Metal-labeled polymer microbeads with control over labeling levels

Abstract

The present disclosure relates to metal-labeled polymeric microbeads, in particular lanthanide-labeled polymeric microbeads, for mass cytometry bead-based assays and multiplexing applications. The polymeric microbeads comprise copolymers that include structural monomers and metal chelating monomers.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Patent Application No. 63 / 161,414, filed March 15, 2021, and U.S. Patent Application No. 63 / 319,608, filed March 14, 2022. The contents of these provisional patent applications are incorporated herein by reference in their entireties.

[0002] FIELD OF THEINVENTION The present disclosure relates to metal-labeled polymeric microbeads, in particular lanthanide-labeled polymeric microbeads, for mass cytometry bead-based assays and multiplexed applications. [Background technology]

[0003] Mass cytometry (MC) is an emerging analytical technique that employs inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) to analyze the signals of isotopic labels on cell and microbead samples. Bead-based applications in mass cytometry require the labeling of microbeads with different metal ions that can be individually identified by MC.

[0004] Bead-based assays are attractive as analytical techniques due to their high sample volume efficiency for multiplexed assays with high throughput capabilities. A variety of applications have been developed using bead-based assay technology, including capture sandwich immunoassays, competitive immunoassays, serology, gene expression profiling and genotyping. 1、2In contrast to classical planar or ELISA techniques, bead-based assays use colloidal suspensions of particles as solid supports for different affinity reagents that can target different molecules as analytes. By coding the beads and generating a library of beads as classifiers for the analytes, the various captured analytes can be tracked by decoding and identifying the individual beads across an experiment. In this way, the analysis of multiple analytes can be performed simultaneously in one assay using a set of beads functionalized with different affinity molecules. Most commercially available bead-based assays use luminescent tags as labels to barcode these beads and examine these classifier beads in a high-throughput manner by flow cytometry. Luminex has marketed a library of 500 separately labeled polystyrene microspheres for flow cytometric bead-based assays, with three different colors at ten different intensity levels and surface functionalities for attachment to bioaffinity reagents. 3-5 .

[0005] Mass cytometry (MC) is an emerging analytical technique that combines the features of flow cytometry and elemental mass spectrometry to characterize single-cell or bead samples. 5 In mass cytometry, cell and bead samples are labeled with heavy metal isotopes and introduced separately into the plasma torch of an inductively coupled plasma time-of-flight mass spectrometer (ICP-TOF-MS) to analyze the signals of the metal isotope labels. MC can accurately analyze different metal isotopes based on their atomic masses without channel overlap and even at low abundances of heavy metal isotopes in biological samples, and MC avoids the complicated signal compensation process associated with flow cytometry. It greatly improves the detection of sample features with enhanced resolution and sensitivity compared to fluorescence-based bead assays. Since a much larger number of heavy metal isotopes can be employed as labels, MC offers great potential for high-throughput cytometric bead-based assays with much higher multiplexing capabilities. 7-8 .

[0006] To develop metal-encoded microbeads for multiplexed MC assays, Abdelrahman et al. introduced the concept of synthesizing a library of microbeads encoded with various metal isotopes at different concentration levels. 9 The theoretical maximum variation (n) of this microbead library can be calculated as follows: n=K M -1 (1) where K is the level of metal concentration level of the microbead (including zero concentration); M is the number of different isotopes encoded in the bead. The term (-1) is a bead with zero metal content of all isotopes, which cannot be detected by MC. Lanthanide isotopes are an attractive choice as mass tags for encoding PS microbeads because of their similar chemical and physical properties, low natural abundance in MC, and their high detection sensitivity. 8 There are 15 lanthanide elements, and these isotopes comprise at least 36 detection channels, from 139 amu to 176 amu, theoretically available to be employed as bead labels. For example, with four concentration levels for each coding isotope, a library of beads with thousands of variants could in principle be created for MC bead-based assays. To ensure the quality of the MC signal, the metal-containing microbeads employed in MC applications need to be sized in the range of about 1-5 μm in diameter with a very narrow particle size distribution. Smaller microbeads tend to contain fewer metal isotopes, which may not be enough to be detected as single events in MC, while larger microbeads may be inconsistent and not completely consumed in the ICP torch.

[0007] The synthesis of polymeric microbeads encoded with several types of heavy metal isotopes has been reported. In the approach adopted by Abdelrahman et al., lanthanide-encoded polystyrene (PS) microbeads were synthesized by multistage dispersion polymerization (DisP) of styrene in the presence of polyvinylpyrrolidone (PVP) as a steric stabilizer and acrylic acid (AA) as a comonomer for metal ion incorporation. With some optimization, Abdelrahman et al. reported that approximately 1x10 8 Lanthanide ion-encoded PS microbeads could be achieved, which produced strong signal intensities in MC with relatively small bead-to-bead variability (RCV<15%), fulfilling the requirements for use as reagents in MC applications. 9、10 .

[0008] Liang et al. later tested the idea of ​​replacing the comonomer AA with methacrylic acid (MAA) and obtained similar results. 11 Other approaches to incorporate metal ions into microbeads have also been explored, including the imbibition of styrene-soluble lanthanide chelates into PS beads by seeded emulsion polymerization and the covalent incorporation of lanthanide nanoparticles onto polymer microbeads by dispersion polymerization. 12、13 However, compared to the AA approach developed by Abdelrahman et al., much lower incorporation efficiencies and higher bead-to-bead metal content variations were observed in these later attempts.

[0009] The AA approach, however, is inadequate for the synthesis of batches of beads with designed different metal contents for MC calibration purposes. This strategy offers poor control of metal incorporation in the bead synthesis, especially when a large number of different metal ions are mixed with the AA in the second stage aliquots. For example, lanthanide ions with smaller ionic radii (e.g., Ho 3+ and Lu 3+ ) are more highly ionized than lanthanides with larger ionic radii (e.g., Ce 3+ and Eu 3+ ) tend to be incorporated into microbeads much more efficiently than 14、15. This lack of control is a time-consuming challenge, especially when attempting to synthesize, for example, a plurality or library of microbeads that are coded with various metal ions and have a predetermined metal content at different levels for each metal.

[0010] Microbeads can be used for a variety of qualitative and quantitative applications, e.g., for analyte detection. Several cytokines are believed to play important roles in COVID-19 pathology. However, current research tools are limited in their ability to fully explore the mechanisms of cytokine production and action. 34 . Cytokines are soluble signaling protein molecules produced by cells at picomolar to nanomolar concentrations to regulate immune responses and modulate cellular activity. They represent an extremely large and diverse group of pro- and anti-inflammatory factors in the body. 35 Deep characterization of cytokines in blood samples can provide important details of the immune response to disease or infection, or to vaccines, therapies, and interventions. Enzyme-linked immunosorbent assay (ELISA) is the most widely used monoplex Ab-based immunoassay method for the analysis of cytokines in biological samples. However, ELISA assays require substantial amounts of sample and are time-consuming when multiple cytokines are analyzed simultaneously. 36、37 . Bead-based immunoassays are technology platforms that provide information-rich, multimetric analysis in a high-throughput setting. Bead-based assays employing fluorescent dyes for signal detection are the current standard test method for multiplexed cytokine analysis, and kits for cytokine analysis are commercially available. In this type of assay, polymer microbeads are labeled with two or three different fluorescent dyes at various concentrations to well-defined intensities. Successful antigen detection is recognized by reporter antibodies (Abs) labeled with dyes with distinct emissions. 38-44However, a recent report from six College of American Pathologists cytokine surveys conducted between 2015 and 2018 described variability in cytokine analysis in a set of four cytokines (IL-1, IL-6, IL-8, and TNFα). 45 This study investigated interlaboratory variability and between-method and even within-the-same laboratory variability. This report highlights some of the current challenges in quantitative cytokine detection. Summary of the Invention

[0011] Described herein are metal-encoded polymeric microbeads that can be synthesized by multi-stage dispersion polymerization and include structural monomers and metal chelating monomers that include a chelating agent that is chelated to a metal prior to being subjected to polymerization. The microbeads include and are formed by polymers that include copolymers. As shown in the embodiments herein, the resulting microbeads have a narrow particle size distribution and have a predetermined amount of different metals. The amount of metal incorporated into the microbeads is substantially consistent from bead to bead, allowing a substantially uniform population of microbeads to be obtained. The metal can be incorporated throughout the microbead as opposed to being present only on the surface. Additionally, as described in some embodiments, the microbeads of the present disclosure can be functionalized on the surface of the microbeads and conjugated to biomolecules such as antibodies or other affinity reagents.

[0012] In particular, provided in certain embodiments are microbeads with substantially controlled metal content and methods of making them. Metal-encoded microbeads include at least one metal element and may include multiple metal elements. The metal elements may include natural abundance isotopes of the element or may include enriched isotopes of one or more elements. As shown in the examples, lanthanide-encoded microbeads were created by employing polymerizable metal-complexes as ligands in a two-step dispersion polymerization. In one example, diethylenetriaminepentaacetic acid (DTPA) was functionalized with a polymerizable monomer by reacting DTPA dianhydride with 4-vinylbenzylamine (VBA). Various lanthanide metal ion complexes of this DTPA derivative were then prepared. Metal-encoded microbeads were synthesized by introducing these polymerizable metal-DTPA complexes into a dispersion polymerization reaction of a monomer such as styrene. The resulting microbeads have extremely small bead-to-bead variations in their size and metal content. Similar metal incorporation efficiencies were observed in bead syntheses using different metal ions complexed to this polymerizable chelator. The metal content in microbeads prepared using this technique was linearly dependent on the metal complex input in the bead synthesis, regardless of the type of metal. Batches of microbeads encoded with four lanthanides at three different concentrations were prepared. These microbeads produced three different levels of MC signal intensity with very good baseline resolution. As an illustrative example, and as described hereinafter, the surface of the microbead batch was a functionalized surface, conjugated to an antibody, and used to detect a target analyte (e.g., a sample biomolecule).

[0013] As shown herein as a representative application of the disclosed microbeads in mass cytometry (MC), a multiplexed bead-based assay was developed for the detection of analytes including cytokines. In this type of assay, classifier beads were labeled with heavy metal isotopes with different metal incorporation levels. Each classifier bead carried a different Ab on its surface. Reporters can be metal or metal oxide nanoparticles (NPs) with appropriate Abs of other bioidentification elements bound to their surface. Samples were stochastically injected into the plasma torch of an inductively coupled plasma time-of-flight mass spectrometer. This instrument is capable of single mass resolution over the range m / z:85 to m / z:209. Thus, as shown, an extremely high level of multiplexing was possible. In the bead-based assay with MC, cytokines were used as exemplary targets. As described herein, 4-plex analysis of a mixture of four cytokines is demonstrated. 9-plex assay is also illustrated as well. 9-plex assay is tested on peripheral blood mononuclear cell (PBMC) samples, comparing unstimulated samples with samples stimulated to promote cytokine secretion.

[0014] Thus, in one aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: Structural monomers, and A copolymer comprising a metal chelating monomer comprising a metal and a chelating agent; The chelating agent coordinates to the metal at at least three sites; and The structural monomer is a copolymer that does not include a chelating agent. The present invention includes metal-encoded microbeads comprising: In another aspect, the present disclosure includes a population of microbeads of the present disclosure. In another aspect, the present disclosure includes a kit comprising a plurality of individual populations of microbeads of the present disclosure.

[0015] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: polymerizing the structural monomers in the presence of a steric stabilizer in a nucleation stage to obtain a first mixture comprising polymerized structural monomers, non-polymerized structural monomers, and a steric stabilizer; mixing the first mixture with a metal chelating monomer comprising a metal and a chelating agent attached to at least one polymerizable end group, the chelating agent coordinating the metal at at least three sites; and the metal chelating monomer is polymerizable with the structural monomer to obtain a second mixture; and polymerizing the second mixture to form a copolymer of the microbeads, the structural monomers not including a chelating agent; The present invention also includes a method for preparing metal-encoded microbeads comprising: In another aspect, the present disclosure includes microbeads prepared by the methods of the present disclosure.

[0016] In another aspect, the present disclosure includes a method of preparing metal-encoded microbeads, the method comprising: providing an aqueous dispersion of swellable seed particles and an anionic surfactant; contacting the aqueous dispersion with monomers comprising structural monomers and metal chelating monomers, the metal chelating monomers comprising a metal and at least one chelating agent attached to the polymerizable terminus, the chelating agent coordinating the metal at at least three sites; diffusing the monomer into the seed particles to form an aqueous dispersion of swollen seed particles; and initiating polymerization of monomers in the aqueous dispersion of swollen seed particles; and the structural monomer does not include a chelating agent. Exemplary embodiments of the present disclosure are further described in connection with the drawings. [Brief description of the drawings]

[0017] [Figure 1] 1 is a schematic diagram showing the H-NMR spectrum of a Na3(DTPA-VBAm2) molecule dissolved in D2O. The structure of a given Na3(DTPA-VBAm2) has labeled protons corresponding to the indicated chemical shifts. [Diagram 2]Figures 2A and 2B are SEM images and size graphs, respectively, of metal-containing microbeads Ce-1 synthesized in the presence of the Ce(DTPA-VBAm2) metal complex added in the second step (d = 2.9 μm, CV = 1.2%). [Diagram 3] FIG. 3B is a series of graphs showing the incorporation efficiency of five metal ions into Y-1, Ce-1, Eu-1, Ho-1, Lu-1 and 5E1 microbeads (FIG. 3A) and into 4E1, 4E2 and 4E3 microbeads (FIG. 3B) using M(DTPA-VBAm2) complexes (M=Y, Ce, Eu, Ho, and Lu). [Figure 4] 4A and 4B are graphs showing the MC signal intensity (FIG. 4A) and metal content (FIG. 4B) of 5-element-coded microbeads (5E1) prepared in the presence of M(DTPA-VBAm2) metal complexes (M=Y, Ce, Eu, Ho, and Lu) in aliquots from the second stage. The error bars in a) and b) represent the RSD of the MC signal intensity and the SD of the metal content calculated from the MC signal intensity, respectively. [Diagram 5] 1 is a graph showing the linear dependence of metal content concentration in microbeads on feed concentration of metal complex in second stage aliquots. The filled symbols are data from preliminary bead syntheses (Y-1, Ce-1, Eu-1, Ho-1, Lu-1, and 5E1). The solid line is a linear regression of these filled data points. The open symbols are data from bead synthesis samples 4E1, 4E2, and 4E3, where the linear relationship observed in the preliminary bead syntheses was used as a basis for bead synthesis design. [Figure 6] Figure 1 shows the MC signal intensities of three different populations of PS microspheres prepared in the presence of M(DTPA-VBAm2) metal complexes (M = Ce, Eu, Ho, and Lu) in the second stage aliquots, representing the MC intensity histograms of 140Ce, 151Eu, 153Eu, 165Ho, and 175Lu, respectively. The x-axis of each figure is the signal intensity of the isotope, and the y-axis is the number of beads normalized to 100. The first, second, and third histograms represent the signals from 4E1, 4E2, and 4E3, respectively. [Figure 7] FIG. 7A is a schematic diagram showing an antigen detection reagent using M(DTPA-VBAm2)-coded microspheres (Eu-1). In this scheme, Eu-coded microbeads surface-functionalized with goat anti-mouse IgG are incubated with 175Lu-labeled mouse IgG as a reporter. The washed microbeads are then examined by MC for both 153Eu and 175Lu as evidence of reporter detection. FIG. 7B represents a histogram of MC measurements generated using the detection reagent of FIG. 7A, showing 175Lu signal intensity for goat anti-mouse (GAM)-modified Eu-1 microbeads (Eu-1 / GAM). In a second experiment, NAv-modified Eu-1 microbeads without GAM (Eu-1 / NAv) produced a weak signal shown in magenta color as a negative control. [Figure 8] Figures 8A and 8B are 1H-NMR spectra of Na3(DTPA-BAm2) and Na3(DTPA-ALAm2), respectively, measured in D2O. Figure 8C is the 1H-NMR spectrum of Na3(DTPA-AmPMAm2). The structures of these molecules given have the labeled protons corresponding to the chemical shifts indicated. [Figure 9] H-NMR (500 MHz) spectra of Ce(DTPA-VBAm2) (FIG. 9A), Ce(DTPA-BAm2) (FIG. 9B), Ce(DTPA-ALAm2) (FIG. 9C), and Ce(DTPA-AmPMAm2) (FIG. 9D) measured in DO. The resonance peaks in the figure are broadened and shifted because Ce(III) is a paramagnetic NMR shift reagent. [Figure 10] H-NMR (500 MHz) spectra of Y(DTPA-VBAm2) (FIG. 10A), Eu(DTPA-VBAm2) (FIG. 10B), Ho(DTPA-VBAm2) (FIG. 10C), and Lu(DTPA-VBAm2) (FIG. 10D) measured in DO. The resonance peaks in b) and c) were broadened and shifted because Eu(III) and Ho(III) are paramagnetic NMR shift reagents. [Figure 11]11A-11D are histograms of MC 140Ce signal intensity counts in Ce-1, Ce-2, Ce-3, and Ce-4 microbeads, respectively. The x-axis is 140Ce signal intensity and the y-axis is number of beads normalized to 100. [Figure 12] Figure 12A is a histogram of isotope signal intensity counts in a Y-1 microbead sample, Figure 12B is a histogram of isotope signal intensity counts in a Eu-1 microbead sample, Figure 12C is a histogram of isotope signal intensity counts in a Ho-1 microbead sample, and Figure 12D is a histogram of isotope signal intensity counts in a Lu-1 microbead sample. The x-axis is isotope signal intensity and the y-axis is number of beads normalized to 100. [Figure 13]Figure 13A is a graph showing the release profile of metal ions Ce3+ (squares), Eu3+ (circles), Ho3+ (upward triangles), and Lu3+ (downward triangles) from 4E3 microbeads (filled symbols and solid line) at 0.5% solids content into pH 3.0 buffer (50 mM sodium acetate) as determined by ICP-MS. Figure 13B is a graph showing the release profile of metal ions Ce3+ (squares), Eu3+ (circles), Ho3+ (upward triangles), and Lu3+ (downward triangles) from 4E3 microbeads (filled symbols and solid line) at 0.5% solids content into pH 7.0 buffer (10 mM ammonium acetate) as determined by ICP-MS. FIG. 13C is a graph showing the release profile of metal ions Ce3+ (squares), Eu3+ (circles), Ho3+ (upward triangles) and Lu3+ (downward triangles) from 4E3 microbeads (filled symbols and solid line) at 0.5% solids content into pH 10.5 buffer (200 mM sodium carbonate / bicarbonate) as determined by ICP-MS. FIG. 13D is a graph showing the release profile of metal ions Ce3+ (squares), Eu3+ (circles), Ho3+ (upward triangles) and Lu3+ (downward triangles) from 4E3 microbeads (filled symbols and solid line) at 0.5% solids content into 1% PVP solution as determined by ICP-MS. For comparison, the open symbols and dashed line show the release profile of metal ions from a microbead batch prepared by the AA technique under the same conditions as the 4E3 DTPA beads. [Figure 14] FIG. 1 is a schematic diagram of a multi-step strategy for functionalizing microbeads with goat anti-mouse (GAM) via silica coating. [Figure 15] FIG. 1 is a schematic diagram of the microbead assay of the present disclosure. [Figure 16] FIG. 1 is a schematic diagram showing an exemplary multiplexed bead-based sandwich immunoassay with MC performed in a 96-well filter plate. [Figure 17]Panel a shows the 140Ce-142Ce isotope dot plot diagram of a mixture of 11 classifier microbeads (C-1-C-11). The ellipses separate singlet events of the 11 microbeads. Panels b-k are dot plot diagrams showing the gating strategy to individually identify C-1-C-11 microbeads by MC. [Figure 18] Histograms of reporter signal intensity for IL-4 classifier beads (C-5) in a series of 4-plex assays of standard solutions with various IL-4 concentrations. (a), (b), and (c) AuNPs were employed as reporters in 4-plex assays of standard solutions containing IL-4 at concentrations of 0, 1.2, and 20 pg / mL, respectively. (d), (e), and (f) NanoGold were employed as reporters in 4-plex assays of standard solutions containing IL-4 at concentrations of 0, 1.2, and 20 pg / mL, respectively. [Figure 19] Calibration curves of two sets of 4-plex assays for (a) IL-4, (b) IL-6, (c) IFNγ, and (d) TNFα. The x-axis of each plot is the analyte concentration, and the y-axis is the median MC signal intensity of NPs attached to the corresponding classifier beads. Two different types of streptavidin-conjugated reporters (AuNPs and NanoGold) were investigated in these 4-plex assays. Results are shown as circles (●) for AuNPs and squares (■) for NanoGold. Negative events with 197Au signal intensity below 1 count / bead were excluded from the statistical analysis of the median intensity. Dose-response curves were generated by fitting the experimental results with a four-parameter logistic regression model. [Figure 20]Calibration curves of four sets of 9-plex assays for (a) IL-1β, (b) IL-4, (c) IL-6, (d) IL-10, (e) IL-18, (f) IFNγ, (g) TNFα, (h) CD163, and (i) CXCL-9 at different concentrations of biotinylated anti-CD163 and anti-CXCL-9 in the detection Ab cocktail are shown. The x-axis of each plot is the analyte concentration. The y-axis of each plot is the median MC signal intensity of AuNPs attached to the corresponding type of classifier beads. To minimize background noise at low analyte concentrations, the concentrations of biotinylated anti-CD163 and anti-CXCL-9 in the detection Ab cocktail were reduced from 2.5 to 2.0, 1.0, and 0.5 μg / mL, while the concentrations of the other detection Abs were kept constant at 2.5 μg / mL in the cocktail. The results of these assays are plotted as filled circles (●) for a concentration of 2.5 μg / mL, filled squares (■) for a concentration of 2.0 μg / mL, filled triangles (▲) for a concentration of 1.0 μg / mL, and filled diamonds (◆) for a concentration of 0.5 μg / mL. Negative events with 197Au signal intensity below 1 count / bead were excluded from the statistical analysis of median intensity. Dose-response curves were generated by fitting the experimental results with a four-parameter logistic regression model. [Figure 21] Histograms showing median 197Au signal intensities of AuNP reporters attached to classifier beads in 9-plex assays for the analysis of (a) IL-1β, (b) IL-4, (c) IL-6, (d) IL-10, (e) IL-18, (f) IFNγ, (g) TNFα, (h) CD163, and (i) CXCL-9 in stimulated and unstimulated PBMC samples at different sample dilution ratios. Solid bars represent the analysis of stimulated samples, and striped bars represent the analysis of unstimulated samples. [Figure 22]Figure 22(a) is an SEM image of C-1 microbeads prepared by two-step DisP. Figure 22(b) is a graph showing the efficiency of six metal ions incorporated into C-1 microbeads using M(DTPA-VBAm2) complexes (M=La, Ce, Pr, Tb, Ho, and Tm). Error bars are standard deviations of triplicate measurements on the same solution. [Diagram 23] Calibration curves for three sets of 4-plex assays for (a) IL-4, (b) IL-6, (c) IFNγ, and (d) TNFα at different reporter (NP) concentrations. The x-axis of each plot is the analyte concentration. The y-axis of each plot is the median MC signal intensity of AuNPs attached to the corresponding classifier beads. Three concentrations of AuNPs were investigated in these 4-plex assays: 200x, 400x, and 800x dilutions from the stock solution. The results are shown as circles (●) for 200x dilution, squares (■) for 400x dilution, and triangles (▲) for 800x dilution. Events with 197Au signal intensity below 1 count / bead were excluded from the statistical analysis of the median intensity. Dose-response curves were generated by fitting the experimental results with a four-parameter logistic regression model. [Figure 24] FIG. 13 summarizes the median MC signal intensities of AuNPs attached to classifier beads in a series of 9-plex assays of blank samples in the absence of analyte molecules (0 pg / mL). [Diagram 25] Calibration curves for 9-plex assays for IL-1β, IL-4, IL-6, IL-10, IL-18, IFNγ, TNFα, CD163 and CXCL-9 using the same assay conditions as for the analysis of stimulated and unstimulated PBMC samples. Dose-response curves were generated by fitting the experimental results with a four-parameter logistic regression model. [Figure 26]21 is a graph showing cytokine concentrations in stimulated and unstimulated PBMC samples calculated based on the dose-response calibration curve of FIG 25. Some measured MC intensity values ​​shown in FIG 21 are less than the minimum value of the 4P-LR modeled calibration curve shown in FIG 25. Concentrations are not calculated from these values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] I. Definition Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure described herein as appropriate, as understood by one of ordinary skill in the art. As used herein, the term "and / or" means that the listed items are present or used individually or in combination. In effect, the term means that "at least one" or "one or more" of the listed items are used or present. The term "and / or" in reference to its pharma-ceutically acceptable salts and / or solvates means that the compounds of the present application are present as individual salts and hydrates of the compounds of the present disclosure, as well as combinations of solvates of, for example, salts of the compounds of the present disclosure. As used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, an embodiment including "a compound" should be understood to present a particular aspect having one compound, or two or more additional compounds. In embodiments that include an "additional" or "second" component, such as an additional or second compound, the second component, as used herein, is chemically distinct from the other or first component. For example, the metal chelated to the second component may be different from the metal chelated to the first component, where the second and first components may have the same chelator. The "third" component is different from the other, first and second components, and further recited or "additional" components are similarly distinct.

