monomer is dissolvable by one or more of chemical means and physical means.

[0249] Embodiment B7. The polymer bead of any one of embodiments B1-B6, wherein the polymer beads comprise a known optical property.

[0250] Embodiment B7.1. The composition of any one of embodiments B 1.1 -B7, wherein each polymer bead exhibits at least one optical property having a quantitative profile that is substantially similar to a quantitative profile of a corresponding optical property of a target cell.

[0251] Embodiment B8. The composition of any one of embodiments B1-B7, wherein the target biomarker is selected from the group consisting of: CD3, CD4, CD8, CD19, CD14, ccr7, CD45, CD45RA, CD27, CD16, CD56, CD127, CD25, CD38, HLA-DR, PD-1, CD28, CD183, CD185, CD57, IFN-gamma, CD20, TCR gamma/delta, TNF alpha, CD69, IL-2, Ki-67, CCR6, CD34, CD45RO, CD161, IgD, CD95, CD117, CD123, CDl lc, IgM, CD39, FoxP3, CD10, CD40L, CD62L, CD194, CD314, IgG, TCR V alpha 7.2, CDl lb, CD21, CD24, IL-4, Biotin, CCR10, CD31, CD44, CD 138, CD294, NKp46, TCR V delta 2, TIGIT, CDlc, CD2, CD7, CD8a, CD15, CD32, CD103, CD107a, CD141, CD158, CD159c, IL-13, IL-21, KLRG1, TIM- 3, CCR5, CD5, CD33, CD45.2, CD80, CD159a (NKG2a), CD244, CD272, CD278, CD337, Granzyme B, Ig Lambda Light Chain, IgA, IL- 17 A, Streptavidin, TCR V delta 1, CD Id, CD26, CD45R (B220), CD64, CD73, CD86, CD94, CD 137, CD 163, CD 193, CTLA-4, CX3CR1, Fc epsilon R1 alpha, IL-22, Lag-3, MIP-1 beta, Perforin, TCR V gamma 9, CDla, CD22, CD36, CD40, CD45R, CD66b, CD85j, CD160, CD172a, CD186, CD226, CD303, CLEC12A, CXCR4, Helios, Ig Kappa Light Chain, IgE, IgGl, IgG3, IL-5, IL-8, IL-21 R, KIR3dl05, KLRC1/2, Ly-6C, Ly-6G, MHC Class II (I-A/I-E), MHC II, TCR alpha/beta, TCR beta, TCR V alpha 24, Akt (pS473), ALDH1A1, Annexin V, Bcl-2, c-Met, CCR7, cdl6/32, cd41a, CD3 epsilon, CD8b, CDl lb/c, CD16/CD32, CD23, CD29, CD43, CD45.1, CD48, CD49b, CD49d, CD66, CD68, CD71, CD85k, CD93, CD99, CD106, CD122, CD133, CD134, CD146, CD150, CD158b, CD158bl/b2, j, CD158e, CD166, CD169, CD184, CD200, CD200 R, CD235a, CD267, CD268, CD273, CD274, CD317, CD324, CD326, CD328, CD336, CD357, CD366, DDR2, eFluor 780 Fix Viability, EGF Receptor, EGFR (pY845), EOMES, EphA2, ERK1/2 (pT202/pY204), F4/80, FCRL5, Flt-3, FVS575V, FVS700, Granzyme A, HER2/ErbB2, Hesl, Hoechst (33342), ICAM-1, IFN-alpha, IgAl, IgAl/IgA2, IgA2, IgG2, IgG4, IL-1 RAcP, IL-6, IL- 10, IL- 12, IL- 17, Integrin alpha 4 beta 7, Isotype Ctrl, KLRC1, KLRC2, Live/Dead Fix Aqua, Ly-6A/Ly-6E, Ly-6G/Ly-6C, Mannose Receptor, MDR1, Met (pY1234/pY1235), MMP-9, NGF Receptor p75, 0RAI1, ORAI2, ORAI3, p53, P2RY12, PARP, cleaved, RT1B, S6 (pS235/pS236), STIM1, STIM2, TCR delta, TCR delta/gamma, TCR V alpha 24 J alpha 18, TCR V beta 11, TCR V gamma 1.1, TCR V gamma 2, TER- 119, TIMP-3, TRAF3, TSLP Receptor, VDAC1, Vimentin, XCR1, and YAP1.

[0252] Embodiment B9. The composition of any one of embodiments B1-B7, wherein the target biomarker is selected from any one of Tables 1-3.

[0253] Embodiment BIO. The composition of any one of embodiments B1-B9, wherein the nucleic acids comprise deoxyribonucleic acids (DNA).

[0254] Embodiment Bl l. The composition of any one of embodiments Bl -BIO, wherein the nucleic acids comprise ribonucleic acids (RNA).

[0255] Embodiment B12. The composition of any one of embodiments Bl-Bl l, wherein the nucleic acid comprises from 1 to about 50, from 2 to about 50, from 3 to about 50, from 4 to about 50, from about 5 to about 50, from about 6 to about 50, from about 7 to about 50, from about 8 to about 50, from about 9 to about 50, from about 10 to about 50, from about 11 to about 50, from about 12 to about 50, from about 13 to about 50, from about 14 to about 50, from about 15 to about 50, from about 16 to about 50, from about 17 to about 50, from about 18 to about 50, from about 19 to about 50, from about 20 to about 50, from about 21 to about 50, from about 22 to about 50, from about 23 to about 50, from about 24 to about 50, from about 25 to about 50, from about 26 to about 50, from about 27 to about 50, from about 28 to about 50, from about 29 to about 50, from about 30 to about 50, from about 31 to about 50, from about 32 to about 50, from about 33 to about 50, from about 34 to about 50, from about 35 to about 50, from about 36 to about 50, from about 37 to about 50, from about 38 to about 50, from about 39 to about 50, from about 40 to about 50, from about 5 to about 45, from about 6 to about 45, from about 7 to about 45, from about 8 to about 45, from about 9 to about 45, from about 10 to about 45, from about 11 to about 45, from about 12 to about 45, from about 13 to about 45, from about 14 to about 45, from about 15 to about 45, from about 16 to about 45, from about 17 to about 45, from about 18 to about 45, from about 19 to about 45, from about 20 to about 45, from about 21 to about 45, from about 22 to about 45, from about 23 to about 45, from about 24 to about 45, from about 25 to about 45, from about 26 to about 45, from about 27 to about 45, from about 28 to about 45, from about 29 to about 45, from about 30 to about 45, from about 31 to about 45, from about 32 to about 45, from about 33 to about 45, from about 34 to about 45, from about 35 to about 45, from about 36 to about 45, from about 37 to about 45, from about 38 to about 45, from about 39 to about 45, from about 40 to about 45, from about 5 to about 40, from about 6 to about 40, from about 7 to about 40, from about 8 to about 40, from about 9 to about 40, from about 10 to about 40, from about 11 to about 40, from about 12 to about 40, from about 13 to about 40, from about 14 to about 40, from about 15 to about 40, from about 16 to about 40, from about 17 to about 40, from about 18 to about 40, from about 19 to about 40, from about 20 to about 40, from about 21 to about 40, from about 22 to about 40, from about 23 to about 40, from about 24 to about 40, from about 25 to about 40, from about 26 to about 40, from about 27 to about 40, from about 28 to about 40, from about 29 to about 40, from about 30 to about 40, from about 5 to about 35, from about 6 to about 35, from about 7 to about 35, from about 8 to about 35, from about 9 to about 35, from about 10 to about 35, from about 11 to about 35, from about 12 to about 35, from about 13 to about 35, from about 14 to about 35, from about 15 to about 35, from about 16 to about 35, from about 17 to about 35, from about 18 to about 35, from about 19 to about 35, from about 20 to about 35, from about 21 to about 35, from about 22 to about 35, from about 23 to about 35, from about 24 to about 35, from about 25 to about 35, from about 26 to about 35, from about 27 to about 35, from about 28 to about 35, from about 29 to about 35, from about 30 to about 35, from about 5 to about 30, from about 6 to about 30, from about 7 to about 30, from about 8 to about 30, from about 9 to about 30, from about 10 to about 30, from about 11 to about 30, from about 12 to about 30, from about 13 to about 30, from about 14 to about 30, from about 15 to about 30, from about 16 to about 30, from about 17 to about 30, from about 18 to about 30, from about 19 to about 30, from about 20 to about 30, from about 21 to about 30, from about 22 to about 30, from about 23 to about 30, from about 24 to about 30, from about 25 to about 30, from about 5 to about 25, from about 6 to about 25, from about 7 to about 25, from about 8 to about 25, from about 9 to about 25, from about 10 to about 25, from about 11 to about 25, from about 12 to about 25, from about 13 to about 25, from about 14 to about 25, from about 15 to about 25, from about 16 to about 25, from about 17 to about 25, from about 18 to about 25, from about 19 to about 25, from about 20 to about 25, from about 5 to about 20, from about 6 to about 20, from about 7 to about 20, from about 8 to about 20, from about 9 to about 20, from about 10 to about 20, from about 11 to about 20, from about 12 to about 20, from about 13 to about 20, from about 14 to about 20, from about 15 to about 20, from about 1 to about 15, from about 2 to about 15, from about 3 