[0019] As used in this disclosure and the claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. The term "consisting of" and its derivatives as used herein are intended to be closed terminology that specifies the presence of stated features, elements, components, groups, integers, and / or steps and also excludes the presence of other unstated features, elements, components, groups, integers, and / or steps. As used herein, the term "consisting essentially of" is intended to specify the presence of stated features, elements, components, groups, integers, and / or steps, as well as those that do not materially affect the basic and novel characteristics of these features, elements, components, groups, integers, and / or steps.

[0020] As used herein, the term "suitable" means that the selection of a particular compound or conditions will depend on the particular synthetic operation being performed, the identity of the molecule being converted, and / or the particular use for the compound, but that the selection is well within the skill of one trained in the art.

[0021] In an embodiment of the present disclosure, the compounds described herein may have at least one asymmetric center. When a compound has two or more asymmetric centers, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any ratio are encompassed within the scope of the present disclosure. Although the stereochemistry of a compound may be as shown in any given compound listed herein, it should be further understood that such compounds may contain a certain amount (e.g., less than 20%, suitably less than 10%, more suitably less than 5%) of compounds of the present disclosure with alternative stereochemistry. Any optical isomers, such as separated, pure or partially purified optical isomers or racemic mixtures thereof, are intended to be encompassed within the scope of the present disclosure. Compounds of the present disclosure may also exist in different tautomeric forms and any tautomeric forms that the compounds form, as well as mixtures thereof, are intended to be included within the scope of the present disclosure.

[0022] This description refers to numerous chemical terms and abbreviations used by those of ordinary skill in the art. Nonetheless, definitions of selected terms are provided for clarity and consistency. The terms "about," "substantially," and "approximately," as used herein, refer to a reasonable amount of deviation from the modified term that does not significantly change the end result. These terms of degree should be interpreted as including at least or up to ±5% deviation from the modified term, unless this deviation would negate the meaning of the word it modifies or the context would suggest otherwise to one of ordinary skill in the art.

[0023] The term "alkyl" as used herein, whether used alone or as part of another group, refers to a straight or branched, saturated alkyl group. The number of carbon atoms that the referenced alkyl group can contain is indicated by the prefix "Cn1-n2". For example, the term C1-10 alkyl refers to an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The term "alkyl" as used herein, whether used alone or as part of another group, refers to a straight or branched, saturated alkyl group, i.e., a saturated carbon chain containing substituents at two of its termini. The number of carbon atoms that a referenced alkylene group can contain is indicated by the prefix "Cn1-n2". For example, the term C2-6 alkylene refers to an alkylene group having 2, 3, 4, 5 or 6 carbon atoms. The term "available," as in "available hydrogen atom" or "available atom," refers to an atom that is contemplated as being available to those of ordinary skill in the art to be substituted with a substituent. The terms "amine" or "amino," as used herein, whether used alone or as part of another group, refer to a group of general formula NR'R", where R' and R" are each independently selected from hydrogen or C1-6 alkyl. The term "cycloalkyl" as used herein, whether used alone or as part of another group, refers to a saturated carbocyclic group containing one or more rings. The number of carbon atoms possible in the cycloalkyl group referred to is indicated by the numerical prefix "Cn1-n2". For example, the term C3-10 cycloalkyl refers to a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The term "aryl" as used herein, whether used alone or as part of another group, refers to a carbocyclic group containing at least one ring. In certain embodiments of the present disclosure, the aryl group contains 6, 9 or 10 carbon atoms, such as phenyl, indanyl or naphthyl. The term "heterocycloalkyl" as used herein, whether used alone or as part of another group, refers to a cyclic group containing at least one non-aromatic ring in which one or more atoms are heteroatoms selected from O, S and N. Heterocycloalkyl groups are either saturated or unsaturated (i.e., containing one or more double bonds). When a heterocycloalkyl group contains the prefix Cn1-n2, the prefix indicates the number of carbon atoms in the corresponding carbocyclic group in which one or more, suitably 1 to 5, ring atoms are replaced with a heteroatom as defined above. The term "heteroaryl," as used herein, whether used alone or as part of another group, refers to a cyclic group containing at least one heteroaromatic ring in which one or more atoms are heteroatoms selected from O, S and N. When a heteroaryl group contains the prefix Cn1-n2, the prefix indicates the number of carbon atoms in the corresponding carbocyclic group in which one or more, suitably 1 to 5, ring atoms are replaced with a heteroatom as defined above.

[0024] All cyclic groups, including aryl and cyclo groups, contain one or more rings (i.e., are polycyclic). When a cyclic group contains more than one ring, the rings may be fused, bridged, spiro-fused, or joined by bonds. A first ring is "fused" to a second ring means that the first ring and the second ring share two adjacent atoms between them. A first ring is "bridged" with a second ring means that the first ring and the second ring share two non-adjacent atoms between them. A first ring is "spirofused" to a second ring means that the first ring and the second ring share one atom between them. The term "halo" as used herein refers to a halogen atom and includes fluoro, chloro, bromo and iodo.

[0025] The term "optionally substituted" refers to a group, structure or molecule that is unsubstituted or substituted with one or more substituents. As used herein, the term "atm" refers to atmospheric pressure. As used herein, the term "MS" refers to mass spectrometry. As used herein, the term "aq." refers to aqueous.

[0026] As used herein, the term "protecting group" or "PG" refers to a chemical moiety that protects or masks reactive portions of a molecule to prevent side reactions at these reactive portions of the molecule while manipulating or reacting another portion of the molecule. After the manipulation or reaction is completed, the protecting group is removed under conditions that do not degrade or decompose the remaining portion of the molecule. The selection of a suitable protecting group can be performed by those skilled in the art. Many conventional protecting groups are known in the art and are described, for example, in "Protective Groups in Organic Chemistry" McOmie, JFW Ed., Plenum Press, 1973, Greene, TW and Wuts, PGM, "Protective Groups in Organic Synthesis", John Wiley & Sons, 3rd Edition, 1999 and Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas).

[0027] In forming copolymers, it can be appreciated that certain polymerizable end groups of metal chelating monomers are better matched or selectively polymerized with certain structural monomers. The reactivity of comonomers can be determined using methods known in the art, for example, by measuring reactivity ratios. For example, vinyl and methyl vinyl react with vinyl ethers, aryl vinyls react with other styrenes, and acrylates react with other acrylates.

[0028] As used herein, the term "EDTA" refers to ethylenediaminetetraacetic acid. As used herein, the term "DTPA" refers to diethylenetriaminepentaacetic acid. As used herein, the term "EGTA" refers to egtazic acid. As used herein, the term "EDDS" refers to ethylenediamine-N,N'-disuccinic acid. As used herein, the term "EDDHA" refers to ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid). As used herein, the term "BAPTA" refers to 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. As used herein, the term "TACN" refers to 1,4,7-triazacyclononane. As used herein, the term "TACD" refers to 1,5,9-triazacyclododecane. As used herein, the term "cyclene" refers to 1,4,7,10-tetraazacyclododecane. The term "cyclam" as used herein refers to 1,4,8,11-tetraazacyclotetradecane. As used herein, the term "(13)aneN4" refers to 1,4,7,10-tetraazacyclotridecane. The term "1,7-diaza-12-crown-4" as used herein refers to 1,7-dioxa-4,10-diazacyclododecane. The term "1,10-diaza-18-crown-6" as used herein refers to 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane. As used herein, the term "DFO" refers to desferrioxamine. As used herein, the terms "TACD-type chelators," "TACN-type chelators," "cyclen-type chelators," and the like, refer to chelators that contain specific base structures (i.e., TACD, TACN, cyclen, etc.), which can be further substituted at available hydrogen atom positions.

[0029] The term "swellable polymer seed" as used herein refers to a polymer particle that can increase in volume. For example, the swellable polymer seed can increase in volume when contacted with a swelling agent. The swelling agent can be, for example, an anionic surfactant and / or an organic compound. When the swellable polymer seed is swollen, compounds such as monomers (e.g., structural monomers and metal chelating monomers), steric stabilizers, and polymerization initiators can diffuse into the interior of the swollen polymer seed. Polymerization of the monomers can then occur.

[0030] The term "substantially oxygen-free conditions" as used herein refers to reaction conditions in which the oxygen content is low or absent. For example, substantially oxygen-free conditions can refer to reaction conditions in which the reaction is carried out under an inert atmosphere, such as a noble gas (e.g., helium, argon) or nitrogen atmosphere. For example, substantially oxygen-free conditions can refer to an oxygen content of about 0 ppm to about 5 ppm, about 0 ppm to about 3 ppm, about 0 ppm to about 2 ppm, or about 0 ppm to about 1 ppm, or about 0.01 ppm to about 2 ppm.

[0031] The term "antibody" as used herein is intended to encompass monoclonal, polyclonal, and chimeric antibodies and binding fragments thereof. Antibodies may be derived from recombinant sources and / or produced in transgenic animals. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating antibodies with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibodies and other fragments can also be synthesized by recombinant techniques. Antibody fragment as used herein refers to binding fragments.

[0032] The term "oligonucleotide" as used herein refers to a nucleic acid comprising a sequence of nucleotide or nucleoside monomers consisting of bases, sugars, and intersugar (backbone) linkages of natural and non-natural origin, including single-stranded and double-stranded molecules, RNA and DNA. Oligonucleotides may comprise non-naturally occurring monomers and may be long in size (e.g., greater than 1000 monomers up to 10K monomers), intermediate in size (e.g., 200 to 1000 nucleotides, inclusive) or short in size, e.g., less than 200 monomers, 100 monomers, 50 monomers. The term "oligonucleotide" encompasses, for example, single-stranded DNA (ssDNA), genomic DNA (gDNA), complementary DNA (cDNA, reverse transcribed from RNA), messenger RNA (mRNA), "antisense oligonucleotides" and "miRNA", as well as oligonucleotide analogs, such as "morpholino oligonucleotides", "phosphorothioate oligonucleotides", or any oligonucleotide or analog thereof known to one of skill in the art.

[0033] The recitations herein of numerical ranges by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are deemed to be modified by the term "about."

[0034] Furthermore, it is intended that the definitions and embodiments described in a particular section are applicable to other embodiments described herein as appropriate, as can be understood by those skilled in the art. For example, in the following sections, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature indicated as being preferred or advantageous.

[0035] For ranges described herein, subranges, such as increments of 0.1 therebetween, are also contemplated. For example, if a range is 0 ppm to 5 ppm, subranges such as 0.1 ppm to about 5 ppm, 0 ppm to about 4.9 ppm, 0.1 ppm to about 4.9 ppm, etc. are also contemplated.

[0036] II. Compounds, Compositions, and Kits of the Disclosure Thus, in one aspect, the present disclosure includes a metal-encoded microbead, the microbead comprising: Structural monomers, and a copolymer comprising a metal chelating monomer comprising a metal and a chelating agent; The chelating agent coordinates the metal at at least three sites; and the structural monomer does not include a chelating agent.

[0037] The microbeads of the present disclosure may include a substantially uniformly distributed metal. For example, metals chelated to metal chelating monomers are not limited to surface attachment, but rather may be found dispersed throughout and / or within the microbeads of the present disclosure.

[0038] In some embodiments, the chelator coordinates the metal at at least 4 sites, at least 5 sites, at least 6 sites, at least 7 sites, or at least 8 sites, for example, DTPA is an octadentate ligand (capable of coordination at 8 sites).

[0039] In some embodiments, the structural monomer does not include any chelating agents (eg, does not include any chelating agents that coordinate a metal at at least two sites). In some embodiments, the structural monomer is metal-free. For example, the structural monomer may not contain a metal by chelation, by covalent bonding (e.g., tellurium in the carbon skeleton of the structural monomer), or possibly by any other means. The structural monomer may not contain a transition metal or a class of transition metals. For example, the structural monomer may not contain a rare earth metal (e.g., lanthanide) and / or a soft metal as described herein. In some embodiments, such as in the case of mass cytometry applications, the metal chelating monomer may contain a heavy metal (e.g., more than 80 amu), while the structural monomer does not contain a heavy metal (e.g., more than 80 amu).

[0040] In some embodiments, the structural monomers are selected from substituted or unsubstituted styrene, α-methylstyrene, acrylic acid and esters and their amides, methacrylic acid and esters and their amides, and derivatives thereof. In one embodiment, the structural monomers are selected from substituted or unsubstituted styrene and / or or combinations thereof.

[0041] In some embodiments, the metal chelating monomer has the formula (I) prior to polymerization: [ka] where the ligand is a chelating agent, L is a linker, X is a polymerizable end group, M is a metal, and n is an integer equal to or greater than 1, and the metal chelating monomer is neutrally charge prior to polymerization. The metal is chelated to the chelating agent of the metal chelating monomer through ionic non-covalent interactions, and thus the metal is incorporated into the microbeads of the present disclosure through non-covalent interactions.

[0042] In some embodiments, L is selected from a bond, a C3-C8 alkylamine, a C3-C8 alkylene, a C3-C8 cycloalkyl, a C3-C8 heterocycloalkyl, a 5- or 6-membered aryl or heteroaryl, an alkylaryl, an alkylheteroaryl, a C3-C8 cycloalkylaryl, a C3-C8 cycloalkylheteroaryl, C(O), —C(O)O, or a mixture thereof. Each of the alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl can be independently unsubstituted or substituted with one or more substituents selected from a C1-C6 alkyl, a C1-C6 alkenyl, a C3-C8 cycloalkyl, a C3-C8 heterocycloalkyl, an amide, an ester, an aryl, a heteroaryl, an alkylaryl, an alkylheteroaryl, a C3-C8 cycloalkylaryl, a C3-C8 cycloalkylheteroaryl, CN, or a mixture thereof. L can be attached to the chelator, for example, by an amide or ester group / bond.

[0043] It may be appreciated that where the structural monomer and the metal chelate monomer have similar hydrophobicity, incorporation or mixing of one into the other is preferred. For example, if the structural monomer is hydrophobic, the hydrophobic metal chelate monomer may have a more favorable interaction with the structural monomer, resulting in more efficient mixing of the two monomers. Thus, in some embodiments, L may be hydrophobic.

[0044] In some embodiments, the polymerizable end group is selected from allyl vinyl, styrene, α-methylstyrene, acrylate esters, methacrylate esters, acrylamide, 2-methylacrylamide, and mixtures thereof, optionally the polymerizable end group is allyl vinyl or styrene, in some embodiments the polymerizable end group is allyl vinyl or vinyl ester.

[0045] The chelator can be, for example, tridentate, in some embodiments, the chelator is tetradentate, pentadentate, hexadentate, heptadentate, or octadentate, and optionally the chelator is hexadentate or octadentate. In some embodiments, the chelator comprises an aminopolyacid moiety, or a derivative thereof. In some embodiments, the derivative of the aminopolyacid moiety comprises an amide of the aminopolyacid moiety. For example, the aminopolyacid moiety can be selected from an aminopolycarboxylic acid, an aminopolyphosphonic acid, or a combination thereof.

[0046] In some embodiments, L is a bond, the chelator is an aminopolyacid, and the polymerizable end group is an allylvinyl. For example, the metal chelating monomer can be a metal coordinated vinylbenzeneiminodiacetic acid or a metal coordinated divinylbenzeneiminodiacetic acid. In other embodiments, the aminopolyacid moiety is one or more substituted oligomers of ethyleneimine, propyleneamine, or mixtures thereof, and the oligomer is substituted with two or more carboxylic acids and / or phosphonic acids. The oligomer can be a crown ether or an azacrown ether. In some embodiments, the aminopolyacid moiety is one or more substituted oligomers of ethylene oxide, ethyleneimine, propylene oxide, propyleneamine, ethanolamine, propanolamine, aminophenol cyclohexanediamine, or mixtures thereof. In yet other embodiments, the oligomer is further substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, halo, alcohol, amine, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.

[0047] In some embodiments, the chelator is selected from DFO, EDTA, DTPA, EGTA, EDDS, EDDHA, BAPTA, H4neunpa, H6phospa, H4CHXoctapa, H4octapa, H2CHXdedpa, H5decapa, Cy-DTPA, Ph-DTPA, TACN type chelators, TACD type chelators, cyclen type chelators, cyclam type chelators, (13)aneN4 type chelators, 1,7-diaza-12-crown-4 type chelators, 1,10-diaza-18-crown-6 type chelators, or derivatives thereof. For example, the TACN-type chelator can be NOTA, NOPO, TRAP, or a derivative of any of these. In some embodiments, the cyclen-type chelator is DOTA or a derivative thereof. In some embodiments, the cyclam-type chelator is selected from TETA, cross-linked TETA, DiAmSar, or derivatives thereof. In another embodiment, the (13)aneN4 type chelator is selected from TRITA or a derivative thereof. In yet other embodiments, the 1,10-diaza-18-crown-6 type chelating agent is selected from MACROPA or a derivative thereof.

[0048] In some embodiments, the chelator is selected from DTPA, Cy-DTPA, Ph-DTPA, or derivatives thereof. For example, a derivative of DTPA can include DTPA having two adjacent carbon atoms joined together with the atom between them to form a 5- or 6-membered ring, optionally a cycloalkyl ring, aryl or heteroaryl ring.