to about 15, from about 4 to about 15, from about 5 to about 15, from about 6 to about 15, from about 7 to about 15, from about 8 to about 15, from about 9 to about 15, or from about 10 to about 15 nucleotides. [0256] Embodiment B13. The composition of any one of embodiments Bl -Bl 2, wherein the UMI comprises from 1 to about 50, from 2 to about 50, from 3 to about 50, from 4 to about 50, from about 5 to about 50, from about 6 to about 50, from about 7 to about 50, from about 8 to about 50, from about 9 to about 50, from about 10 to about 50, from about 11 to about 50, from about 12 to about 50, from about 13 to about 50, from about 14 to about 50, from about 15 to about 50, from about 16 to about 50, from about 17 to about 50, from about 18 to about 50, from about 19 to about 50, from about 20 to about 50, from about 21 to about 50, from about 22 to about 50, from about 23 to about 50, from about 24 to about 50, from about 25 to about 50, from about 26 to about 50, from about 27 to about 50, from about 28 to about 50, from about 29 to about 50, from about 30 to about 50, from about 31 to about 50, from about 32 to about 50, from about 33 to about 50, from about 34 to about 50, from about 35 to about 50, from about 36 to about 50, from about 37 to about 50, from about 38 to about 50, from about 39 to about 50, from about 40 to about 50, from about 5 to about 45, from about 6 to about 45, from about 7 to about 45, from about 8 to about 45, from about 9 to about 45, from about 10 to about 45, from about 11 to about 45, from about 12 to about 45, from about 13 to about 45, from about 14 to about 45, from about 15 to about 45, from about 16 to about 45, from about 17 to about 45, from about 18 to about 45, from about 19 to about 45, from about 20 to about 45, from about 21 to about 45, from about 22 to about 45, from about 23 to about 45, from about 24 to about 45, from about 25 to about 45, from about 26 to about 45, from about 27 to about 45, from about 28 to about 45, from about 29 to about 45, from about 30 to about 45, from about 31 to about 45, from about 32 to about 45, from about 33 to about 45, from about 34 to about 45, from about 35 to about 45, from about 36 to about 45, from about 37 to about 45, from about 38 to about 45, from about 39 to about 45, from about 40 to about 45, from about 5 to about 40, from about 6 to about 40, from about 7 to about 40, from about 8 to about 40, from about 9 to about 40, from about 10 to about 40, from about 11 to about 40, from about 12 to about 40, from about 13 to about 40, from about 14 to about 40, from about 15 to about 40, from about 16 to about 40, from about 17 to about 40, from about 18 to about 40, from about 19 to about 40, from about 20 to about 40, from about 21 to about 40, from about 22 to about 40, from about 23 to about 40, from about 24 to about 40, from about 25 to about 40, from about 26 to about 40, from about 27 to about 40, from about 28 to about 40, from about 29 to about 40, from about 30 to about 40, from about 5 to about 35, from about 6 to about 35, from about 7 to about 35, from about 8 to about 35, from about 9 to about 35, from about 10 to about 35, from about 11 to about 35, from about 12 to about 35, from about 13 to about 35, from about 14 to about 35, from about 15 to about 35, from about 16 to about 35, from about 17 to about 35, from about 18 to about 35, from about 19 to about 35, from about 20 to about 35, from about 21 to about 35, from about 22 to about 35, from about 23 to about 35, from about 24 to about 35, from about 25 to about 35, from about 26 to about 35, from about 27 to about 35, from about 28 to about 35, from about 29 to about 35, from about 30 to about 35, from about 5 to about 30, from about 6 to about 30, from about 7 to about 30, from about 8 to about 30, from about 9 to about 30, from about 10 to about 30, from about 11 to about 30, from about 12 to about 30, from about 13 to about 30, from about 14 to about 30, from about 15 to about 30, from about 16 to about 30, from about 17 to about 30, from about 18 to about 30, from about 19 to about 30, from about 20 to about 30, from about 21 to about 30, from about 22 to about 30, from about 23 to about 30, from about 24 to about 30, from about 25 to about 30, from about 5 to about 25, from about 6 to about 25, from about 7 to about 25, from about 8 to about 25, from about 9 to about 25, from about 10 to about 25, from about 11 to about 25, from about 12 to about 25, from about 13 to about 25, from about 14 to about 25, from about 15 to about 25, from about 16 to about 25, from about 17 to about 25, from about 18 to about 25, from about 19 to about 25, from about 20 to about 25, from about 5 to about 20, from about 6 to about 20, from about 7 to about 20, from about 8 to about 20, from about 9 to about 20, from about 10 to about 20, from about 11 to about 20, from about 12 to about 20, from about 13 to about 20, from about 14 to about 20, from about 15 to about 20, from about 1 to about 15, from about 2 to about 15, from about 3 to about 15, from about 4 to about 15, from about 5 to about 15, from about 6 to about 15, from about 7 to about 15, from about 8 to about 15, from about 9 to about 15, or from about 10 to about 15 nucleotides.