[0049] Prior to polymerization, the monomers described herein are unreacted, i.e., the monomers comprise a polymerizable end group that can participate in polymerization. In some embodiments, prior to polymerization, the metal chelating monomer is: [ka] where L and X are as described herein.

[0050] In another embodiment, prior to polymerization, the metal chelating monomer is: [ka] TIFF2024511743000005.tif53132, or a mixture thereof. For example, the mixture may include one or more of the above metal chelating monomers having different metals.

[0051] In other embodiments, the chelating agent comprises a porphyrin or a phthalocyanine. For example, the chelator can be a substituted or unsubstituted porphyrin.

[0052] In some embodiments, the porphyrin and the phthalocyanine are each independently substituted with C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, carboxylic acid, or mixtures thereof.

[0053] When the chelating agent is or includes a porphyrin or phthalocyanine, the metal can be a soft metal. For example, the soft metal can be cadmium, cobalt, copper, iron, zinc, nickel, tin, osmium, palladium, platinum, gold, thallium, mercury, or lead, including isotopes thereof, as well as mixtures thereof. In some embodiments, the soft metal has an atomic mass of 80 amu or greater.

[0054] In some embodiments, prior to polymerization, the metal chelating monomer comprises: [ka] or a mixture thereof, where n is an integer from 1 to 4. For example, L can be aniline. In some embodiments, n is 2 or at least 2.

[0055] In other embodiments, the metal chelating monomer is: [ka] or mixtures thereof.

[0056] The microbeads of the present disclosure may also further comprise a steric stabilizer, which may be PVP, polyvinyl alcohol, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polyacrylic acid, water-soluble homopolymers of acrylic acid esters, water-soluble homopolymers of methacrylic acid esters, water-soluble homopolymers of acrylamide, homopolymers of methacrylamide, water-soluble copolymer steric stabilizers, or mixtures thereof.

[0057] In some embodiments, the water-soluble copolymer steric stabilizer is selected from copolymers of acrylic acid esters, methacrylic acid esters, acrylamides or methacrylamides with methyl acrylate and / or ethyl acrylate, or mixtures thereof. The copolymers may also be crosslinked, such as is achieved when the metal chelating monomers and / or the structural monomers have two or more polymerizable groups. For example, the microbeads of the present disclosure may include multiple metals, for example, each metal may be incorporated by the same or different types of metal chelating monomers. The metal may be multiple metals. In some embodiments, the plurality of metals comprises one or more enriched isotopes.

[0058] In some embodiments, the multiple metals comprises at least two metals, at least three metals, or at least four metals. In other embodiments, the amount of each metal in the plurality of metals is within about 20% or about 10% of the amount of another metal in the plurality of metals, for example, this can be determined by mass cytometry for a population of microbeads or for a single microbead. The metal can be, for example, a transition metal (i.e., a metal in groups 3-12 of the periodic table, or a metal in the lanthanide or actinide series). The metal can be, for example, indium, bismuth, or a rare earth metal. The rare earth metal can be, for example, a lanthanide metal, yttrium, or mixtures thereof. In some embodiments, the metal is indium, bismuth, a soft metal, or a rare earth metal. The soft metal can be cadmium, cobalt, copper, iron, zinc, nickel, tin, osmium, palladium, platinum, gold, thallium, mercury, lead, and isotopes thereof, and mixtures thereof. In other embodiments, the metal comprises a rare earth metal selected from Y, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, their respective isotopes, and mixtures thereof. In yet other embodiments, the metal is selected from La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, their respective isotopes, and mixtures thereof. In yet other embodiments, the rare earth metal is 89Y, 139La, 136Ce, 138Ce, 140Ce, 142Ce, 141Pr, 142Nd, 143Nd, 145Nd, 146Nd, 148Nd, 145Pm, 144Sm, 149Sm, 150Sm, 152Sm, 154Sm, 151Eu, 153Eu, 154Gd, 155Gd, 156Gd, 157Gd, 158Gd, 160 The cations are selected from Gd, 152Gd, 159Tb, 156Dy, 158Dy, 160Dy, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 162Er, 164Er, 166Er, 167Er, 168Er, 170Er, 169Tm, 168Yb, 170Yb, 171Yb, 172Yb, 173Yb, 174Yb, 176Yb, 175Lu, or mixtures thereof. The metal may be substantially uniformly distributed throughout the microbead, or it may be compartmentalized, for example within the microbead.

[0059] In some embodiments, the microbeads have a glass transition temperature (e.g., as measured by differential scanning calorimetry (DSC)) of about or above 60° C., optionally about or above 70° C., about or above 80° C., about or above 90° C., about or above 100° C., about or above 115° C., about or above 125° C., or about or above 135° C. For example, DSC results are obtained on the second or third heating scan at a scan rate of about 10° C. / min to about 20° C. / min.

[0060] In other embodiments, the microbeads have a diameter of about 0.6 μm to about 20 μm, about 1 μm to about 15 μm, about 2 μm to about 10 μm, or about 2 μm to about 6 μm. In some embodiments, the microbeads of the present disclosure have a size suitable for mass cytometry. In yet another embodiment, the microbeads are colloidally stable in water.For example, the microbeads of the present application are substantially stable when stored in a buffer and / or physiological medium.For example, substantially stable in a buffer and / or physiological medium means that there is no significant leakage or less than 1% leakage of metals when stored in a buffer and / or physiological medium.

[0061] In some embodiments, the surface of the microbeads comprises functionalization for attachment to biomolecules. The functionalization can be introduced by adding a coating layer. In some embodiments, the attachment is a covalent attachment. For example, as shown in the examples, microbeads can be coated with silica and functionalized with reactive functional groups such as carboxylic acid groups. Biomolecules can be added to the microbeads. Biomolecules can be classified as proteins, oligonucleotides, lipids, carbohydrates, or small molecules. Alternatively or additionally, biomolecules can be classified by their function. Biomolecules are not particularly limited, and various functionalizations can be used to conjugate biomolecules to microbeads. For example, oligonucleotides can be single-stranded DNA molecules, cDNAs that hybridize to target nucleic acid analytes (e.g., sample nucleic acid biomolecules) under optionally stringent conditions, or oligonucleotides can be aptamers. For example, biomolecules can be oligonucleotides that specifically hybridize to target oligonucleotides, such as target mRNAs endogenous to the sample (e.g., hybridize to sample oligonucleotides). Hybridization can be of a sequence that is more than 8, more than 10, more than 15, or more than 20 nucleotides.

[0062] In certain aspects, biomolecules can be classified according to their function. For example, a biomolecule can be an affinity reagent, an antigen (e.g., an analyte that is specifically bound by an affinity reagent), or an enzyme substrate. An affinity reagent can be an antibody (e.g., or a fragment thereof), an aptamer, a receptor (e.g., or a portion thereof), or any other biomolecule that specifically binds a target (e.g., avidin, such as streptavidin, which specifically binds biotin). For example, a bead can be coupled with an antibody and used to detect the presence of its target antigen in a sample, such as a cytokine, a viral protein, a cancer biomarker, etc. In certain methods and kits, a microbead can be functionalized with avidin for attachment of another biomolecule functionalized with biotin (e.g., to make the bead compatible with any of a number of different assays). An antigen can be a protein (or a peptide sequence thereof) that contains an epitope that is specifically bound by an affinity reagent, such as an antibody. For example, a bead can be attached to a viral antigen (such as a viral protein sequence) and used to detect the presence of an antibody in a sample that specifically binds the viral antigen, as further described herein. An enzyme substrate can be any substrate that is acted upon by a particular enzyme, such as an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. For example, the substrate can be a protein (e.g., or a peptide sequence thereof) that is a substrate for an enzyme, such as a protease, a phosphatase, a kinase, a methyltransferase, a demethylase, etc. Non-protein substrates include, for example, a double-stranded oligonucleotide that includes a restriction sequence that can be cleaved by a restriction enzyme or a site for DNA repair (such as a nick), an oligonucleotide sequence that includes a targeting sequence by a DNA methyltransferase, or any non-protein substrate known to those skilled in the art. For example, a bead can be attached to the substrate, exposed to a sample that includes an enzyme that modifies the substrate, and the modification (or lack thereof) of the substrate can be detected (e.g., as described further herein).

[0063] In some embodiments, the affinity reagent is or comprises an antibody or a binding fragment thereof. The antibody may be, for example, a biotinylated antibody or binding fragment, and may be directly or indirectly attached to a microbead. For example, as shown in the Examples, neutravidin (NAv) was covalently bound to COOH groups on silica coated on the surface of microbeads by EDC / NHS coupling, and biotinylated antibodies were attached to the surface of NAv-modified microbeads by strong biotin-avidin affinity.

[0064] Thus, the affinity reagent can be avidin or related biotin-binding molecules such as streptavidin, neutravidin and captAvidin. Microbeads containing such affinity reagents can be customized with biotinylated antibodies specific for the target analyte of interest.

[0065] In some embodiments, the affinity reagents, which are optionally antibodies, are specific for cytokines, optionally chemokines, interferons, lymphokines, monokines, interleukins such as IL-1-36, tumor necrosis factors and colony stimulating factors. The antibodies may also be specific for pathogenic proteins such as viral, bacterial or fungal pathogens. Such microbeads can be used for the detection of the presence of such pathogens or their products in a sample, such as an environmental or patient sample. In another embodiment, the antigen is a viral antigen. Such microbeads containing viral antigens can be used, for example, to detect viral antibodies in patient samples, as well as antigens from other pathogens, etc.

[0066] In yet another embodiment, the copolymer of the microbead further comprises a third monomer, the third monomer being present at least on the surface of the microbead and comprising at least one reactive functional group. In some embodiments, the microbead is functionalized with at least one reactive functional group contained in the third monomer, for example, by three-stage dispersion polymerization. In some embodiments, the third monomer is substituted or unsubstituted acrylic acid or methacrylic acid. An example of three-stage dispersion polymerization is described in Abdelrahman et al., JACS, 2009, 15276, the contents of which are incorporated herein by reference.

[0067] For example, the surface of the microbeads can be functionalized with reactive functional groups. In some embodiments, the reactive functional groups are on the silicon dioxide coating. In other embodiments, the reactive functional group is selected from an amine, a thiol, an alcohol, an aldehyde, a carboxylic acid, an epoxide, a vinyl, an alkyne, a maleimide, or a click chemistry moiety (e.g., dibenzocyclooctyne (DBCO), azide, transcyclooctene or tetrazine, or derivatives thereof), or mixtures thereof. In yet other embodiments, the functionalization comprises a coating of silicon dioxide on the surface of the microbeads, and optionally the functionalization further comprises functionalizing the silicon dioxide coating. In some embodiments, the surface of the microbeads is functionalized with reactive functional groups. In other embodiments, the reactive functional groups are on a silicon dioxide coating. For example, the reactive functional groups can be selected from amines, thiols, alcohols, aldehydes, carboxylic acids, epoxides, vinyls, alkynes, maleimides, or mixtures thereof.

[0068] It is understood that the functionalization of silicon dioxide coating can be carried out using methods known in the art.For example, the method of functionalizing microbeads with silicon dioxide coating is described in US Patent Application Publication No. 2019 / 0091647.The contents of this patent application publication are incorporated herein by reference.

[0069] In some embodiments, the attachment to the biomolecule is a non-covalent attachment. In other embodiments, the surface of the microbeads is functionalized with avidin, streptavidin, neutravidin, or a mixture thereof. In yet other embodiments, the surface of the microbeads is conjugated to biomolecules.

[0070] In some embodiments, the metal provides a barcode that identifies the biomolecule. In some embodiments, the internal structure of the microbead comprises a copolymer. In other embodiments, the metal chelating monomer chelates a single metal atom and does not chelate multiple metal atoms.

[0071] In some embodiments, the microbeads comprise a polymer seed that does not include a metal chelating monomer, hi some embodiments, the polymer seed has an interior space formed by swelling the polymer seed, and the copolymer is present within at least the interior space of the polymer seed. In some embodiments, the polymer seed comprises a structural monomer, and optionally the structural monomer of the polymer seed is of the same structure as the structural monomer of the copolymer. In another aspect, the present disclosure includes a population of microbeads of the present disclosure.

[0072] In some embodiments, the population has a size distribution with a coefficient of variation (CV) of about 10% or less than 10%. In other embodiments, the coefficient of variation is less than 5%.

[0073] In some embodiments, each microbead comprises multiple metals, and the average amount across the population of microbeads of each metal of the multiple metals is about 10% or within 10% of the average amount of another metal of the multiple metals. In other embodiments, the plurality of metals includes one or more enriched isotopes. In some embodiments, the amount of each metal in one microbead of the population of microbeads is about 20% or within 20%, or about 10% or within 10%, or about 5% or within 5% of the amount of the same metal in another microbead of the population of microbeads. In other embodiments, the amount of each metal in the population of microbeads has a distribution with a coefficient of variation of about 20% or less than 20%. In yet other embodiments, the amount of each metal in the population of microbeads has a distribution with a coefficient of variation of about 10% or less than 10%. In some embodiments, the microbeads in a population of microbeads contain substantially the same amount of the same metal. In some embodiments, the same metal is more than one metal, and the microbeads include each metal of the more than one metal in substantially the same amount.

[0074] A further embodiment is a composition comprising a microbead or a plurality of microbeads.The composition can be, for example, a buffer, for example, buffered to a pH of about 7.The buffer can include a buffering agent such as ammonium acetate.The composition can also include PVP. In certain embodiments, the composition is an aqueous colloidal suspension. The composition may include one or more ingredients selected from stabilizers, preservatives, buffers, and mixtures thereof. In another aspect, the present disclosure includes kits comprising multiple individual populations and / or compositions of microbeads of the present disclosure.

[0075] In some embodiments, each population of microbeads is distinguishable from another population of microbeads based on the metal or metals of the microbeads. In some embodiments, the microbeads of at least one population (eg, each population) of microbeads comprise a metal or metals that is different from the metal or metals of the microbeads of another population of microbeads. In some embodiments, the microbeads of at least one population (eg, each population) of microbeads comprise a plurality of metals in a ratio that differs from the microbeads of another population of microbeads. In some embodiments, the microbeads in each population of microbeads are conjugated to a different biomolecule, for example the microbeads can be used as barcoding agents for biomolecules attached to the microbeads by virtue of the metal or metals in the microbeads. In another aspect, the present disclosure includes microbeads prepared by the methods of the present disclosure.

[0076] In some embodiments, the structural monomers and the metal chelating monomers may be copolymerized by any of the methods described herein. Of note, the structural monomers and / or metal chelating monomers used in the methods described herein may have any of the aspects described in this section.

[0077] The kits of the present disclosure may include any of the microbeads or populations of microbeads described herein. The microbeads or kits thereof may also include additional aspects for mass spectrometry assays, as further described herein. Aspects of the present disclosure also include additional methods, such as mass spectrometry assays, as further described herein.

[0078] III. Methods of the Disclosure In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: polymerizing the structural monomers in the presence of a steric stabilizer in a nucleation stage to obtain a first mixture comprising polymerized structural monomers, unpolymerized structural monomers, and a steric stabilizer; combining the first mixture with a metal chelating monomer comprising a metal and a chelating agent attached to at least one polymerizable end group to obtain a second mixture, combining said chelating agent with at least three sites to coordinate the metal; and said metal chelating monomer is polymerizable with said structural monomer; and polymerizing the second mixture to form a copolymer of the microbeads; The structural monomer is polymerized without a chelating agent; The present invention also includes a method for preparing metal-encoded microbeads comprising:

[0079] It can be understood that for more efficient polymerization, the structural monomers and the metal chelating monomers need to be soluble in the reaction medium. Furthermore, it can be understood that if the monomers are substituted with, for example, substituents that interfere with polymerization (e.g., halo, amine, alcohol), the substituents may be temporarily protected with protecting groups known in the art before and / or during polymerization. After polymerization, the protecting groups may be selectively removed and the substituents may be selectively deprotected using methods known in the art.

[0080] In some embodiments, the metal is a plurality of metals. In other embodiments, the structural monomers are polymerized in the nucleation stage to about 5% to about 20% completion based on the structural monomers. In yet other embodiments, polymerization of the second mixture is carried out to from 75% to about 100% completion, from about 80% to about 99% completion, from about 85% to about 95% completion, or from about 85% to about 93% completion based on structural monomers.

[0081] In some embodiments, structural monomers are as defined herein. In some embodiments, the metal chelating monomer is as defined herein. In some embodiments, the steric stabilizer is as defined herein. In some embodiments, the metal is as defined herein.

[0082] In some embodiments, the method further comprises functionalizing the microbeads. In some embodiments, the functionalization of the microbeads comprises: mixing the polymerized second mixture with a third monomer to obtain a third mixture, the third monomer including a reactive functional group; and polymerizing the third mixture; Includes. For example, the reactive functional group can be selected from amine, thiol, alcohol, aldehyde, carboxylic acid, epoxide, vinyl, alkyne, maleimide, or or mixture thereof. It is understood that certain reactive functional groups may interfere with the polymerization process and can be protected using a protecting group known in the art before and / or during polymerization. For example, amines and thiols can be protected by a protecting group. For example, monomers substituted with reactive functional groups can be used in protected form, so that the reactive functional groups will not interfere with the polymerization process. Optionally, the protected reactive functional groups can be deprotected using methods known in the art.

[0083] In some embodiments, the third monomer is selected from optionally substituted acrylic acid, optionally substituted methacrylic acid, and mixtures thereof.

[0084] In another embodiment, the functionalization of the microbeads comprises coating the microbeads with silicon dioxide. In another embodiment, the functionalization of the microbeads further comprises functionalization of the silicon dioxide coating. For example, functionalization of the silicon dioxide coating may include reacting the silicon dioxide coating with an organosilane, which may, for example, be selected from chlorosilanes, alkoxysilanes, derivatives thereof, and mixtures thereof. In some embodiments, the reaction of the silicon dioxide coating with the organosilane is carried out in the presence of a catalyst. For example, the catalyst can be selected from ammonia, hydroxides, organic amines, or mixtures thereof. In some embodiments, the organosilane comprises a reactive functional group. In some embodiments, the reactive functional groups are selected from amines, thiols, alcohols, aldehydes, carboxylic acids, epoxides, vinyls, alkynes, maleimides, or or mixtures thereof. In some embodiments, the organosilane is APTES.

[0085] In other embodiments, the method further comprises conjugating the microbeads to the biomolecule. In some embodiments, the biomolecule is as defined herein. In some embodiments, the microbeads have a diameter of about 0.6 μm to about 20 μm, about 1 μm to about 15 μm, about 2 μm to about 10 μm, or about 2 μm to about 6 μm. In some embodiments, the microbeads are microbeads of the present disclosure. In some embodiments, the internal structure of the microbead comprises a copolymer.

[0086] In another aspect, the present disclosure includes a method of preparing metal-encoded microbeads, the method comprising: providing an aqueous dispersion of swellable seed particles and an anionic surfactant; contacting the aqueous dispersion with monomers including structural monomers and metal chelating monomers, the metal chelating monomers including a metal and at least one chelating agent attached to the polymerizable terminus, the chelating agent coordinating the metal at at least three sites; and diffusing the monomer into the seed particles to form an aqueous dispersion of swollen seed particles; and initiating polymerization of monomers in the aqueous dispersion of swollen seed particles, the structural monomers not including a chelating agent; Includes.

[0087] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: providing an aqueous dispersion comprising a swellable polymer seed, an organic compound, an anionic surfactant, and optionally an organic solvent in which the organic compound is soluble; Diffusing an organic compound into a swellable polymer seed; contacting the aqueous dispersion with a mixture comprising a structural monomer and a metal chelate monomer, optionally wherein the mixture further comprises a steric stabilizer and / or a polymerization initiator; and polymerizing the mixture to obtain a copolymer of microbeads; The present invention also includes a method for preparing metal-encoded microbeads, comprising: Organic compounds are those with a molecular weight of less than 5000 Da and a pH of 10 at 25°C. -2 It has a water solubility of less than g / L.

[0088] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: preparing a swellable polymer seed by emulsion polymerization, wherein an anionic surfactant is used as an emulsifier under substantially oxygen-free conditions; contacting the swellable polymer seeds with an aqueous dispersion comprising an organic compound, an anionic surfactant, and optionally an organic solvent in which the organic compound is soluble; Diffusing an organic compound into a swellable polymer seed; contacting the aqueous dispersion with a mixture comprising a structural monomer and a metal chelate monomer, optionally wherein the mixture further comprises a steric stabilizer and / or a polymerization initiator; and polymerizing the mixture to obtain a copolymer of microbeads; The present invention also includes a method for preparing metal-encoded microbeads comprising: Organic compounds are those with a molecular weight of less than 5000 Da and a pH of 10 at 25°C. -2 It has a water solubility of less than g / L.

[0089] In some embodiments, the structural monomers are selected from the group consisting of acrylic monomers, methacrylate monomers, and vinyl monomers selected from the group consisting of styrene, divinylbenzene (DVB), ethylvinylbenzene, vinylpyridine, aminostyrene, methylstyrene, dimethylstyrene, ethylstyrene, ethylmethylstyrene, p-chlorostyrene, and 2,4-dichlorostyrene. In some embodiments, the aqueous dispersion of swollen seed particles further comprises a steric stabilizer. In some embodiments, the steric stabilizer is polyvinylpyrrolidone. In some embodiments, providing the aqueous dispersion of swellable seed particles comprises preparing monodisperse swellable seed particles by emulsion polymerization. In some embodiments, the aqueous dispersion of swellable seed particles has a molecular weight of less than 5000 Daltons and a viscosity of less than 10 at 25° C. -2 an organic compound having a water solubility of less than 1 g / L; and optionally an organic solvent in which the organic compound is soluble. In some embodiments, the swellable seed particles are monodisperse swellable seed oligomeric particles. For example, the anionic surfactant may be an alkyl sulfate or an alkyl sulfonate. In some embodiments, the anionic surfactant is 8-16 The anionic surfactant is an alkyl sulfate or sulfonic acid or their salt. For example, the anionic surfactant can be decyl sulfate, dodecyl sulfate, decyl sulfonic acid, dodecyl sulfonic acid or their salt. In some embodiments, the anionic surfactant is sodium dodecyl sulfate or sodium decyl sulfate. In some embodiments, structural monomers are as defined herein. In some embodiments, the metal chelating monomer is as defined herein. In some embodiments, the organic compound is a polymerization initiator. In some embodiments, the polymerization initiator is a peroxide, an azo compound, or a mixture thereof. In some embodiments, the organic solvent is a non-polymerizable solvent selected from an alcohol, an ether, a ketone, a dialkyl sulfoxide (eg, DMSO), a dialkyl formamide (eg, DMF), or a mixture thereof.

[0090] IV. Synthesis of Monomers of the Present Disclosure The monomers of the present disclosure can be prepared by various synthetic methods. The selection of certain structural features and / or substituents may affect the selection of one method over another. The selection of a particular preparation method for a given monomer is within the skill of the art. Some starting materials for preparing the compounds described in the present disclosure are available from commercial chemical sources. Other starting materials, such as those described below, are easily prepared from available precursors using straightforward conversions well known in the art. In the following scheme showing the preparation of the second monomer of the present application, all variables are as defined in the present description unless otherwise specified.

[0091] Compounds of formula (I) can be prepared, for example, by the methods shown in the schemes below. In the structural formulas shown below, the variables are as defined in formula (I) unless otherwise specified. Those skilled in the art will understand that many of the reactions shown in the schemes below may be sensitive to oxygen and / or water, and will know to carry out the reactions under anhydrous, inert atmospheres as necessary. Reaction temperatures and times are presented for illustrative purposes only, and may be varied to optimize yields, as will be understood by those skilled in the art.

[0092] Thus, in one embodiment, a compound of formula I is prepared as shown in Scheme A. A chelator or ligand of formula A is attached to one or more linkers having a polymerizable end group of formula B to form a metal chelate capable monomer of formula C. A metal D can then be chelated to the monomer of formula C to form a metal chelating monomer of formula I. Scheme A [ka] In some embodiments, the ligand of formula A may contain one or more carboxylic acid groups. Thus, step a in Scheme A can be carried out according to Scheme B. The ligand of formula E can be attached to one or more linkers bearing a polymerizable group of formula B by amide bond formation or esterification to obtain a metal-chelating monomer of formula F. It is understood that amide bond formation can be carried out using methods known in the art, for example by formation of an activated ester. Scheme B [ka] It is understood that the metal chelation step b can be carried out using methods known in the art. For example, in some embodiments, the metal chelate monomer of formula C or F and the metal of formula I can each be dissolved in a suitable solvent. The suitable solvent can be selected by one of skill in the art and can include water. The metal of formula D can be a salt of the metal, for example, a halide salt of the metal. The metal chelate monomer and the metal solution can be mixed together. For example, the pH of the resulting mixture can be monitored and adjusted to a suitable pH. In some embodiments, a suitable pH includes about 5.5 to about 7.5, or about 6. The resulting mixture can be stirred until the metal chelate monomer of formula I is produced.

[0093] It should be understood that throughout the methods described herein, suitable protecting groups, where appropriate, are added to and subsequently removed from various reactants and intermediates in a manner readily understood by those skilled in the art. Conventional procedures for using such protecting groups, as well as examples of suitable protecting groups, are described, for example, in "Protective Groups in Organic Synthesis", TW Green, PG M Huts, Wiley-Interscience, New York, (1999). It should also be understood that the conversion of a group or substituent to another group or substituent by chemical manipulation can be performed on any intermediate or final product on the synthetic route to the final product, where the possible transformation methods are limited only by the inherent incompatibility that the molecule has at that stage with the conditions or reagents used to transform other functional groups. Such inherent incompatibilities, and how to circumvent them by performing the appropriate transformations and synthetic steps in a suitable order, will be readily understood by those skilled in the art. Examples of transformations are provided herein, and it should be understood that the transformations described are not limited only to the general groups or substituents for which the transformations are exemplified. References and descriptions of other suitable transformations are given in "Comprehensive Organic Transformations-A Guide to Functional Group Preparations" RC Larock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions can be found in organic chemistry textbooks, such as "Advanced Organic Chemistry", March, 4th Edition, McGraw Hill (1992) or "Organic Synthesis", Smith, McGraw Hill, (1994). Techniques for purification of intermediates and final products include, for example, normal and reverse phase chromatography, on columns or spinning plates, recrystallization, distillation, and liquid-liquid or solid-liquid extraction, and will be readily understood by those skilled in the art.

[0094] V. Mass Spectrometry and Kits The microbeads and kits described above may include additional aspects for use in mass spectrometry assays. Described below are methods and kits for mass spectrometry assays, which may include or use any suitable microbeads or kits described elsewhere herein. Suitable assays include assays for detecting target or sample biomolecules, such as target oligonucleotides, proteins (e.g., cytokines, cancer biomarkers, or antibodies against specific antigens), or enzymes (e.g., kinases, phosphatases, proteases, or any other enzymes of interest that modify substrate biomolecules attached to microbeads). The assays may use hybridization and / or sandwich ELISA formats to detect target sample biomolecules. Suitable assays also include cell analysis (e.g., using microbeads as standards for calibration, normalization, or quantification in mass cytometry assays).

[0095] The microbeads (and kits or methods thereof) of the present disclosure may be used for optical detection (e.g., fluorescence-based detection). Alternatively, the microbeads (and kits or methods thereof) of the present disclosure may be analyzed by elemental analysis (e.g., mass spectrometry). Thus, the microbeads may exhibit low fluorescence (e.g., may have a fluorescence quantum yield of less than 0.2, less than 0.1, less than 0.05, or less than 0.02 across the visible and / or UV range) but still be suitable for analysis by mass spectrometry (e.g., atomic mass spectrometry, such as by ICP-MS) or another form of elemental analysis (e.g., ICP-OES, X-ray dispersive spectroscopy).

[0096] The assay methods and kits described herein may be for the analysis of samples by mass spectrometry. Suitable samples include any biological sample, such as a cell sample (e.g., a suspension of cells or tissue sections) or a biological fluid (e.g., containing a sample biomolecule in suspension). The sample may be a cell line, cell culture supernatant, or may be taken from an organism, such as a human, rodent, or other mammal. The sample may be a blood sample, such as whole blood, serum, plasma, or peripheral blood mononuclear cells (PBMCs). The sample may be a saliva sample, a nasal swab, a resection (e.g., a biopsy of a solid tissue). The sample may include whole cells, or may be homogenized (e.g., from a lysate) from a sample containing whole cells. In certain embodiments, the sample may be a purified sample biomolecule that is characterized by the assay or kit described herein. For example, the assay or kit may screen for potential sample biomolecules that are drug candidates. It is worth noting that additional steps may be performed in any of the methods described herein to remove unbound sample biomolecules from the microbeads, unbound reporters from the microbeads, or unbound mass-tagged antibodies from the cells.

[0097] In imaging mass cytometry (IMC), the sample may be a tissue section that is labeled with mass tags (e.g., by mass-tagged antibodies) for analysis by mass spectrometry (e.g., LA-ICP-MS or SIMS). In suspension mass cytometry, particles in suspension (such as cells and / or beads) may be introduced into a mass cytometer (e.g., an ICP-MS system) for analysis.

[0098] The mass spectrometry of the present application can be atomic mass spectrometry. The mass spectrometer of the present application can detect multiple mass channels simultaneously. Different mass channels can correspond to metals or their isotopes from different mass tags. Such a simultaneous mass spectrometer can be, for example, a time-of-flight mass spectrometer (TOF-MS) or a magnetic sector mass spectrometer. The mass spectrometer can atomize the sample for atomic mass spectrometry. For example, the sample can be atomized using ICP, laser ablation ICP, secondary ions (e.g., for secondary ion mass spectrometry (SIMS)) or any suitable ionization source. The mass cytometer can include ion optics to specifically detect metals from the mass tags and / or microbeads described herein and remove, for example, lighter atoms (e.g., endogenous elements such as C, N, O, and light metals, and, if the mass spectrometer is an ICP system, optionally additional plasma gas elements such as argon and argon dimers). Exemplary mass cytometers are further described in U.S. Patent Application Publication Nos. 20050218319 and 20160049283, which are incorporated herein by reference.

[0099] An exemplary assay scheme is shown in FIG. 15. The copolymer microbeads of the present disclosure may include a barcode. The barcode may be multiple metals or concentrated metal isotopes chelated by a metal chelating monomer. The barcode may be an assay barcode (i.e., a barcode that identifies the sample biomolecule that the microbead is functionalized to bind). Alternatively or additionally, the microbeads may include a sample barcode that is used to identify the sample with which the microbeads have been or will be mixed. The microbeads may be attached (e.g., covalently attached) to a biomolecule (e.g., a capture biomolecule) that specifically binds to the sample biomolecule. The biomolecule may include an affinity reagent, such as an antibody that specifically binds a sample antigen biomolecule of interest, such as, for example, a viral particle, a cytokine, a cancer biomarker, etc. The biomolecule may include an oligonucleotide, such as, for example, an ssDNA oligonucleotide, having a sequence that specifically hybridizes to a sample oligonucleotide biomolecule of interest. Mixing a plurality of such beads with a sample may allow the biomolecule of each bead to bind to their respective sample biomolecule, as shown in FIG. 15. Additionally, as shown in FIG. 15, a reporter may be attached to the sample biomolecule (e.g., before, after, or during the step of binding the sample biomolecule to the biomolecule attached to the bead). The reporter may include a mass tag. Thus, the method of the present disclosure may include binding the sample biomolecule to a biomolecule (e.g., a capture biomolecule) attached to a barcoded microbead, and may further include binding a mass-tagged reporter to the sample biomolecule. The barcode and mass tag may then be detected by atomic mass cytometry as described herein. Similarly, certain kits described herein optionally include barcoded microbeads attached to biomolecules that specifically bind the sample biomolecule, and the kit may further include a reporter that binds the mass tag to the sample biomolecule.

[0100] In some embodiments, the kit comprises a population of microbeads, or a separate population of multiple microbeads, as described in any other embodiment herein. In certain aspects, each population of microbeads can be distinguished from another population of microbeads based on the metal or metals of the microbeads (e.g., by atomic mass spectrometry). For example, the microbeads of at least one population of microbeads (e.g., each population) may contain a metal or metals that is different from the metal or metals of the microbeads of another population of microbeads, or may contain metals in a different ratio than the microbeads of another population of microbeads.

[0101] In some embodiments, the microbeads of each population of microbeads in the kit are conjugated to different biomolecules. In some embodiments, the kit may further include a reporter comprising a mass tag, such as a reporter capable of specifically binding a sample biomolecule that is specifically bound by at least one different biomolecule. The different biomolecules attached to the microbeads of the kit may specifically bind to different sample biomolecules. The sample biomolecule may be any biomolecule described herein present in the sample. Such a sample biomolecule may be a protein (e.g., or a peptide thereof), an oligonucleotide, a lipid, a carbohydrate, or a small molecule. The protein may be, for example, an antibody or a cytokine. The oligonucleotide may be a genomic DNA sequence, a cDNA sequence, or an RNA sequence, such as an mRNA sequence. The oligonucleotide may also be a DNA RNA hybrid and / or may include one or more modified residues.

[0102] The sample biomolecule may comprise an oligonucleotide (e.g., RNA), and at least one different biomolecule of the microbead may be an oligonucleotide (e.g., ssDNA) that specifically hybridizes to the sample biomolecule (e.g., target analyte). For example, the reporter comprises a plurality of oligonucleotides that hybridize to indirectly bind the plurality of mass-tagged oligonucleotides to the sample biomolecule. Alternatively, the reporter comprising the mass tag may directly hybridize to the sample biomolecule. In certain aspects, the sample biomolecule may be a biomolecule other than an oligonucleotide, such as an antigen (e.g., a protein such as a cytokine), and the reporter may comprise an oligonucleotide conjugated to an antibody, where the antibody can bind to the antigen, and the oligonucleotide conjugated to the antibody is directly or indirectly (e.g., by hybridization) bound to the reporter's mass tag oligonucleotide. Such indirect hybridization may allow the binding of a plurality of mass tags to the sample biomolecule, such as by hairpin chain reaction, branched in situ hybridization, or any other suitable method. Thus, in some embodiments, the reporters described herein may (or may not) include a separate biomolecular system that binds the mass tags together with the sample biomolecules.

[0103] In some embodiments, the at least one distinct biomolecule attached to the microbead is an antibody (e.g., or a fragment thereof, such as a nanobody). For example, the sample biomolecule is a virus particle, where the at least one distinct biomolecule is a first antibody that specifically binds the virus particle, and the reporter comprises a second antibody that specifically binds the virus particle.

[0104] In some embodiments, at least one sample biomolecule is a cytokine, e.g., at least one different biomolecule attached to the microbead is a first antibody that specifically binds the cytokine, and the reporter comprises a second antibody that specifically binds the cytokine. In some embodiments, the cytokine is selected from IL-18, IL-1F4, TNFα, IL-6, IFNγ, IL-4, CD163, CXCL-9 / MIG, IL-10, IL-1β, and combinations thereof.

[0105] In some embodiments, at least one sample biomolecule is a cancer biomarker (e.g., prostate specific antigen), at least one distinct biomolecule is a first antibody that specifically binds the cancer biomarker, and the reporter comprises a second antibody that specifically binds the cancer biomarker.

[0106] In some embodiments, the at least one distinct biomolecule comprises a viral antigen, the sample biomolecule is an antibody that specifically binds the viral antigen, and the reporter comprises a secondary antibody that binds to the sample biomolecule.

[0107] The methods and kits of the present disclosure may include a plurality of different reporters, each of which is capable of binding a sample biomolecule that is specifically bound by a different biomolecule. Each of the plurality of different reporters includes the same mass tag (detected in the same mass channel) or different mass tags (detected in different mass channels). The mass tags may include metal nanoparticles, such as metal nanocrystals (e.g., nanogold particles), quantum dots, polymer nanoparticles, and the like. The nanoparticles may be 100 nm or less than 100 nm, less than 50 nm, less than 20 nm, less than 10 nm in diameter, for example, between 2 nm and 100 nm or between 5 nm and 50 nm in diameter. The mass tags may include metal chelating polymers, such as linear or branched polymers that include metal-binding (e.g., metal chelating) pendant groups. Other mass tags are also within the scope of the present disclosure, such as organotellurium polymer mass tags, in which tellurium atoms are covalently bonded to carbon atoms of the polymer. The mass tags may have one or more metal elements or enriched isotopes thereof. Mass tags may be conjugated to biomolecules (eg, reporter biomolecules that bind to sample biomolecules) by any suitable binding means described herein or known to those of skill in the art.

[0108] In some embodiments, the biomolecule attached to the microbead is an enzyme substrate for the sample biomolecule. The disclosed method or kit may further include a reporter that specifically binds (e.g., can specifically bind) to the enzyme substrate when the enzyme substrate is modified by the sample biomolecule, or a reporter that specifically binds (e.g., can specifically bind) to the enzyme substrate when the enzyme substrate is not modified by the sample biomolecule. For example, the enzyme substrate may include a kinase substrate, the sample biomolecule (e.g., target analyte) may include a kinase that phosphorylates the kinase substrate, and the reporter may be a phosphorylation-specific antibody that binds the phosphorylated substrate. In another example, the enzyme substrate includes a mass tag that is removed when the enzyme substrate is modified by the sample biomolecule, such as a peptide sequence that is cleaved by the sample biomolecule that is a protease. In some embodiments, the absence of a signal from the mass tag may thus indicate the presence of the sample biomolecule enzyme. An example of an enzyme assay is described in U.S. Patent Application Publication No. 20070190588, which is incorporated herein by reference.

[0109] In some embodiments of the kits and methods of the present disclosure, microbeads from multiple different populations are present in a mixture (in a first mixture of microbeads). Some embodiments may further include a second mixture of microbeads that includes the same biomolecules as the first mixture of microbeads, where the microbeads of the first mixture include a different sample barcode than the microbeads of the second mixture. The sample barcodes are, for example, present inside the microbeads (e.g., may be a subset of metals chelated by the metal chelating monomers of the microbeads of the first and second mixtures). Alternatively, in that case, the sample barcodes may be present on the surface of the microbeads of the first and second mixtures. The method or kit may include multiple sample barcodes in separate compartments that are functionalized to bind to the surface of microbeads from multiple different populations (e.g., where the sample barcodes of each compartment are applied to microbeads of different mixtures). The sample barcoded microbeads may be mixed together (e.g., after mixing with their respective samples, but optionally before mixing with the reporter) and then analyzed by mass spectrometry. The mass spectrum from the sample barcode of each microbead can thereby be used to identify the sample it originated from.

[0110] In certain embodiments, the kit or method may further include a panel of mass-tagged antibodies in a mutual mixture, at least some of the antibodies of the panel being specific for a cell surface marker (e.g., used to bind a cell surface protein in a cell sample). The plurality of antibodies may be mixed with the microbeads of the kit, for example, in a buffer solution or in lyophilized form (e.g., less than 5% or less than 1% water by volume). The plurality (e.g., each) of the mass-tagged antibodies contains the same metal as that chelated by the metal chelating monomer of the microbeads of the kit. However, the cells and microbeads described herein may be distinguishable by atomic mass spectrometry. For example, the cells described herein may contain one or more metals (or enriched isotopes thereof), such as iridium intercalators, that bind to the DNA of the cells, which cells of natural origin do not normally contain. The microbeads may be analyzed together with the cells as mass standards and / or to detect biomolecules (e.g., cytokines, antibodies, cancer biomarkers) in a sample solution (e.g., cell culture supernatant or serum).

[0111] In some embodiments, a kit comprising one or more populations of microbeads described herein further comprises a steric stabilizer (e.g., in admixture with the microbeads of the kit). As described herein, the steric stabilizer can be polyvinylpyrrolidone (PVP) at 0.05%, 0.1%, 0.2%, 0.5%, or greater than 1% by weight, such as from about 0.05% to about 10%, from about 0.1% to about 5%, from about 0.2% to about 2%, etc. Alternatively or additionally, the microbeads of the kit can be in a solution buffered at a pH of from about 4 to about 10 (inclusive), from about 5 to about 9 (inclusive), from about 6 to about 8 (inclusive), a pH greater than about 3, a pH greater than about 4, or a pH less than about 10.

[0112] In some embodiments, the microbeads of the kits of the present disclosure are lyophilized, e.g., the microbeads of the kits are less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, or about 0.05% to about 5% moisture by weight.

[0113] In some embodiments, the microbeads are fused to a solid support for calibration (e.g., as reference particles), normalization, or quantification in imaging mass spectrometry (or imaging mass cytometry), as further described herein. The solid support can be any suitable support, such as a slide (e.g., a microscope slide, such as a clear glass or quartz slide) or an adhesive film (for application to a microscope slide in any of the methods of the present invention). The solid support can further include a biological sample.

[0114] In some embodiments, the solid support comprises at least 2, for example at least 3, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 2000, at least 5000, or at least 10000 fused microbeads. The microbeads (reference particles) may all be the same, or the microbeads may differ in metal or its content. When different microbeads are used, there are usually multiple respective populations of microbeads.

[0115] The microbeads are dispersed on the solid support such that almost all the microbeads are individually (i.e., discretely) located on the solid support, so that each fused microbead can be individually identified and sampled. Those skilled in the art will understand that the solid support may further include some fused microbeads aggregated on the sample carrier, and therefore these aggregates may not be suitable for sampling for calibration and normalization of signal intensity. For example, up to about 2%, e.g., up to about 5%, up to about 8%, up to about 10%, up to about 15%, or up to about 20% of the fused microbeads may be aggregated on the solid support. In other words, at least about 80%, e.g., at least about 85%, at least about 90%, at least about 92%, or at least about 95% of the microbeads may be individually separated. Optical interrogation can identify which positions of the solid support have discrete microbeads and guide the acquisition by the imaging mass spectrometer.

[0116] The step of fusing at least one microbead to the solid support may include heating the solid support. In some embodiments, the step of fusing at least one microbead to the sample carrier or solid support includes heating the sample carrier or solid support at a temperature above the glass transition temperature of the microbead and then cooling the sample carrier or solid support below the glass transition temperature of the microbead. In other words, fusing at least one microbead to the sample carrier or solid support is performed by vitrification. In some embodiments, the sample carrier or solid support is heated to a maximum temperature of up to 300°C, for example up to 275°C, up to 250°C, up to 225°C, or up to 200°C.