[0257] Embodiment B14. A composition comprising a first population of polymer beads of any one of embodiments B 1.1 -Bl 3 and a second population of polymer beads of any one of embodiments B 1.1 -B 13.

[0258] Embodiment Bl 5. The composition of embodiment B14, wherein the first population of polymer beads exhibits an optical property that is distinct from the corresponding optical property of the second population of polymer beads.

[0259] Embodiment Bl 6. The composition of embodiment B7 or Bl 5, wherein the optical property comprises side scatter (SSC).

[0260] Embodiment Bl 6.1. The composition of embodiment Bl 6, wherein the polymer beads comprise scatter-modulating additives.

[0261] Embodiment Bl 6.2. The composition of embodiment Bl 6.1, wherein the scattermodulating additives comprises one or more of a nanoparticle, a colloidal silica, an encapsulated material, and a chemical side-group. [0262] Embodiment Bl 7. The composition of embodiment B7 or Bl 5, wherein the optical property comprises forward scatter.

[0263] Embodiment B 17.1. The composition of embodiment B 17, wherein the forward scatter is defined by a refractive index (RI) of each of the polymer beads.

[0264] Embodiment Bl 7.2. The composition of embodiment Bl 7.1, comprising polymer beads with an RI of greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.1 0, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0265] Embodiment B17.3. The composition of embodiment B17.1, comprising polymer beads with an RI of about 1.10 to about 3.0, or about 1.15 to about 3.0, or about 1.20 to about 3.0, or about 1.25 to about 3.0, or about 1.30 to about 3.0, or about 1.35 to about 3.0, or about 1.4 to about 3.0, or about 1.45 to about 3.0, or about 1.50 to about 3.0, or about 1.6 to about 3.0, or about 1.7 to about 3.0, or about 1.8 to about 3.0, or about 1.9 to about 3.0, or about 2.0 to about 3.0.

[0266] Embodiment Bl 7.4. The composition of embodiment Bl 7.1, comprising polymer beads with an RI of less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.

[0267] Embodiment Bl 8. The composition of any one of any one of embodiments B7-B17.4, wherein the optical property comprises forward scatter and side scatter.

[0268] Embodiment Bl 9. The composition of any one of embodiments B 1.1 -Bl 8, wherein the polymer bead has an average diameter ranging from about 1 pm to about 20 pm.

[0269] Embodiment B20. The composition of any one of embodiments B 1.1 -Bl 8, wherein the polymer bead has an average diameter ranging from about 5 pm to about 40 pm. [0270] Embodiment B21. The composition of any one of embodiments B 1.1 -Bl 8, wherein the polymer bead has an average diameter ranging from about 5 pm to about 10 pm.