[0117] The kits of any of the embodiments described herein may further include one or more buffers (e.g., PBS, red blood cell lysis buffer, detergent buffer, staining buffer, and / or buffers for reconstituting lyophilized reagents such as lyophilized microbeads, lyophilized reporters, lyophilized antibodies), anticoagulants (e.g., for treating blood samples), fixation reagents, permeabilization reagents, or any reagents for carrying out the methods or examples described herein.

[0118] The disclosed methods include analyzing any of the microbeads described herein by mass spectrometry. For example, the microbeads (e.g., one or more populations of microbeads) can be elemental standards used to calibrate a mass spectrometer and / or for normalization (e.g., normalization of mass spectra obtained from mass tags) or quantification (quantification of the number of antibodies in a cell or pixel).

[0119] In some embodiments, a method of mass spectrometry includes mixing separate populations of microbeads of the present disclosure with a sample, where the microbeads of each population of microbeads are bound to a different biomolecule, and where each population of microbeads is distinguished from another population of microbeads based on the metal or metals of the microbead; binding different sample biomolecules of the sample to different biomolecules of the separate populations of microbeads; binding a reporter directly or indirectly to each different sample biomolecule, where the reporter bound to each different sample biomolecule comprises a mass tag; and detecting the metals and mass tags of the individual microbeads by mass spectrometry.

[0120] Such methods may further include attaching different biomolecules to the microbeads of the distinct populations of microbeads prior to mixing the distinct populations of microbeads with the sample. The one or more sample biomolecules may include oligonucleotides, antibodies (or other affinity reagents), cytokines, cancer biomarkers (e.g., one or more prostate specific antigens), and the like.

[0121] In some embodiments, the method of mass spectrometry uses a kit of any of the embodiments described herein and further comprises mixing separate populations of microbeads with a sample, where the microbeads of each population of microbeads are bound to a different biomolecule, and where each population of microbeads is distinguished from another population of microbeads based on the metal or metals of the microbead; binding different sample biomolecules of the sample to different biomolecules of the separate populations of microbeads; binding a reporter directly or indirectly to each different sample biomolecule, where the reporter bound to each of the different sample biomolecules comprises a mass tag; and detecting the metals and mass tags of the individual microbeads by mass spectrometry.

[0122] In some embodiments, the kit or method of mass spectrometry of cell samples using any of the microbeads described herein comprises: providing a plurality of mass-tagged antibodies, each of which is conjugated to a polymeric mass tag that chelates a metal or a plurality of metals of their enriched isotopes; contacting the sample with the plurality of mass-tagged antibodies; detecting the mass-tagged antibodies bound to the sample and the microbeads by mass spectrometry. The microbeads and cell samples can be combined before analysis by mass spectrometry, or can be analyzed in separate sample experiments. The microbeads can be used to assay one or more sample biomolecules. Alternatively or additionally, the microbeads can be elemental standards used for calibration, normalization and / or quantification, as further described herein.

[0123] The microbeads can be of a single population with consistent size and / or amount of one or more metals as described herein. Alternatively, the microbeads can be of different populations, each characterized by a different set of metals, combination of metals, and / or amount of metals. For example, a set of microbead populations has metals from more than 10 distinct elements (e.g., more than 30 distinct isotopic masses) together. For example, the microbeads of the present application can provide signals in more than 4, more than 6, more than 10, more than 20, or more than 30 mass channels (e.g., atomic mass channels greater than 80 amu) together. The microbeads can be added together with cells in suspension mass cytometry workflow, or provided on the same carrier as tissue sections or cell smears in imaging mass cytometry workflow. The microbeads can be used in the assays described herein or can be standards (e.g., giving signals in most or all mass channels in which the mass tags detected by mass cytometry are detected). Mass spectra of microbeads having different amounts of the same metal (e.g., or enriched isotopes thereof) can be used to generate curves (e.g., curves of signal intensity versus known amounts of metal) for use in calibration, normalization, or quantification, as described further herein.

[0124] The microbeads may be used to calibrate the mass spectrometer used in any of the detection steps described herein. The calibration may be one or more of mass resolution, mass calibration, dual count calibration, pre-xy and xy optimization, detector voltage, gas calibration or current calibration. Mass resolution ensures that there is sufficient separation between ions of different masses and may be based in part on the shape of the peak from a particular isotope. Mass resolution above a certain value may indicate a pass. Mass calibration may include automatic adjustment to check the value of one or more mass channels (e.g., values ​​from metals of microbead standards) and then calculate TOF values ​​for additional mass channels, and / or align the correct ion to the detection channel so that the full signal of each ion is collected. Dual count calibration may determine the dual count factor (dual slope to correlate pulse counts and intensities) (dual count factor converts analog signals to ion count signals). This correlation can be important when, for example, ion concentration increases and pulses overlap during a cellular or microbead event. XY optimization is the process by which the optimal alignment of the torch and vacuum boundary is determined to provide maximum signal (e.g., from a metal in microbead standard) to the mass channel. Optimizing the alignment of the system is important for maximum transmission of ions into the vacuum boundary. Detector voltage calibration uses dual count calibration to determine the detector voltage that provides the best signal while ensuring detector life. The optimal detector voltage can be achieved when the dual count coefficient is 0.03±0.003. The detector voltage should not be more positive than −1,100V. Gas and / or current calibration can optimize the propellant and makeup gas flows (and optionally additional gas flows) with the maximum mass channel signal (e.g., from a metal in microbead standard) that can be achieved by varying the make-up and nebulizer gas flows while controlling oxide formation.This ensures that the plasma temperature is optimal in the system and that minimal metal oxides are formed. The current applied at the vacuum boundary is gradually increased to promote the movement of the ion cloud through the boundary. The value that gives the highest signal can then be selected. Calibration can be performed during the sample experiment (e.g., to account for variations in sensitivity).

[0125] The microbeads can be used as standards to normalize mass spectrometry signals obtained from mass tags (e.g., mass tags of a sample described herein) based on mass spectrometry signals obtained from microbeads (e.g., obtained from microbeads containing one or more of the same metals as at least one mass tag, or from similar mass spectra, or from calibration curves generated from multiple microbead populations containing different amounts of metals). Each tag can provide a signal in one or more mass channels. Such normalization of mass tag signals can be to individual cells or assay microbead events (e.g., in suspension mass cytometry), or to individual cells, assay microbeads, or pixels (e.g., in imaging mass cytometry). For example, individual cells of a cell smear can be detected by IMC, or cells of a solid tissue section algorithmically segmented based on membrane staining, and mass tag signals across single cells can be normalized or quantified as described herein. The mass tag signal of a cell or pixel can be normalized to the signal from a microbead detected within the time interval of the cell or pixel, for example, within 10,000 seconds, within 5,000 seconds, within 2,000 seconds, within 1,000 seconds, within 500 seconds, within 200 seconds, or within 100 seconds of when the cell or pixel is detected. Alternatively or additionally, if the microbeads used as a standard include a separate population of microbeads with different metals, the normalization of the mass tag signal can be based on one or more microbeads whose metals are detected in the same mass channel as the mass tag. Alternatively or additionally, if the microbeads used as a standard include a separate population of microbeads with different metal amounts, the normalization of the signal from a mass tag containing the same metal can be based on one or more microbeads that give similar signal intensities (e.g., 10-fold or less difference, 5-fold or less difference, 2-fold or less difference, etc.) to the same mass channel. As described herein, the metal can be an enriched isotope.Therefore, a microbead standard may be used that includes a population of microbeads with different metals and / or different metal amounts. Such normalization may be similar to the EQ4™ beads provided by Fluidigm to normalize mass cytometry data (e.g., normalized data of FCS files obtained by mass cytometry). However, the microbeads of the present disclosure may provide signals of more than 4, more than 6, more than 10, more than 20, or more than 30 mass channels together (e.g., atomic mass channels greater than 80 amu).

[0126] Microbeads can be used as a standard to quantify the amount of one or more mass-tagged antibodies (or other mass-tagged biomolecules), such as when a known (or estimated) number of metal atoms from the mass tag are bound to the antibody (or other biomolecule). For example, the number of metal atoms bound to a mass-tagged antibody (or other biomolecule), which may have one or more mass tag entities, can be determined by starting from a known number of metals per mass tag (e.g., on a polymer mass tag) and a UV / visible spectrum or ICP-MS signal characteristic; and further analyzing the proportion of mass-tagged antibodies (or other biomolecules) by UV or visible spectrum or ICP-MS. Such analysis can be performed on fractions obtained by fast protein liquid chromatography. Quantifying the mass-tagged antibodies can thus be based on the average number of metal atoms for each mass-tagged antibody and the mass spectrometry signal detected from the mass-tagged antibodies and microbeads. The microbeads can have a known number of metal atoms (e.g., determined as described herein). The microbeads and the mass-tagged antibodies (or other biomolecule) can contain the same metal. Thus, quantification of mass-tagged antibodies can be calculated as the number of metals in the microbead multiplied by the ratio of the signal from the mass-tagged antibody (or other biomolecule) to the metal signal from the microbead, divided by the average number of metal atoms per antibody (or other biomolecule). Quantification can be performed on individual cells, assay microbeads, or pixels.

[0127] Embodiments of the present disclosure include computer readable media configured to perform one or more of the calibration, normalization, or quantification described herein.

[0128] In some embodiments, the detecting step may include inductively coupled plasma mass spectrometry (ICP-MS), such as for suspension mass cytometry or imaging mass spectrometry. Mass spectrometry may be by simultaneous mass spectrometry, such as a time-of-flight mass spectrometer (TOF-MS) or a magnetic sector mass spectrometer. In some embodiments, detecting the microbeads can be by imaging mass spectrometry (e.g., imaging mass cytometry). The microbeads (e.g., microbead standards) can be fused (e.g., melted) to a solid surface prior to the detection step. Imaging mass spectrometry can be, for example, by laser ablation ICP-MS or secondary ion mass spectrometry (SIMS).

[0129] Biological samples may include any sample of biological properties that require analysis. For example, samples may include biomolecules, tissues, body fluids, and animal, plant, fungal, or bacterial cells. They may also include molecules of viral origin. Exemplary samples include, but are not limited to, sputum, blood, blood cells (e.g., PBMCs), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissue, such as frozen sections taken for histological purposes. Another exemplary source of biological samples is virus and animal, plant, bacterial, fungal cell cultures, where gene expression states may be manipulated to investigate intragenic associations. In some cases, other samples, such as artificial samples, may be investigated. Certain aspects of the present disclosure are particularly useful when investigating samples of human origin, and are particularly useful when investigating samples of human peripheral blood.

[0130] Mass tagged oligonucleotide can be directly or indirectly hybridized to target oligonucleotide.For example, one or more intermediate oligonucleotides provide scaffolds to which multiple mass tagged oligonucleotides can hybridize, thereby amplifying signals.Some embodiments of the present application therefore include oligonucleotides for hybridization-based signal amplification.

[0131] In some embodiments, the sample biomolecule can be a target oligonucleotide, such as a DNA or RNA molecule (such as coding RNA, small interfering RNA, or microRNA) of a cell or bead. The target oligonucleotide can be single-stranded. The target oligonucleotide can have a known specific sequence (or homology to a known specific sequence).

[0132] In some embodiments, the reporter may comprise a non-oligonucleotide biomolecule, such as an antibody or derivative thereof, which may be conjugated to an oligonucleotide, such as a synthetic single-stranded DNA oligonucleotide containing a known sequence. In such cases, both the antibody and the oligonucleotide may be referred to as part of the reporter.

[0133] The signal from the target oligonucleotide or the reporter comprising the non-oligonucleotide biomolecule conjugated to the oligonucleotide can be amplified by hybridization scheme. Hybridization can be branched or linear. In certain embodiments, the polymerase extends the first oligonucleotide along the template to provide additional sites for the attachment of element tags, such as additional hybridization sites for element-tagged oligonucleotides. Mass-tagged oligonucleotides include a single label atom or include polymers that include multiple label atoms, and are sometimes called reporters. Mass-tagged oligonucleotides can include label atoms, such as heavy metal atoms, in the chemical structure of the oligonucleotide itself.

[0134] Signal amplification offers a unique benefit to bead-based assays, in which the same reporter tag (labeled metal element or isotope) can be amplified and used across different beads and their target analytes.

[0135] If the assay biomolecule is an oligonucleotide, the element-tagged reporter oligonucleotide may hybridize to another portion of the target RNA or DNA, thereby providing a signal when the target RNA or DNA is bound to the bead. If the assay biomolecule is an affinity reagent, such as an antibody that binds the analyte at a first epitope, the element-tagged reporter affinity reagent (e.g., a reporter antibody) may bind to another epitope on the analyte, thereby providing a signal when the target analyte is bound to the bead. The analyte may be further bound by a reporter, such as an element-tagged reporter antibody or oligonucleotide. The reporter may include a highly sensitive (e.g., high intensity) element tag that provides a very large number of isotopes (e.g., 50, 100, 200, 500, 1000 copies of a single isotope), thereby allowing detection of a smaller number of target analytes bound to the assay bead. Such a highly sensitive element tag may include a nanoparticle (e.g., including a metal nanocrystal surface functionalized to bind a biomolecule such as an antibody or oligonucleotide) or a hyperbranched polymer. For example, multiple reporter biomolecules containing the same nanogold particle element tag will provide high signal, taking advantage of the fact that different reporter biomolecules containing the same element tag can be identified by assay barcodes (e.g., of beads that display the analyte for which they are specific). In certain embodiments, nanoparticle tags (e.g., gold nanoparticles) can be coupled to reporter probes via biotin-avidin (e.g., biotin-streptavidin) interactions. For example, nanoparticles (e.g., gold nanoparticles) can be conjugated to streptavidin. Reporters may also contain low-sensitivity element tags that provide low-abundance isotopes (e.g., 100, 50, 30, 20, 10, or 5 copies of an isotope) that are different from the most abundant isotope, thereby allowing detection / quantification of the amount of such abundant analytes where the most abundant isotope would saturate the detector.In certain embodiments, the high and low abundance isotopes have a mass difference (e.g., greater than 5, 10, 20, 30, 40 or 50 amu) such that saturation of the detector by the high abundance isotope does not affect detection of the low abundance isotope. Reporters for different analytes (e.g., including antibodies that bind to target analytes of different assay beads) can contain the same isotope or combination of isotopes, as the analytes are identified by the unique assay barcodes of the beads.

[0136] In certain embodiments, the reporter may comprise a reporter system that provides signal amplification by binding multiple entities of elemental tags with a single entity of target analyte (e.g., sample biomolecule). Signal amplification may be by enzyme deposition, hybridization (e.g., branched hybridization, stranded hybridization, and / or hybridization of multiple reporter oligonucleotides to a single-length intermediate oligonucleotide), extension (e.g., single extension, rolling circle extension), and / or a series of branched conjugation. In certain embodiments, multiple (e.g., all) analytes detected by assay beads may be detected by the same reporter system. In certain embodiments, the signal amplification reporter system may have high sensitivity elemental tags. For example, an element tag containing an enzyme substrate moiety can be deposited onto a bead (or a molecule attached to a bead) from solution by an enzyme attached to a reporter biomolecule. Such a reaction can be covalent binding by a tyramide element tag acted upon by horseradish peroxidase bound to a reporter biomolecule.

[0137] Some embodiments include hybridization schemes whereby multiple element-tagged oligonucleotides hybridize indirectly (through one or more oligonucleotide intermediates) to a single oligonucleotide target. For example, the oligonucleotide target may be a target RNA or DNA (e.g., gDNA or cDNA) sequence, or may be an oligonucleotide present on a reporter antibody.

[0138] As described herein, mass cytometry may allow for sufficient detection channels (mass channels) to detect both the sample and assay barcode in the bead while allowing additional channels for reporters (to detect assay targets). Thus, the bead assays described herein may be barcoded samples and / or assays for use in mass cytometry. For example, multiple different conditions (e.g., enzymes or drug candidates such as agonists or antagonists of one or more enzymes) may be applied to a biological sample and their effects on multiple targets may be detected with enzyme assay beads. Individual conditions may be identified with a common sample barcode across different assay beads exposed to the same condition. The assay barcoded beads may be combined prior to analysis, e.g., prior to exposure to the conditions. The sample barcoded beads may be combined prior to analysis.

[0139] In certain embodiments, the enzyme can be a protein that modifies DNA, such as a protease, kinase, phosphatase, or DNA methyltransferase. The target is the substrate that is acted upon by the enzyme, and the reporter (e.g., a reporter biomolecule as described herein) can bind the target (e.g., a sample biomolecule) only before or after it is acted upon by the enzyme. For example, a phospho-specific antibody that detects a phosphorylated form of a protein target can be increased in abundance when acted upon by a kinase enzyme, or decreased in abundance when acted upon by a phosphatase. If the enzyme is a protease, the reporter can be attached to the end of a peptide substrate and released from attachment to the bead when the substrate is cleaved (whereby a decrease in the reporter element tag indicates an increase in protease activity).

[0140] Sample barcodes can be used to indicate which of a number of enzymes (or their agonists / antagonists) were tested in a particular assay. For example, candidate enzymes, agonists, and antagonists are added to a biological fluid such as a cell lysate, and the sample is then contacted with assay barcoded beads to detect the activity of the enzyme. Alternatively, the candidates can be administered to cells (e.g., directly or by genetic engineering) or to an organism such as a patient or mammalian test subject, and the sample taken from that source is contacted with the assay beads. Sample barcoding allows many such candidates to be screened simultaneously. In either case, sample barcodes can be added as described herein for beads to identify the candidates. For example, more than 10, more than 20, more than 50, more than 100, more than 500, or more than 1000 separate samples can be barcoded. For example, 12 distinct isotopes in 6 unique combinations give 924 distinct combinations (e.g., for barcoding up to 924 samples). Another 12 distinct isotopes can be used for barcoding close to 1000 assays. Thus, more than 10, 20, 50, 100, 500, or 1000 distinct assay beads can be barcoded (e.g., beads that detect the amount of different substrates acted upon by candidate agents). At least one channel will be left for substrate detection by reporters, as described herein. This allows for unconventional screening with immediate readout by mass cytometry.

[0141] Post-translational modification of proteins is carried out by enzymes in living cells. Known post-translational modifications include protein phosphorylation and dephosphorylation, as well as methylation, prenylation, sulfation, and ubiquitination. The presence or absence of phosphate groups on proteins, particularly enzymes, is known to play a regulatory role in many biochemical and signal transduction pathways.

[0142] Bead-based kinase assays for mass cytometry are discussed in U.S. Patent Application Publication No. 20070190588, which is incorporated herein by reference and summarized below. However, such bead-based assays have never been proposed for both sample and assay barcoding, which offers advantages for screening and is uniquely enabled by the high complexity of mass cytometry.

[0143] Kinase function is to transfer a phosphate group (phosphorylation) from a high-energy donor molecule, such as ATP, to a specific target molecule (substrate). Enzymes that remove phosphate groups from targets are known as phosphatases. The largest group of kinases are protein kinases, which act on specific proteins and regulate their activity. A variety of other kinases act on small molecules (lipids, carbohydrates, amino acids, nucleotides, etc.) and are often named after their substrates, including adenylate kinase, creatine kinase, pyruvate kinase, hexokinase, nucleotide diphosphate kinase, and thymidine kinase.

[0144] Protein kinases catalyze the transfer of phosphate from adenosine triphosphate (ATP) to a target peptide or protein substrate at serine, threonine, or tyrosine residues. Protein kinases are distinguished by their ability to phosphorylate substrates on distinct sequences. Commercially available kinases are either active (phosphorylated by the supplier) or inactive and require phosphorylation by another kinase.

[0145] Protein phosphatases hydrolyze phosphate monoesters at phosphoserine, phosphothreonine, or phosphotyrosine residues into protein or peptide molecules with a phosphate ion and a free hydroxyl group. This action is the opposite of that of protein kinases. Examples include protein tyrosine phosphatases, which hydrolyze phosphotyrosine residues, alkaline phosphatase, serine / threonine phosphatase, and inositol monophosphate degrading enzymes.

[0146] Another aspect of the present disclosure is to provide a kit for the detection and measurement of an element in a sample, the element to be measured comprising an element tag attached to a phosphorylated substrate, an element of a metal ion coordination compound, and an element of a uniquely labeled support, the kit comprising an element tag for directly tagging the phosphorylated substrate; a number of phosphorylated substrates; the uniquely labeled support; a metal ion coordination compound; and optionally a phosphatase, a phosphatase buffer, and ADP.

[0147] Another aspect of the present disclosure is to provide a method for a kinase assay, the method comprising: incubating ATP, at least one kinase, a free metal ion coordinating compound, and a number of unphosphorylated substrates immobilized on an element-labeled support such that a single type of unphosphorylated substrate is attached to a single type of element-labeled support under conditions that allow the kinase to phosphorylate the substrate; separating the number of phosphorylated substrates immobilized on the element-labeled support having the attached metal ion coordinating compound from the free metal ion coordinating compound and the number of immobilized unphosphorylated substrates; and measuring the number of phosphorylated substrates immobilized on the element-labeled support having the attached metal ion coordinating compound by elemental analysis.

[0148] Another aspect of the present disclosure is to provide a kit for the detection and measurement of an element in a sample, the element to be measured comprising an element tag attached to a non-phosphorylated substrate and a metal ion coordinating compound, the kit comprising: an element tag for directly tagging the non-phosphorylated substrate; a non-phosphorylated substrate; a solid support; a metal ion coordinating compound; and optionally a kinase, a kinase buffer, and ATP.

[0149] Pharmacological modulation of enzymes has become a major component in identifying potential therapeutics. Proteases are a subclass of proteolytic enzymes that have recently been shown to play important roles in signal transduction pathways, the dysregulation of which can lead to cancer, cardiovascular disease, and neurological disorders. Of the approximately 400 known proteases, several dozen have been investigated as potential drug candidates. Small molecule inhibitors of proteases are today considered useful therapeutic lead compounds for the treatment of degenerative diseases, the treatment of cancer, and as antibacterial, antiviral, and antifungal agents. Bead-based protease assays for mass cytometry are discussed in U.S. Patent Application Publication No. 20170023583, which is incorporated herein by reference and summarized below. However, such bead-based assays have never been proposed for both sample and assay barcoding, which offers advantages for screening and is uniquely enabled by the high complexity of mass cytometry.

[0150] There is a need for robust, sensitive, and quantitative enzyme assays that allow for the simultaneous measurement of multiple enzymatic reactions, that would allow for the conservation of valuable biological samples and reagents, and that could achieve high throughput, reduced assay times, and reduced overall costs of enzyme analysis.

[0151] One aspect of the present invention is a method for detecting protease activity in a biological fluid. The method may include attaching a coded bead to a first amino acid of a peptide substrate to form an immobilized peptide substrate, the peptide substrate comprising a first amino acid and a last amino acid and being a substrate for a protease enzyme; attaching an elemental tag to the last amino acid of the peptide substrate to form a tagged peptide substrate; incubating the immobilized tagged peptide substrate with the biological fluid; and detecting the elemental tag and the coded bead in the biological fluid by elemental analysis.

[0152] Encoded microbeads, as discussed herein, can be used for both assay and sample barcoding.

[0153] The protease assay kit includes an assay-coded bead attached to the first amino acid of a peptide substrate (immobilized peptide substrate), the peptide substrate includes the first and last amino acids, and can be a substrate for a protease enzyme. An element tag can be attached to or near the last amino acid of the peptide substrate to form a tagged peptide substrate. The coded microbeads can be both assay and sample barcoded, as discussed herein.

[0154] A mixture of assay beads may collectively target at least 5, 10, 20, 50, 100, 200, 500, 1000 or more analytes (sample biomolecules). In certain embodiments, a sample barcode may identify assay barcode beads and / or cells from at least 5, 10, 20, 50, or 100 samples.

[0155] Sample barcoding reagents for cells may include element-tagged antibodies (that bind across multiple cell types or the majority of cells in a sample), element tags functionalized to non-specifically bind to cells (e.g., by covalent interactions), and / or metals in solution. Sample barcoding reagents for cells may further include reagents to place the sample barcode in the cells (e.g., cell permeabilization reagents such as DMSO, detergents or alcohols, etc.). Sample barcoding reagents for assay barcoded beads may be present within the beads, on the surface of the beads, or applied to the beads. If applied to the beads, the sample barcoding reagent may include functional groups described herein for binding to the surface of the beads (e.g., for binding to functional groups presented by the beads or for binding to blocking reagents present on the bead surface). Sample barcoding reagents for a given sample may include a unique combination of isotopes specific to that sample. In certain embodiments, cells and assay barcoded beads from the same sample (e.g., individual blood samples) may be labeled with the same assay barcode. The same assay barcode used for labeling cells and beads may contain the same combination of isotopes and / or the same means of attachment (e.g., functional groups).

[0156] The sample barcoding reagent may be provided in a mixture with or together with an antibody panel, such as a lyophilized antibody panel. The sample barcoding reagent may be provided in a mixture with or together with the assay barcoded beads. The assay barcoded beads may be provided in a mixture with or together with the antibody panel. The assay barcoded beads and the sample barcoding reagent may be provided in a mixture with or together with the lyophilized antibody panel (e.g., where the sample barcoding reagent binds both the assay barcoded beads and the cells in the sample). In certain embodiments above, the sample barcoding reagent may be present in, on, or provided together with the sample and / or assay barcoded beads. In some cases, the barcoding reagents can be provided in a pre-configured form by preparing the barcoding reagents with multiple unique combinations of assay barcodes and sample barcodes. In such cases, each unique barcoding reagent can be stored in a separate container, such as a separate well of a well plate. As an example, a well plate can be configured such that all wells along a particular column (or row) share the same assay barcode, while all wells along a particular row (or column) share the same sample barcode. In another example, a well plate can be configured such that each filled well contains a barcoding reagent with a specific unique sample barcode and a variety of combinations of multiple assay barcodes. Thus, a first well can contain barcoding reagents all with a first sample barcode but each with a different assay barcode, and a second well can contain barcoding reagents all with a second barcode but each with a different assay barcode. In some cases, the pre-configured barcoding reagents can require the production of thousands of groups of unique beads. In some cases, the barcoding reagents (e.g., beads) can be provided in a semi-configured form by preparing barcoding reagents with surfaces functionalized to bind unique assay barcodes and sample barcodes. In such cases, each group of barcoding reagents can be conjugated to a biomolecule (e.g., an antibody) that has a targeting function associated with the assay associated with that group of barcoding reagent's assay barcode.

[0157] If semi-configured barcoding reagents are provided, the sample barcodes can be bound to the barcoding reagents prior to combining the barcoding reagents with the sample. In one example, different barcoding reagents can be mixed together and then distributed across a series of vessels (e.g., wells of a well plate). A unique sample barcode can then be added to each vessel, and the product can be mixed with a unique sample to perform an assay for that sample that allows the assay barcode to be identified and simultaneously label the sample with the sample barcode.

[0158] If a semi-configured barcoding reagent is provided, the sample barcode can be bound to the barcoding reagent after combining the barcoding reagent and the sample. In one example, the semi-configured barcoding reagent can be provided together or mixed together. The barcoding reagent can then be added to each of the series of samples. Separately, a unique sample barcode can be mixed with each of the series of samples before or after the barcoding reagent is added. The sample barcode can label the barcoding reagent and / or cells or particles of the sample.

[0159] In one case, the barcoding reagent may include assay barcoded beads functionalized with polydopamine for attachment of capture antibodies. Another molecule (e.g., avidin) can be added together with the capture antibodies. After the capture antibodies are added to the assay barcoded beads, the beads may be mixed and aliquoted for each sample. In the case of sample barcodes, a unique combination of element tags functionalized (e.g., with biotin) to bind molecules can be added. In some cases, elemental analysis can be performed on an individual particle basis, known as particle elemental analysis. Particle elemental analysis involves measuring the elemental composition of individual particles (e.g., per cell) using a mass spectrometer-based flow cytometer or the like. Certain aspects of the present disclosure utilize particle elemental analysis on a cell-by-cell basis, which may be known as cytometric elemental analysis. In some cases, elemental analysis can be performed on a bulk basis, known as bulk elemental analysis or solution elemental analysis. Bulk elemental analysis involves measuring the elemental composition of the entire volume of a sample.

[0160] Elemental analysis can be used to interrogate samples, such as biological samples. If a sample is labeled with a known elemental tag, detection of the elemental tag during elemental analysis can indicate a property of the sample that is associated with the elemental tag.

[0161] As shown herein, mass cytometry is any method of detecting elemental tags (mass tags) in biological samples, such as simultaneously detecting multiple distinguishable mass tags with single-cell resolution. Mass cytometry can include the analysis of mass tagged beads separated from cells or added to cells. Any of the present kits and methods can include or be adapted to mass cytometry. Mass cytometry includes suspension mass cytometry and imaging mass cytometry (IMC).

[0162] Suspension mass cytometry involves the analysis of suspended elementally tagged cells and / or beads by mass spectrometry (e.g., by atomic mass spectrometry) and is described in U.S. published patent applications, including U.S. Patent Application Publication Nos. 20050218319, 20150183895, and 20150122991, all of which are incorporated herein by reference.

[0163] Imaging mass cytometry (IMC) includes imaging mass spectrometry (e.g., imaging atomic mass spectrometry) of any elementally tagged biological sample, such as tissue sections or cell smears. IMC can atomize and ionize mass tags of cell samples by one or more of laser irradiation, ion beam irradiation, electron beam emission, and / or inductively coupled plasma. Mass cytometry can simultaneously detect distinct mass tags from single cells, for example, by time of flight (TOF) or magnetic sector mass spectrometry (MS). Examples of mass cytometry include suspension mass cytometry where cells flow in, as well as ICP-MS and imaging mass cytometry where cell samples (e.g., tissue sections) are sampled, for example, by laser ablation (LA-ICP-MS) or by primary ion beam (e.g., in the case of SIMS). Laser-based IMC is described in US Patent Publication Nos. 20160131635, 20170148619, 20180306695, and 20180306695, all of which are incorporated herein by reference. In certain embodiments, when the sample is a cell smear for analysis by IMC, the cells can be processed as described herein, for example, by staining with a freeze-dried panel, sample barcoding, and / or assay barcoding. Similarly, the assay beads described herein can be analyzed by IMC separately or in a mixture with the cells.

[0164] Mass tags can be sampled, atomized and ionized before elemental analysis. For example, mass tags in biological samples can be sampled, atomized and / or ionized by irradiation, such as laser beam, ion beam or electron beam. Alternatively or additionally, mass tags can be atomized and ionized by plasma, such as inductively coupled plasma (ICP). In suspension mass cytometry, a whole cell containing mass tags can be flowed into an ICP-MS, such as an ICP-TOF-MS. In imaging mass cytometry, certain irradiation can remove (and possibly ionize and atomize) a portion (e.g., pixel, region of interest) of a solid biological sample, such as a tissue sample, containing mass tags. Examples of IMC include LA-ICP-MS and SIMS-MS of mass tagged samples. In certain embodiments, ion optics can deplete ions other than the isotope of the mass tag. For example, ion optics can remove lighter ions (e.g., C, N, O), organic molecular ions. In ICP applications, the ion optics may filter out gases such as Ar and / or Xe, such as with a high-pass type quadrupole filter, etc. In certain embodiments, IMC may provide images of mass tags (e.g., targets bound to mass tags) with cellular or subcellular resolution.