EXAMPLES

Example 1: Generation of Hydrogel Beads

[0271] Photomasks for UV lithography were sourced from CADart Services Inc. and were designed using AutoCad (AutoDesk, Inc.). SU-8 photo resist (Microchem, Inc.) was photo crosslinked on 4" silicon wafers using a collimated UV light source (OAI, Inc.) to create masters for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sigma Aldrich, Inc.) was prepared and formed using standard published methods for soft lithography and microfluidic device fabrication (See, McDonald JC, et al., 2000, Electrophoresis 21 :27-40).

[0272] Droplets were formed using flow-focusing geometry where two oil channels focus a central stream of aqueous monomer solution to break off droplets in a water-in-oil emulsion. A fluorocarbon-oil (Novec 7500 3M, Inc.) was used as the outer, continuous phase liquid for droplet formation. To stabilize droplets before polymerization, a surfactant was added at 0.5% w/w to the oil phase (ammonium carboxylate salt of Krytox 157 FSH, Dupont). To make the basic polyacrylamide gel bead, a central phase of an aqueous monomer solution containing N- acrylamide (1-20% w/v), a cross-linker that allows for the hydrogel to be lysed (N,N'- bis(acryloyl)cystamine, bis(2-methacryloyl)oxyethyl disulfide, allyl disulfide, polyethylene glycol (PEG) N-hydroxysuccinimide (NHS) ester disulfide, acryloyl-PEG-disulfide-PEG- acryloyl, or succinimidyl 3-(2-pyridyldithio)propionate, dicumyl alcohol dimethacrylate, dicumyl alcohol diacrylate, 2,5-dimethyl-2,5-hexanediol dimethacrylate, acylhydrazone, or 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane), an accelerator, and ammonium persulfate (1% w/v) was used. An accelerator, (N,N,N’,N’ tetramethylethylenediamine (2% vol%) was added to the oil-phase in order to trigger hydrogel bead polymerization after droplet formation. [0273] Co-monomers may be added to the basic gel formulation to add functionality. Allylamine provided primary amine groups for secondary labeling after gel formation. Forward scatter may be modulated by adjusting the refractive index of the gel by adding co-monomers allyl acrylate and allyl methacrylate. Side scattering of the droplets may be tuned by adding a colloidal suspension of silica nanoparticles and/or PMMA (poly(methyl methacrylate)) particles (-100 nm) to the central aqueous phase prior to polymerization.

[0274] Stoichiometric multiplexing of the hydrogel beads was achieved by utilizing comonomers containing chemically orthogonal side groups (amine, carboxyl, maleimide, epoxide, alkyne, etc.) for secondary labeling.

[0275] Droplets were formed at an average rate of 5 kHz and were collected in the fluorocarbon oil phase. Polymerization was completed at 50 °C for 30 minutes, and the resulting hydrogel beads were washed from the oil into an aqueous solution. Example 2: Hydrogel Beads Mimic the Forward and Side Scatter Profiles of Various Cell Types

[0276] This example describes the optional tuning of hydrogel beads to match the optical properties of one or more target cells. Different types of cells (e.g., granulocytes, monocytes, and lymphocytes) exhibit different optical-scatter properties (e.g., forward scatter and side scatter). In some embodiments, the optical properties of the hydrogel beads are tuned to mimic specific cell types. Tuning of hydrogels can be carried out via methods described in U.S. 10,753,846. Briefly, as depicted in Figs. 1-3, hydrogel beads are tuned in multiple dimensions to match specific cell types. Cells are deconvolved using combinations of optical parameters such as FSC and SSC (Fig. 2), and/or secondary markers. Hydrogel beads are further functionalized with stoichiometrically tuned ratios of specific chemical side-groups and secondary labels allowing the bead to precisely match the target cell without suffering from the biological noise associated with fixed cell lines. (Fig. 2).

Example 3: A method to quantitate the number of biomarkers on a cell, bead, or surface is developed.

[0277] Accurately quantitating the number of biomarkers bound to a sample (e.g., a cell, bead, or surface) is difficult. For flow cytometry, an understanding of the number of antigens bound to the sample facilitates selection of a fluorophore for flow cytometry. For example, brighter fluorophores should be used to detect biomarkers that are present on the sample in a lower density, whereas dimmer fluorophores should be used to detect biomarkers that are present on the sample in a higher density.