[0165] The present disclosure also provides the following embodiments: Embodiment 1. Structural monomers, and Metal chelating monomers containing a metal and a chelating agent A copolymer comprising: The chelating agent coordinates the metal at at least three sites; and The structural monomer is a copolymer that does not include a chelating agent. Metal-encoded microbeads comprising: Embodiment 2. The microbeads of embodiment 1, wherein the structural monomer is selected from substituted or unsubstituted styrene, α-methylstyrene, acrylic acid and esters and amides thereof, methacrylic acid and esters and amides thereof, and derivatives thereof, and optionally the structural monomer is styrene. Embodiment 3. The metal chelating monomer has the formula (I) prior to polymerization: [ka] 3. The microbead of embodiment 1 or 2, having the structure: wherein the ligand is a chelating agent, L is a linker, X is a polymerizable end group, M is a metal, and n is an integer equal to or greater than 1, and wherein the metal chelating monomer is neutrally charged prior to polymerization. Embodiment 4. L is selected from a bond, a C3-C8 alkylamine, a C3-C8 alkylene, a C3-C8 cycloalkyl, a C3-C8 heterocycloalkyl, a 5- or 6-membered aryl or heteroaryl, an alkylaryl, an alkylheteroaryl, a C3-C8 cycloalkylaryl, a C3-C8 cycloalkylheteroaryl, C(O), —C(O)O, or a mixture thereof, where optionally each of the alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl is independently unsubstituted or substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, an amide, an ester, an aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or a mixture thereof. 4. The microbead of embodiment 3. Embodiment 5. The microbead of embodiment 3 or 4, wherein L is attached to the chelating agent via an amide or ester. Embodiment 6. Microbeads according to any one of embodiments 3 to 5, wherein the polymerizable end group is selected from allyl vinyl, styrene, α-methylstyrene, acrylate esters, methacrylate esters, acrylamide, 2-methylacrylamide, and mixtures thereof, and optionally the polymerizable end group is allyl vinyl or styrene. Embodiment 7. A microbead according to any one of embodiments 1 to 6, wherein the chelating agent is tetradentate, pentadentate, hexadentate, heptadentate, or octadentate, optionally wherein the chelating agent is hexadentate or octadentate. Embodiment 8. A microbead according to any one of embodiments 1 to 7, wherein the chelating agent comprises an aminopolyacid moiety, or a derivative thereof. Embodiment 9. The microbead of embodiment 8, wherein the aminopolyacid moiety is selected from an aminopolycarboxylic acid, an aminopolyphosphonic acid, or a combination thereof. Embodiment 10. Microbeads according to embodiment 8 or 9, wherein the aminopolyacid moiety is one or more substituted oligomers of ethyleneimine, propyleneamine, or mixtures thereof, the oligomer being substituted with two or more carboxylic acids and / or phosphonic acids, and optionally the oligomer is a crown ether or azacrown ether. Embodiment 11. The microbead of embodiment 10, wherein the oligomer is further substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof. Embodiment 12. Microbeads according to any one of embodiments 1 to 11, wherein the chelating agent is selected from DFO, EDTA, DTPA, EGTA, EDDS, EDDHA, BAPTA, H4neunpa, H6phospa, H4CHXoctapa, H4octapa, H2CHXdedpa, H5decapa, Cy-DTPA, Ph-DTPA, TACN type chelating agents, TACD type chelating agents, cyclen type chelating agents, cyclam type chelating agents, (13)aneN4 type chelating agents, 1,7-diaza-12-crown-4 type chelating agents, 1,10-diaza-18-crown-6 type chelating agents, or derivatives thereof. Embodiment 13. A microbead according to embodiment 12, wherein the TACN type chelator is selected from NOTA, NOPO, TRAP, or derivatives thereof. Embodiment 14. The microbead of embodiment 12, wherein the cyclen-type chelator is selected from DOTA or a derivative thereof. Embodiment 15. The microbead of embodiment 12, wherein the cyclam-type chelator is selected from TETA, cross-linked TETA, DiAmSar, or derivatives thereof. Embodiment 16. (13) A microbead according to embodiment 12, wherein the aneN4 type chelator is selected from TRITA or a derivative thereof. Embodiment 17. The microbeads of embodiment 12, wherein the 1,10-diaza-18-crown-6 type chelating agent is selected from MACROPA or a derivative thereof. Embodiment 18. The microbeads of embodiment 12, wherein the chelating agent is selected from DTPA, Cy-DTPA, Ph-DTPA, or derivatives thereof. Embodiment 19. The microbead of embodiment 18, wherein the derivative of DTPA comprises DTPA in which two adjacent carbon atoms are bonded together with the atom between them to form a 5- or 6-membered ring, optionally a cycloalkyl ring, an aryl or a heteroaryl ring. Embodiment 20. Prior to polymerization, the metal chelating monomer is: [ka] 19. The microbead according to embodiment 18, wherein L and X are as defined in any one of embodiments 4 to 6. Embodiment 21. The metal chelating monomer is: [ka] TIFF2024511743000013.tif55132, or a mixture thereof. Embodiment 22. Microbeads according to any one of embodiments 1 to 9, wherein the chelating agent comprises a porphyrin or a phthalocyanine. Embodiment 23. The microbeads of embodiment 22, wherein the chelating agent is a substituted or unsubstituted porphyrin. Embodiment 24. Prior to polymerization, the metal chelating monomer is: [ka] or a mixture thereof, wherein n is an integer from 1 to 4. Embodiment 25. The microbead of embodiment 24, wherein L is aniline. Embodiment 26. The microbead of embodiment 24 or 25, wherein n is at least 2. Embodiment 27. The metal chelating monomer is: [ka] 4. The microbead of embodiment 3, selected from: Embodiment 28. The microbeads of any one of embodiments 1 to 27, further comprising a steric stabilizer, optionally selected from PVP, polyvinyl alcohol, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polyacrylic acid, water-soluble homopolymers of acrylic acid esters, water-soluble homopolymers of methacrylic acid esters, water-soluble homopolymers of acrylamide, homopolymers of methacrylamide, water-soluble copolymer steric stabilizers, or mixtures thereof. Embodiment 29. Microbeads according to embodiment 28, wherein the water-soluble copolymer steric stabilizer is selected from copolymers of acrylic acid esters, methacrylic acid esters, acrylamides or methacrylamides with methyl acrylate and / or ethyl acrylate, or mixtures thereof. Embodiment 30. A microbead according to any one of embodiments 1 to 29, wherein the copolymer is crosslinked. Embodiment 31. A microbead according to any one of embodiments 1 to 30, wherein the metal is a plurality of metals. Embodiment 32. The microbead of embodiment 31, wherein the plurality of metals comprises one or more enriched isotopes. Embodiment 33. The microbead of embodiment 31 or 32, wherein the multiple metals comprise at least two metals, at least three metals, or at least four metals. Embodiment 34. A microbead according to any one of embodiments 31 to 33, wherein the amount of each metal in the plurality of metals is within about 20% or about 10% of the amount of another metal in the plurality of metals. Embodiment 35. Microbeads according to any one of embodiments 1 to 34, wherein the metal comprises indium, bismuth, or a rare earth metal, optionally wherein the rare earth metal is selected from a lanthanide metal, yttrium, or a mixture thereof. Embodiment 36. The microbead of embodiment 35, wherein the metal comprises a rare earth metal selected from Y, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, their respective isotopes, and mixtures thereof. Embodiment 37. The rare earth metal is 89Y, 139La, 136Ce, 138Ce, 140Ce, 142Ce, 141Pr, 142Nd, 143Nd, 145Nd, 146Nd, 148Nd, 145Pm, 144Sm, 149Sm, 150Sm, 152Sm, 154Sm, 151Eu, 153Eu, 154Gd, 155Gd, 156Gd, 157Gd, 158Gd, 160Gd, 152Gd, 159 37. The microbeads of embodiment 36, wherein the microbeads are selected from Tb, 156Dy, 158Dy, 160Dy, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 162Er, 164Er, 166Er, 167Er, 168Er, 170Er, 169Tm, 168Yb, 170Yb, 171Yb, 172Yb, 173Yb, 174Yb, 176Yb, 175Lu, or mixtures thereof. Embodiment 38. A microbead according to any one of embodiments 1 to 37, wherein the metal is dispersed throughout the microbead. Embodiment 39. Microbeads according to any one of embodiments 1 to 38, wherein the microbeads have a glass transition temperature of about or above 60°C, optionally about or above 70°C, about or above 80°C, about or above 90°C, about or above 100°C, about or above 115°C, about or above 125°C, or about or above 135°C. Embodiment 40. The microbeads according to any one of embodiments 1 to 39, wherein the microbeads have a diameter of about 0.6 μm to about 20 μm, about 1 μm to about 15 μm, about 2 μm to about 10 μm, or about 2 μm to about 6 μm. Embodiment 41. A microbead according to any one of embodiments 1 to 40, wherein the microbead is colloidally stable in water. Embodiment 42. Microbeads according to any one of embodiments 1 to 41, wherein the surface of the microbeads comprises a functionalization for attachment to biomolecules. Embodiment 43. The microbead of embodiment 42, wherein the attachment is a covalent attachment. Embodiment 44. A microbead according to embodiment 42 or 43, wherein the biomolecule is selected from a protein, an oligonucleotide, a small molecule, a lipid, a carbohydrate, or a mixture thereof. Embodiment 45. A microbead according to embodiment 42 or 43, wherein the biomolecule is an affinity reagent, optionally wherein the affinity reagent is an antibody. Embodiment 46. Microbeads according to embodiment 45, wherein the antibodies are specific for cytokines, optionally chemokines, interferons, lymphokines, monokines, interleukins such as IL-1-36, tumor necrosis factors and colony stimulating factors. Embodiment 47. The microbead of embodiment 44, wherein the antigen is a viral antigen. Embodiment 48. A microbead according to any one of embodiments 42 to 47, wherein the functionalization comprises a coating of silicon dioxide on the surface of the microbead, and optionally the functionalization further comprises functionalizing the silicon dioxide coating. Embodiment 49. A microbead according to embodiment 42, wherein the attachment to the biomolecule is a non-covalent attachment. Embodiment 50. A microbead according to any one of embodiments 42 to 49, wherein the surface of the microbead is functionalized with avidin, streptavidin, neutravidin, or a mixture thereof. Embodiment 51. A microbead according to any one of embodiments 42 to 50, wherein the surface of the microbead is conjugated to a biomolecule. Embodiment 52. A microbead according to any one of embodiments 42 to 51, wherein the metal provides a barcode that identifies the biomolecule. Embodiment 53. A population of microbeads as defined in any one of embodiments 1 to 52. Embodiment 54. A population of microbeads according to embodiment 53, wherein the population has a particle size distribution with a coefficient of variation (CV) of about 10% or less than 10%. Embodiment 55. A population of microbeads according to embodiment 54, wherein the coefficient of variation is less than 5%. Embodiment 56. A population of microbeads according to any one of embodiments 53 to 55, wherein each microbead comprises multiple metals and the average amount across the population of microbeads of each metal of the multiple metals is about 10% or within 10% of the average amount of another metal of the multiple metals. Embodiment 57. The population of microbeads of embodiment 56, wherein the plurality of metals comprises one or more enriched isotopes. Embodiment 58. A population of microbeads according to any one of embodiments 53 to 57, wherein the amount of each metal in one microbead of the population of microbeads is within about 20% or 20%, or within about 10% or 10%, or within about 5% or 5% of the amount of the same metal in another microbead of the population of microbeads. Embodiment 59. A population of microbeads according to any one of embodiments 53 to 57, wherein the amount of each metal in the population of microbeads has a distribution with a coefficient of variation of about 20% or less than 20%. Embodiment 60. A population of microbeads according to embodiment 59, wherein the amount of each metal in the population of microbeads has a distribution with a coefficient of variation of about 10% or less than 10%. Embodiment 61. A population of microbeads according to any one of embodiments 58 to 60, wherein the microbeads of the population of microbeads contain substantially the same amount of the same metal. Embodiment 62. The population of microbeads of embodiment 61, wherein the same metal is a plurality of metals and the microbead comprises substantially the same amount of each metal of the plurality of metals. Embodiment 63. A kit comprising a plurality of distinct populations of microbeads as defined in any one of embodiments 53 to 62. Embodiment 64. The kit of embodiment 63, wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or metals of the microbeads. Embodiment 65. The kit of embodiment 63 or 64, wherein the microbeads of at least one population of microbeads comprise a metal or metals that differ from the metal or metals of the microbeads of another population of microbeads. Embodiment 66. The kit of embodiment 63 or 64, wherein the microbeads of at least one population of microbeads comprise a plurality of metals in a ratio different from that of the microbeads of another population of microbeads. Embodiment 67. A kit according to any one of embodiments 64 to 66, wherein the microbeads of each population of microbeads are conjugated to a different biomolecule. 68. polymerizing the structural monomers in the presence of a steric stabilizer in a nucleation stage to obtain a first mixture comprising polymerized structural monomers, non-polymerized structural monomers, and a steric stabilizer; combining the first mixture with a metal chelating monomer comprising a metal and a chelating agent attached to at least one polymerizable end group to obtain a second mixture; combining said chelating agent with at least three sites to coordinate the metal; and said metal chelating monomer is polymerizable with said structural monomer; and polymerizing the second mixture to form a copolymer of the microbeads; The structural monomer is polymerized without a chelating agent; 23. A method for preparing metal-encoded microbeads comprising: Embodiment 69. The method of embodiment 68, wherein the metal is a plurality of metals. Embodiment 70. The method of embodiment 68 or 69, wherein the structural monomer is polymerized in the nucleation stage to about 5% to about 20% completion based on the structural monomer. Embodiment 71. The method of any one of embodiments 68-70, wherein polymerization of the second mixture occurs to from 75% to about 100% completion, from about 80% to about 99% completion, from about 85% to about 95% completion, or from about 85% to about 93% completion based on the structural monomers. Embodiment 72. The method of any one of embodiments 68 to 71, wherein the structural monomer is as defined in embodiment 2 or 3. Embodiment 73. The method of any one of embodiments 68 to 72, wherein the metal chelating monomer is as defined in any one of embodiments 2, and 4 to 26. Embodiment 74. The method of any one of embodiments 68 to 73, wherein the steric stabilizer is as defined in embodiment 27. Embodiment 75. The method of any one of embodiments 68 to 74, wherein the metal is as defined in any one of embodiments 29 to 35. Embodiment 76. The method according to any one of embodiments 68 to 75, wherein the method further comprises functionalizing the microbeads. Embodiment 77. The functionalization of microbeads is mixing the polymerized second mixture with a third monomer to obtain a third mixture, the third monomer including a reactive functional group; and polymerizing the third mixture; 77. The method of embodiment 76, comprising: Embodiment 78. The method of embodiment 77, wherein the reactive functional group is selected from an alcohol, an aldehyde, a carboxylic acid, an epoxide, a vinyl, an alkyne, a maleimide, or a mixture thereof. Embodiment 79. The method of embodiment 76, wherein the functionalization of the microbeads comprises coating the microbeads with silicon dioxide. Embodiment 80. The method of embodiment 76, wherein the functionalization of the microbeads further comprises functionalizing the silicon dioxide coating. Embodiment 81. The method of any one of embodiments 68 to 80, further comprising conjugating microbeads to the biomolecule. Embodiment 82. The method according to embodiment 81, wherein the biomolecule is as defined in any one of embodiments 44 to 47. Embodiment 83. The method of any one of embodiments 68 to 82, wherein the microbeads have a diameter of about 0.6 μm to about 20 μm, about 1 μm to about 15 μm, about 2 μm to about 10 μm, or about 2 μm to about 6 μm. Embodiment 84. The method of any one of embodiments 68 to 83, wherein the microbeads are as defined in any one of embodiments 1 to 52. Embodiment 85. A microbead prepared by the method of any one of embodiments 68 to 83. Embodiment 86. A microbead according to any one of embodiments 1 to 52, wherein the internal structure of the microbead comprises a copolymer. Embodiment 87. A microbead according to any one of embodiments 1 to 52, and 86, wherein the metal chelating monomer chelates a single metal atom and does not chelate multiple metal atoms. Embodiment 88. A microbead according to any one of embodiments 1 to 52, and 86 and 87, wherein the microbead comprises a polymer seed that does not contain a metal chelating monomer. Embodiment 89. The microbead of embodiment 88, wherein the polymer seed comprises a structural monomer, and optionally the structural monomer of the polymer seed is identical in structure to the structural monomer of the copolymer. Embodiment 90 The method of embodiment 68, wherein the internal structure of the microbead comprises a copolymer. Embodiment 91. A method for preparing metal-encoded microbeads, comprising: providing an aqueous dispersion of swellable seed particles and an anionic surfactant; contacting the aqueous dispersion with monomers comprising structural monomers and metal chelating monomers, the metal chelating monomers comprising a metal and at least one chelating agent attached to a polymerizable terminus, the chelating agent coordinating the metal at at least three sites, and the structural monomers being free of the chelating agent; diffusing a monomer into the seed particles to form an aqueous dispersion of swollen seed particles; and initiating polymerization of monomers in the aqueous dispersion of swollen seed particles; A method comprising: Embodiment 92. The method of embodiment 91, wherein the structural monomer is selected from the group consisting of acrylic monomers, methacrylate monomers and vinyl monomers selected from the group consisting of styrene, divinylbenzene (DVB), ethylvinylbenzene, vinylpyridine, aminostyrene, methylstyrene, dimethylstyrene, ethylstyrene, ethylmethylstyrene, p-chlorostyrene and 2,4-dichlorostyrene. Embodiment 93. The method of embodiment 91 or 92, wherein the aqueous dispersion of swollen seed particles further comprises a steric stabilizer. Embodiment 94. The method of embodiment 93, wherein the steric stabilizer is polyvinylpyrrolidone. Embodiment 95. The method of any one of embodiments 91-94, wherein providing an aqueous dispersion of swellable seed particles comprises preparing monodisperse swellable seed particles by emulsion polymerization. Embodiment 96. The aqueous dispersion of swellable seed particles has a molecular weight of less than 5000 Daltons and a viscosity of 10 at 25° C. -2 96. The method of any one of embodiments 91-95, further comprising: an organic compound having a water solubility of less than 100 g / L; and, optionally, an organic solvent in which said organic compound is soluble. Embodiment 97. The method of any one of embodiments 91 to 96, wherein the swellable seed particles are monodisperse swellable seed oligomeric particles. Embodiment 98. The method of any one of embodiments 91 to 97, wherein the anionic surfactant is sodium dodecyl sulfate. Embodiment 99. The method of any one of embodiments 91 to 98, wherein the structural monomer is as defined in embodiment 2. Embodiment 100. The method of any one of embodiments 91 to 99, wherein the metal chelating monomer is as defined in any one of embodiments 1, and 3 to 27. Embodiment 101. The kit of embodiment 67, further comprising a reporter comprising a mass tag, wherein the reporter specifically binds a sample biomolecule that is specifically bound by at least one distinct biomolecule. Embodiment 102. The kit of embodiment 101, wherein the sample biomolecule is an oligonucleotide and at least one different biomolecule is an oligonucleotide that specifically hybridizes to the sample biomolecule. Embodiment 103. The kit of embodiment 102, wherein the reporter comprises a plurality of oligonucleotides that hybridize to indirectly bind the plurality of mass-tagged oligonucleotides to the sample biomolecules. Embodiment 104. A kit according to any one of embodiments 101 to 103, wherein at least one different biological molecule is an affinity reagent, such as an antibody. Embodiment 105. The kit of embodiment 104, wherein the sample biomolecule is a virus particle, the at least one different biomolecule is a first antibody that specifically binds the virus particle, and the reporter comprises a second antibody that specifically binds the virus particle. Embodiment 106. The kit of embodiment 104, wherein the sample biomolecule is a cytokine, the at least one different biomolecule is a first antibody that specifically binds the cytokine, and the reporter comprises a second antibody that specifically binds the cytokine. Embodiment 107. The kit of embodiment 104, wherein the sample biomolecule is a cancer biomarker, the at least one different biomolecule is a first antibody that specifically binds the cancer biomarker, and the reporter comprises a second antibody that specifically binds the cancer biomarker. Embodiment 108. A kit described in any one of embodiments 101 to 107, wherein at least one different biological molecule includes a viral antigen, the sample biological molecule is an antibody that specifically binds the viral antigen, and the reporter includes a secondary antibody that binds to the sample biological molecule. Embodiment 109. A kit according to any one of embodiments 101 to 108, further comprising a plurality of different reporters, each different reporter binding a sample biomolecule that is specifically bound by a different biomolecule. Embodiment 110. The kit of embodiment 109, wherein each of the multiple different reporters comprises the same mass tag. Embodiment 111. A kit according to any one of embodiments 101 to 110, wherein the mass tag comprises a metal nanoparticle. Embodiment 112. A kit according to any one of embodiments 101 to 110, wherein the mass tag comprises a metal chelating polymer. Embodiment 113. The kit of embodiment 67, wherein at least one biomolecule is an enzyme substrate for a sample biomolecule. Embodiment 114. The kit according to embodiment 113, further comprising a reporter that specifically binds to the enzyme substrate when it is modified by a sample biomolecule. Embodiment 115. The kit of embodiment 113, further comprising a reporter that specifically binds to the enzyme substrate if it is not modified by a sample biomolecule. Embodiment 116. The kit of embodiment 115, wherein the enzyme substrate comprises a mass tag that is removed when the enzyme substrate is modified by a sample biomolecule. Embodiment 117. A kit according to any one of embodiments 101 to 116, wherein the microbeads from a plurality of different populations are present in a first mixture of microbeads. Embodiment 118. The kit of embodiment 117, further comprising a second mixture of microbeads comprising the same biomolecules as the first mixture of microbeads, wherein the microbeads of the first mixture comprise a different sample barcode than the microbeads of the second mixture. Embodiment 119. The kit of embodiment 118, wherein the sample barcode is present inside a microbead. Embodiment 120. The kit of embodiment 119, wherein the sample barcode is a subset of the metals chelated by the metal chelating monomers of the microbeads of the first and second mixtures. Embodiment 121. The kit of embodiment 118, wherein the sample barcodes are present on the surface of the microbeads of the first and second mixtures. Embodiment 122. The kit of embodiment 118, further comprising a plurality of sample barcodes in separate compartments that are functionalized to bind to the surface of microbeads from a plurality of different populations. Embodiment 123. A kit according to any one of embodiments 101 to 122, further comprising a panel of mass-tagged antibodies in a mutual mixture, at least some of the antibodies of the panel being specific for a cell surface marker. Embodiment 124. The kit of embodiment 123, wherein the multiple antibodies are mixed with the microbeads of the kit. Embodiment 125. The kit of embodiment 123 or 124, wherein at least some of the mass-tagged antibodies contain the same metal as that chelated by the metal chelating monomers of the microbeads of the kit. Embodiment 126. A kit according to any one of embodiments 101 to 125, further comprising a steric stabilizer mixed with the microbeads of the kit. Embodiment 127. The kit according to embodiment 126, wherein the steric stabilizer is polyvinylpyrrolidone. Embodiment 128. A kit according to any one of embodiments 101 to 127, wherein the microbeads of the kit are present in a solution buffered at a pH of 5 and 9 or from 5 to 9. Embodiment 129. A kit according to any one of embodiments 101 to 125, wherein the microbeads are freeze-dried. Embodiment 130. A kit according to any one of embodiments 101 to 125, wherein the microbeads are fused to a solid support. Embodiment 131. The kit of embodiment 130, wherein the solid support is a microscope slide. Embodiment 132. The kit of embodiment 130, wherein the solid support is an adhesive membrane. Embodiment 133. A kit according to any one of embodiments 101 to 132, further comprising one or more buffers, anticoagulants, fixation reagents, and permeabilization reagents. Embodiment 134. A method comprising detecting a population of microbeads according to any one of embodiments 53 to 62. Embodiment 135. The method of embodiment 134, further comprising calibrating a mass spectrometer used to detect the microbeads based on a mass spectrum obtained from the microbeads. Embodiment 136 The method of embodiment 134, further comprising normalizing the mass spectrometry signals obtained from the plurality of mass tags based on a mass spectrum obtained from the microbeads. EMBODIMENT 137. mixing distinct populations of microbeads according to any one of embodiments 53 to 62 with a sample, wherein the microbeads of each population of microbeads are conjugated to different biomolecules, and each population of microbeads is distinguished from another population of microbeads based on the metal or metals of the microbead; Binding different sample biomolecules of a sample to different biomolecules of a distinct population of microbeads; conjugating, directly or indirectly, a reporter to each of the distinct sample biomolecules, wherein the reporter attached to each of the distinct sample biomolecules comprises a mass tag; and detecting the metal and mass tags of the individual microbeads by mass spectrometry; A method of mass spectrometry comprising: Embodiment 138. The method of embodiment 137, further comprising attaching different biomolecules to the microbeads of the distinct populations of microbeads prior to the step of mixing the distinct populations of microbeads with the sample. Embodiment 139. The method of embodiment 137 or 138, wherein the different sample biomolecules comprise oligonucleotides. Embodiment 140. The method of any one of embodiments 137 to 139, wherein the different sample biomolecules include antibodies. Embodiment 141. The method of any one of embodiments 137 to 140, wherein the different sample biomolecules include cytokines. Embodiment 142. The method of any one of embodiments 137 to 141, wherein the different sample biomolecules include cancer biomarkers. EMBODIMENT 143. mixing distinct populations of microbeads with the sample, wherein the microbeads of each population of microbeads are conjugated to a different biomolecule, and each population of microbeads is distinguished from another population of microbeads based on the metal or metals of the microbead; binding different sample biomolecules of a sample to different biomolecules of a distinct population of microbeads; conjugating, directly or indirectly, a reporter to each of the distinct sample biomolecules, wherein the reporter attached to each of the distinct sample biomolecules comprises a mass tag; and detecting the metal and mass tags of the individual microbeads by mass spectrometry; A method of mass spectrometry using a kit according to any one of embodiments 101 to 133, comprising: EMBODIMENT 144. providing a plurality of mass-tagged antibodies, each of the mass-tagged antibodies being attached to a polymeric mass tag that chelates multiple atoms of a metal or enriched isotope thereof; contacting the sample with a plurality of mass-tagged antibodies; detecting the mass-tagged antibodies bound to the sample and the microbeads by mass spectrometry; A method for mass spectrometry of a cell sample using microbeads according to any one of embodiments 63 to 74, comprising: Embodiment 145. The method of embodiment 144, further comprising calibrating a mass spectrometer used in the detection step, wherein the calibrating is based on a mass spectrometer signal obtained from the microbeads. Embodiment 146 The method of embodiment 144, further comprising normalizing the mass spectrometry signal obtained from the mass tag based on the mass spectrometry signal obtained from the microbeads. Embodiment 147. The method of embodiment 144, further comprising quantifying the mass-tagged antibodies based on the average number of metal atoms for each mass-tagged antibody and the detected mass spectrometry signals from the mass-tagged antibodies and the microbeads. Embodiment 148. The method of embodiment 146 or 147, wherein the microbead and the mass-tagged antibody contain the same metal. Embodiment 149. A method according to any one of embodiments 134 to 148, wherein the detecting step comprises imaging mass cytometry. Embodiment 150. The method of embodiment 149, further comprising quantifying or normalizing mass-tagged antibodies bound at individual pixels or to individual cells based on mass spectrometry signals detected from the microbeads. Embodiment 151 The method of embodiment 149 or 151, wherein the microbeads are melted to the solid surface prior to the detecting step. Embodiment 152. The method of any one of embodiments 134 to 151, wherein the detecting step comprises suspension mass cytometry. Embodiment 153. The method of any one of embodiments 134 to 152, wherein the detecting step comprises inductively coupled plasma mass spectrometry (ICP-MS). Embodiment 154. A method according to any one of embodiments 149 to 151, wherein the detecting step includes laser ablation ICP-MS or secondary ion mass spectrometry (SIMS). Embodiment 155. The method of any one of embodiments 134 to 154, wherein the mass spectrometry is time-of-flight mass spectrometry (TOF-MS). The above disclosure broadly describes the present disclosure. A more complete understanding can be obtained by reference to the following specific examples. These examples are set forth solely for the purpose of illustrating the present disclosure and are not intended to limit the scope of the present invention. Changes in form and substitution of equivalents are contemplated as suggested by the circumstances or as may become expedient. Although specific terms are used in this specification, such terms are intended to be descriptive and not limiting.

[0166] Working Example The following non-limiting examples are illustrative of the present disclosure. Example 1 Polystyrene (PS) microbeads with one or more lanthanides chelated with DTPA Metal-encoded microbeads were synthesized by introducing a polymerizable metal-DTPA complex into a dispersion polymerization reaction of styrene in ethanol as a second-stage aliquot. The synthesis of the components and microbeads as well as the materials used are described.

[0167] material Diethylenetriaminepentaacetic dianhydride (DTPA dianhydride, 98%), 2,2-azobis(2-methylpropionitrile) (AIBN, 98%), polyvinylpyrrolidone (PVP, Mw approx. 55 kDa), Triton X305 (TX305, 70% aqueous solution), benzylamine (BA, 99%), N-(3-aminopropyl)methacrylamide hydrochloride (APMAm, 98%), triethylamine (TEA, 99%), sodium acetate (anhydrous, ≥99%), ammonium acetate (≥98%), sodium carbonate (≥99%), hydrogen peroxide solution (H2O2, 30% in H2O), and yttrium(III) chloride hexahydrate (YCl3· Metal salts with purity ≥99.99% (trace metals standard) including cerium(III) chloride heptahydrate (CeCl3 7H2O,), europium(III) chloride hexahydrate (EuCl3 6H2O), holmium(III) chloride hexahydrate (HoCl3 6H2O), and lutetium(III) chloride hexahydrate (LuCl3 6H2O), as well as deuterium oxide (D2O, 99.9%), were purchased from Sigma-Aldrich. 4-Vinylbenzylamine (VBA, ≥92%) was provided by TCI America. Absolute ethanol (EtOH) was prepared from commercially available alcohol. Nitric acid (trace metal grade, 68-69%), sulfuric acid (trace metal grade), sodium hydroxide and phosphate buffered saline (1xPBS solution, Fisher BioReagents) were purchased from Fisher Scientific. All above chemicals were used without further purification. Styrene (St, Sigma-Aldrich, ≥99%) was purified by passing through an alumina (Sigma-Aldrich, activated, neutral packed column) packed column. Single element standard solutions for inductively coupled plasma mass spectrometry (ICP-MS) calibration were purchased from PerkinElmer (Pure Plus). On average, 1000 mg / ml of ... 89 Y(69x10 6 ), 115 In(43x10 6 ), 140 Ce(17x106 ), 151 Eu(10x10 6 ), 153 EU(11x10 6 ), 165 Ho(5.8x10 6 ), 175 Lu(7.5x10 6 ), and 209 Bi(5.8x10 6 ) for mass cytometry (MC) calibration and normalization, as described by Liu et al., 2020 15 The polymerization conditions were as described in. EQ™ 4 element (EQ4) mass cytometry (MC) calibration beads were kindly provided by Fluidigm Canada. Deionized water with a minimum resistivity of 18 MΩ·cm was produced by a Millipore purification system. Compressed nitrogen (99.998%, Praxair) was used as a protective atmosphere for the polymerization reactions.