[0278] An assay to evaluate the number of biomarkers present on a sample (e.g., a hydrogel bead) is developed. The sample is incubated with a population of nucleic acids that are bound to a linker. Each nucleic acid comprises a unique molecular identifier (UMI) and a primer binding site. An exemplary nucleic acid has the nucleic acid sequence: 5’- GGAGTCTCGTGGGCTCGGNNNNNNNNNNNNNNNNNNCGCCCATCTAATACA TCCAATCTC AGTAGACGCTGCCGACGAAT-3’ (SEQ ID NO: 1), where the underlined sequence is the UMI and a primer can bind to any of the bolded sections of the sequence.

[0279] The sample is washed to remove nucleic acid that does not bind to the sample. A single bead is isolated. The bound nucleic acids are eluted from the sample and contacted with a primer that binds to the primer binding site. The nucleic acids are amplified using polymerase chain reaction (PCR) reaction. The PCR reaction amplifies the unique molecular identifier sequences attached to the antibodies. The PCR reaction is sequenced in a sequencing machine (e.g., an Illumina® MISEQ®, NOVASEQ®, or HISEQ® sequencing machine). The number of distinct UMIs that are detected by sequencing corresponds to the number of antigens in the sample.

[0280] The PCR reaction of multiple samples may be sequenced at the same time by labeling each sequence with a barcode.

INCORPORATION BY REFERENCE

[0282] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. Further, U.S. Patent No. 9,915,598, issued on March 13, 2018, and entitled: Hydrogel Particles with Tunable Optical Properties, is hereby incorporated by reference in its entirety for all purposes. Further, U.S. Patent No. 9,714,897, issued on July 25, 2017, and entitled: Hydrogel Particles with Tunable Optical Properties and Methods for Using the Same, is hereby incorporated by reference in its entirety for all purposes. Further, U.S. Patent No. 11,313,782, issued on April 26, 2022, and entitled: Compositions and Methods for Cell-Like Calibration Particles, is hereby incorporated by reference in its entirety for all purposes. Further, U.S. Publication No. 2023/0067460 published on March 2, 2023, and entitled: Hydrogel Particles as Feeder Cells and Synthetic Antigen Presenting Cells, is hereby incorporated by reference in its entirety for all purposes. Further, International Application No. PCT/US2023/066684, filed on May 5, 2023, and entitled: Engineered Particles as Red Blood Cell Mimics and Compositions Containing Same for Hematology, is hereby incorporated by reference in its entirety for all purposes. Further, International Publication No. WO2021/226036, published on November 11, 2021, and entitled: Compositions and Methods for Passive Optical Barcoding for Multiplexed Assays, is hereby incorporated by reference in its entirety for all purposes. Further, International Application No. PCT/US2023/067893, filed on June 2, 2023, and entitled: Apoptotic Cell Mimic, is hereby incorporated by reference in its entirety for all purposes. Further, International Application No. PCT/US2023/072659, filed on August 22, 2023, and entitled: Compositions and Methods for Single Well Multiplexed Calibration and Compensation, is hereby incorporated by reference in its entirety for all purposes. The following documents are also incorporated by reference in their entireties for all purposes: van Buggenum, J. A., Gerlach, J.P., Eising, S., Schoonen, L, van Eijl, R. A., Tanis, S. E. et al. (2016). A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR. Sci Rep, 6, 22675; van Buggenum, J. A. G., Gerlach, J. P., Tanis, S. E. J., Hogeweg, M., Jansen, P. W. T. C., Middelwijk, J. et aL (2018). Immunodetection by sequencing enables large-scale high- dimensional phenotyping in cells. Nat Commun, 9(1), 2384; Reimegard, J., Danielsson, M., Tarbier, M., Schuster, J., Baskaran, S., Panagiotou, S. et al. (2019). Combined mRNA and protein single cell analysis in a dynamic cellular system using SPARC; and Brofelth, M., Ekstrand, A. I., Gour, S., Jansson, R., Hedhammar, M., Elleby, B. et al. (2020). Multiplex profiling of serum proteins in solution using barcoded antibody fragments and next generation sequencing. Commun Biol, 3(1), 339.