[0168] Synthesis of functional DTPA-bis(amide) derivatives To incorporate DTPA metal complexes into polystyrene (PS) microbeads, DTPA was first functionalized by reacting DTPA dianhydride with 4-vinylbenzylamine, benzylamine, allylamine, or aminopropylmethacrylamide (R-NH2=VBA, BA, ALA, or APMAm) in a 1:2 stoichiometric ratio. The synthetic method was adapted from Zhang et al. for DTPA-bis(vinylbenzylamide) (DTPA-VBAm2) with some modifications. 16 DTPA-bis(benzylamide) (DTPA-BAm2) was reported by Aime et al. 17 and for DTPA-bis(allylamide) (DTPA-ALAm2) by Shuhendler et al. 18 The protocol was developed from that reported by. In a typical experiment for preparing DTPA-VBAm2, DTPA dianhydride (1 mmol) was mixed with 4-vinylbenzylamine (2 mmol) in anhydrous DMSO (5.0 mL) at room temperature and stirred overnight. NaOH (1 M, 3 equiv.) in ethanol was added to the reaction to form the trisodium salt of DTPA-VBAm2, and the reaction mixture was subsequently diluted with 45 mL of acetone to precipitate the DTPA salt. The precipitated DTPA salt was then collected by precipitation and redissolved in ethanol. Three cycles of dissolution-precipitation-precipitation were performed to purify the product. The product was dried under reduced pressure at room temperature overnight to remove residual solvent and analyzed by proton nuclear magnetic resonance (NMR) analysis using a Varian 500 MHz spectrometer (Agilent) in D2O solution at room temperature. 1 The compound was characterized by H-NMR.

[0169] Synthesis of metal complexes of DTPA-bis(amide) derivatives Metal complexes of DTPA-bis(amide) derivatives (M(DTPA-R2)) used for bead synthesis were prepared by loading metal ions onto DTPA-R2 in aqueous solution. In a typical metal chelation experiment, 0.30 mmol of DTPA-R2 trisodium salt and an equimolar amount of metal chloride (0.30 mmol) were dissolved separately in water (3 mL). The metal salt solution was then added to the DTPA derivative solution, while the pH was monitored and adjusted to 5.0-6.0 with 0.01 M HCl and 0.01 M NaOH solutions. 3+ During the chelation of the ions to DTPA-VBAm2, the clear solution of the mixture slowly became opaque with the addition of the metal chloride solution. During the experiment, 1-2 mL of ethanol was added to the mixture to keep the solution clear. Each mixture was stirred at room temperature for 3 hours. The M(DTPA-R2) complexes were isolated by acetone precipitation and sedimentation. It is noted that the unchelated metal ions are soluble in acetone-water mixtures, while the metal complexes of DTPA derivatives have low solubility in acetone. The precipitates were then dissolved in ethanol and precipitated with acetone for further purification. The final products of metal complexes were collected by precipitation and dried overnight at room temperature under reduced pressure. 1The metal complexes were characterized using H-NMR.

[0170] Two-stage dispersion polymerization Two-stage dispersion polymerization (2-stage DisP) was used to prepare metal-encoded polystyrene (PS) microbeads in the presence of polyvinylpyrrolidone (PVP) as a steric stabilizer and a DTPA derivative metal complex as a metal ligand. After initiating the polymerization of styrene in ethanol in the presence of PVP, a desired amount of a warm solution of the DTPA metal complex in ethanol was introduced as a second-stage aliquot at 2 h. The reaction was stopped 24 h after initiation with a styrene conversion of over 90%. Table 1 describes a typical formulation of this 2-stage DisP used to prepare the microbeads. [Table 1]

[0171] Example 2 Twelve batches of bead synthesis were carried out using the methods and materials of Example 1 and various amounts of M(DTPA-R2) complex as the feedstock in the second step. These complexes were modified with different functional groups and loaded with different types of metal ions, as described in Table 2. [Table 2] After the reaction was terminated, the microbead dispersion was washed by two cycles of precipitation-redispersion with absolute ethanol and four with water to remove free stabilizer, unreacted monomer, and any smaller diameter particles. These washed microbead dispersions were used for MC characterization and an aliquot was freeze-dried to measure the solids content.

[0172] Example 3 Surface modification and secondary antibody attachment The microbeads were coated with a silica shell and conjugated to a secondary antibody.

[0173] Materials for Silica Coating and Bioconjugation Tetraethyl orthosilicate (TEOS, 99%), (3-aminopropyl)triethoxysilane (APTES, 99%), succinic anhydride (99%), anhydrous dimethyl sulfoxide (DMSO, 99.9%) were purchased from Sigma-Alderich. Ammonia solution 25% (NH4OH), MES buffer (0.5 M, pH 5.5), phosphate buffered saline (1xPBS, pH 7.4), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), NeutraAvidin (NAv), biotin-xx-goat anti-mouse (H+L IgG) and bovine serum albumin (BSA) were ordered from Thermofisher Scientific. MaxPar® 175Lu-labeled mouse anti-anti-TNFα (human) (clone MAb11), cell staining buffer and cell acquisition solution were kindly provided by Fluidigm Canada.

[0174] Preparation of metal-encoded microbeads functionalized with goat anti-mouse by silica coating Metal-encoded microbeads (Eu-1) were first coated with silica (SiO2) by the Stober method. Typically, an aliquot of the bead dispersion containing 50 mg of beads (solid) was washed by sedimentation-redispersion cycles with 5 mL of ethanol-ammonia solution (ethanol:ammonia=99:1 by volume) in a centrifuge tube. TEOS (75 μL) was added to the bead dispersion to initiate the silica condensation reaction. The reaction was stirred (40 rpm, ambient temperature) on a sample rotator for 20 min and quenched by four sedimentation-redispersion cycles with 98% by volume ethanol (5 mL). An aliquot of the silica-coated Eu-1 bead (Eu-1 / SiO2) dispersion (1 mL, approximately 10 mg solid) was treated in a sonication bath for 5 min and further functionalized with amino groups (NH2) by adding APTES (5 μL) to the Eu-1 / SiO2 bead dispersion. The NH2 coating reaction was stirred in an oven at 40°C for 20 h and quenched by 4 cycles of precipitation-redispersion washing with absolute ethanol (1 mL). To convert the functional groups on the NH2 modified microbeads (Eu-1 / NH2) to carboxyl groups (COOH), an aliquot of the freshly prepared Eu-1 / NH2 microbead dispersion (800 μL, approximately 8 mg solid) was washed with an ethanol-TEA solution (1 mL, containing 0.2 vol.% TEA) for 4 precipitation-redispersion cycles, followed by the addition of a freshly prepared succinic anhydride solution in DMSO (100 μL, 10 wt.% succinic anhydride). The COOH modification reaction was stirred overnight at 40°C on a sample rotator (40 rpm) and then terminated by 4 cycles of precipitation-redispersion washing with water (800 μL).

[0175] NAv was then conjugated to COOH-modified Eu-1 beads (Eu-1 / COOH) by EDC / NHS coupling. For the conjugation reaction, an aliquot of Eu-1 / COOH dispersion (100 μL, approx. 1% solids) was washed four times with MES buffer (100 μL. 0.1 M, pH 5.5) to exchange the solvent. The EDC / NHS activation reaction was initiated by adding EDC / NHS solution (100 μL) containing EDC (8 mg) and NHS (12 mg) in MES buffer to the washed Eu-1 / COOH dispersion. After 15 min of incubation, the reaction solution was quickly switched to 1xPBS buffer (100 μL; pH 7.4) by two cycles of centrifugation washing, followed by the addition of NAv solution (50 μL, 2 mg / mL). The NAv conjugation reaction was incubated for 4 hours, followed by 4 cycles of centrifugal washes with PBS buffer (100 μL) to remove unbound NAv.

[0176] Goat anti-mouse (GAM) secondary antibody was attached to Eu-1 microbeads by incubating NAv-modified Eu-1 beads (approximately 2 million beads) in BSA-PBS solution (100 μL, 0.5 wt% BSA in PBS) containing biotin-xx-GAM (20 μg) for 2 h. Excess GAM was then removed by 4 cycles of centrifugation washing with BSA-PBS solution (100 μL).

[0177] Example 4 The size and size distribution, as well as the metal and metal distribution, of the microbeads prepared using the methods and materials of Examples 1 and 2 were characterized.

[0178] Characterization of microbead size and size distribution Scanning electron microscopy (SEM) images of the microbead dispersions were collected using a Hitachi S-5200 microscope to characterize the microbead diameter and diameter distribution. Typically, 2 μL of the diluted bead dispersion was dropped onto a 300 mesh formvar / carbon coated copper grid and allowed to dry. The microbead diameter was measured manually from multiple SEM images using ImageJ software. The mean diameter, standard deviation (SD) and coefficient of variation (CV, see Equation 2) were calculated based on at least 300 measurements.

number

[0179] Acid digestion of microbeads The microbead dispersion was digested with sulfuric acid and H2O2 prior to ICP-MS measurements. In a typical digestion experiment, sulfuric acid (500 μL) and microbead dispersion (100 μL, 0.5-2% solid content) were mixed in a 20 mL glass vial. The microbead dispersion in sulfuric acid was then heated to 250 °C on a hot plate and held for 40 min with magnetic stirring, followed by the addition of a 30% H2O2 solution (50 μL). For ICP-MS analysis, the digestion solution was then diluted with 2% HNO3.

[0180] Measurement equipment Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). ICP-MS (iCAP-Q, Thermo Scientific) was used to quantify the metal ion concentrations in the samples. The samples were analyzed in kinetic energy discrimination (KED) measurement mode. The elemental standard solutions were serially diluted with 2% HNO3 to a series of concentrations of 40, 20, 10, 1 and 0.1 ppb as calibration solutions. Based on the calibration fitting curves, the metal contents in each solution sample were determined. The detection limits of the elements of interest were estimated to be less than 10 ppt.

[0181] Mass cytometry (MC). The metal content of the microbeads was characterized on a bead-by-bead basis by a mass cytometry system (Helios® CyTOF, Fluidigm). Liu et al., 202015 A sample of seven-element coded microbeads (7E1), reported by et al., was employed as an internal standard and mixed with the diluted microbead dispersion sample. The mixture consisting of 7E1 reference beads and microbead sample was then introduced into the MC system at a rate of 30 μL / min, and the beads were introduced individually, but stochastically, into the ICP. After signal acquisition, singlet events were identified and gated on the dot plot generated by the MC. The mean, median, robust standard deviation (RSD) and robust coefficient of variation (RCV) of the singlet signals were reported as non-normalized raw data. Robust statistics provide an alternative approach to classical statistical estimators such as mean, SD, and CV.

[0182] result The styrene conversion of the microbeads prepared using the methods of Examples 1-3 was greater than 90%. Four different types of DTPA derivatives were prepared, as shown in Scheme 1 below, and their incorporation efficiency was tested using Ce(III)-DTPA complexes as described below.

[0183] Synthesis of functional DTPA-R2 derivatives To covalently incorporate metal complexes of DTPA into PS microbeads, DTPA was modified with functional groups that could react with styrene during DisP. In this design, two of the five carboxylates on DTPA were replaced with functional groups, so that the remaining three carboxylates in the functionalized DTPA derivative could form charge-neutral complexes with lanthanide(III) ions, which promoted the binding stability and ethanol solubility of their metal complexes in DisP.

[0184] The metal complexes were synthesized by the methodology outlined in Scheme 1. DTPA dianhydrides react efficiently with primary amines to produce DTPA bisamides. 21、22Bis(amide) derivatives of DTPA are octadentate chelators that bind strongly to lanthanide ions. The bond between metal ions and DTPA bisamides is expected to be highly stable against metal dissociation and exchange during bead synthesis when different types of metal complexes of DTPA-bisamides are mixed in the reaction. Scheme 1. Synthesis of DTPA derivative-metal complexes for two-step dispersion polymerization. The metal chelator DTPA was functionalized by reacting DTPA anhydride with amine-containing functional molecules in DMSO. Different types of metal ions were loaded onto the DTPA derivatives at pH 5-6. [ka]

[0185] From the benzyl carbon of DPTA-bisbenzylamide (DTPA-BAm2) or the allylic carbon of DTPA-bisallylamine (DTPA-ALAm2) 25、26 Two DTPA-bisamides capable of reacting with styrene by chain transfer via hydrogen abstraction were prepared from DTPA-bis-4-vinylbenzylamide (DTPA-allyl Am2), whose 4-vinylphenyl group would be expected to have a similar reactivity with styrene. 27-29 The resulting product is 1 Characterization by 1 H-NMR confirmed their structures.

[0186] Figure 1 shows the structure of Na3(DTPA-VBAm2). 1 8A and B show distinct chemical shifts of the protons in the vinyl group at 5.3 (b, 2H), 5.8 (a, 2H), and 6.8 (c, 2H). The chemical shift at 4.4 ppm (f, 4H) indicates the formation of an amide bond. The incorporation of protons from the vinyl, benzyl, and DTPA moieties confirms the bis-substitution of the vinylbenzyl group in each DTPA molecule. 1 The H-NMR spectrum was 18、19、30The spectra match those of DTPA-BAm2 and DTPA-ALAm2 reported in, respectively, demonstrating the successful synthesis of these two DTPA-bis(amide) derivatives.

[0187] Examination of metal incorporation into PS microbeads with Ce(DTPA-R2) metal complexes Synthesis of Ce(DTPA-R2) metal complexes. To investigate the incorporation activity of different DTPA derivatives, Ce complexes of Ce(DTPA-VBAm2), Ce(DTPA-BAm2), and Ce(DTPA-ALAm2) were prepared. The metal complexes were synthesized by adding an aqueous solution of CeCl3 to DTPA-R2 in water at pH 5.0-6.0 in a 1:1 molar ratio. The Ce(DTPA-R2) complexes were precipitated by the addition of acetone and then eluted. 1 The metal chelation was verified by characterization using H-NMR. 1 The H-NMR spectrum is shown in Figures 9A-C. 3+ Since Ce is a paramagnetic NMR shift reagent, the chemical shifts of the protons from these Ce complexes are somewhat shifted and broadened. 3+ Crystal structures of several M(DTPA-BAm2) complexes (M = In, Y, and Lu) have been reported by several researchers using X-ray diffraction to confirm the metal coordination in the complexes. 17、30 Metal coordination to the DTPA-R2 chelators described herein may be similar to the reported M(DTPA-BAm2) complexes due to the similarity of material properties.

[0188] 2-DisP with Ce(DTPA-R2) metal complexes. The incorporation efficiency of three Ce(DTPA-R2) complexes into PS microbeads was investigated. Equimolar amounts of Ce(DTPA-VBAm2), Ce(DTPA-BAm2) and Ce(DTPA-ALAm2) were dissolved in ethanol and introduced as second-stage aliquots into Ce-1, Ce-2 and Ce-3 bead syntheses, respectively, as listed in Table 2. From each of these syntheses, colloidally stable microbeads were obtained that retained the stability of their colloidal state in water. The average diameter of Ce-1 microbeads was 2.9 μm with a narrow size distribution (CV=1.2%), as shown in Figure 2. The Ce-2 and Ce-3 microbeads were also uniformly sized (CV < 1.5%), as summarized in Table 3, but had slightly smaller average diameters (d = 2.1 and 2.5 μm, respectively), possibly due to the chain transfer effect of the benzyl and allyl groups that inhibited the chain propagation of PS. 31 .

[0189] Metal ion incorporation during the reaction was measured by ICP-MS. The total metal ion content in the reaction was determined after digestion of the sample with H2SO4 + H2O2. The microbeads in the reaction stock were separated by centrifugation. The metal content in the supernatant was also analyzed by ICP-MS to quantify the metal ions remaining in solution. Liu et al., 2020 15 The metal incorporation efficiency of the bead synthesis was calculated based on the difference between the unincorporated metal content in the supernatant and the total metal content in the reaction as described by. This metal incorporation efficiency reflects the Ce complex incorporation into the PS beads because Ce was supplied to the reaction as a Ce-DTPA complex, and the Ce ions in all three Ce complexes are expected to remain chelated with the DTPA derivative during the bead synthesis due to their relatively high binding stability. 23 .

[0190] The Ce incorporation efficiencies of the three microbead syntheses are shown in Table 3. In the Ce-1 synthesis, 74% of the Ce(DTPA-VBAm2) complex added to the reaction was incorporated into the PS beads. However, 97% of Ce(DTPA-BAm2) and Ce(DTPA-ALAm2) remained in solution after the reaction. In other words, only 2.7% of the Ce(DTPA-BAm2) and 2.8% of the Ce(DTPA-ALAm2) complexes added to Ce-2 and Ce-3 were incorporated, respectively.

[0191] As a complementary measure of Ce content, Ce-1, Ce-2 and Ce-3 microbeads were diluted with known amounts of 100% Ce as quantification standards. 140 Ce-containing calibration microbeads were used to investigate the Ce content in the sample beads by MC on a bead-by-bead basis. The Ce content in the sample beads was evaluated by comparing the signal intensities between the sample beads and the calibration beads, assuming that the MC signal intensities from different microbeads are proportional to the metal content in the same measurement. 15 Table 3 summarizes the Ce content of the microbeads of these three batches as measured by MC.

[0192] In MC, Ce-1 microbeads showed sharp and intense activity with a median intensity of 6000 counts per bead and an RCV of 6.2% (see FIG. 11A). 140 The Ce signal was generated (see FIG. 11). The Ce content in the Ce-1 microbeads was calculated to be 5.44×10 per bead. 7 ions (see Table 3). In contrast, Ce-2 and Ce-3 microbeads were much weaker. 140 The Ce signal intensity is shown (FIGS. 11B and C, respectively). The Ce content for the Ce-2 and Ce-3 microbead samples was 1.6×10 6 pcs and 1.6x10 6 The results were virtually identical for the number of Ce ions / bead.

[0193] To confirm the mechanism of incorporation of the polymerizable Ce complex during the bead synthesis, another Ce complex, Ce(DTPA-AmPMAm2), was prepared employing methacrylamide as the functional group capable of copolymerizing with styrene. As in the preparation of Ce(DTPA-VBAm2), the Ce(DTPA-AmPMAm2) complex was prepared by first reacting DTPA dianhydride with APMAm in DMSO, and then co-polymerizing Ce in water at pH 5–6. 3+ The compound was synthesized by chelating ions. 1 H-NMR spectra confirmed the structures of Na3(DTPA-AmPMAm2) and Ce(DTPA-AmPMAm2), respectively. During the Ce chelation experiments, Ce(DTPA-AmPMAm2) appeared to be more hydrophilic than Ce(DTPA-VBAm2), because Ce(DTPA-AmPMAm2) was completely soluble in the chelation solution without the addition of ethanol. Ce-4 microbeads were prepared using an equal amount of Ce(DTPA-AmPMAm2) complex as a ligand under similar conditions to Ce-1, Ce-2 and Ce-3 syntheses. As a result, Ce-4 microbeads also showed a bright chromaticity with a mean diameter of 2.9 μm with a narrow size distribution (CV=1.3%), a median intensity at 4000 counts per bead and an RCV of 5.7%. 140 A Ce signal was generated (see FIG. 11D).

[0194] Both the Ce(DTPA-VBAm2) and Ce(DTPA-AmPMAm2) complexes were efficiently incorporated into PS beads during DisP due to the reactive copolymerization between vinylbenzylamide / methacrylamide and styrene. The much less efficient incorporation of the Ce(DTPA-BAm2) and Ce(DTPA-ALAm2) complexes is attributed to the low reactivity between benzylamide / allylamide as chain transfer agent and styrene under the bead synthesis conditions. [Table 3] The DTPA-VBAm2 chelator was evaluated for the incorporation of other lanthanide metal ions into PS microbeads.

[0195] Preparation of metal-encoded microbeads using M(DTPA-VBAm2) metal complexes Four other metal ions (Y 3+ ,EU 3+ , Ho 3+ and Lu 3+ Complexes of DTPA-VBAm2 with Ce(DTPA-VBAm2) were prepared. Their synthesis employed a method similar to that described above for Ce(DTPA-VBAm2). Complexes of Y(DTPA-VBAm2), Eu(DTPA-VBAm2), Ho(DTPA-VBAm2) and Lu(DTPA-VBAm2) 1 The H-NMR spectra are shown in Figures 10A to 10D, respectively. 3+ and Ho 3+ is a paramagnetic NMR shift reagent. The chemical shifts of the NMR spectra of Eu(DTPA-VBAm2) and Ho(DTPA-VBAm2) show a shift profile similar to that of Ce(DTPA-VBAm2). In contrast, Y 3+ and Lu 3+ is diamagnetic, and the spectra of Y(DTPA-VBAm2) and Lu(DTPA-VBAm2) in Figure 10S and D show no shift effect. The chemical shifts of the protons of the vinylbenzyl functional group in these spectra (5-8 ppm) are similar to those of the chelating agent, whereas the chemical shifts of the protons of DTPA in the aliphatic region (1-4 ppm) are broadened, which is probably due to the interconversion of different DTPA-VBAm2 isomers associated with metal chelation. 30、32It should be noted that in metal chelation experiments, Ho(DTPA-VBAm2) and Lu(DTPA-VBAm2) were less soluble in ethanol but more soluble in water than Ce(DTPA-VBAm2). Without intending to be bound by theory, this observation may be due to the fact that the polarity of the Ho(DTPA-VBAm2) and Lu(DTPA-VBAm2) complexes is higher than that of Ce(DTPA-VBAm2), possibly resulting in stronger binding of Ho and Lu to the DTPA chelator. 23 It is understood that it is preferred that all metal complexes are completely dissolved in the second stage aliquot during bead synthesis.

[0196] To investigate the incorporation of these metal complexes in microbeads, a series of microbeads (Y-1, Eu-1, Ho-1 and Lu-1) containing a single metal element (Y, Eu, Ho and Lu, respectively) was prepared in each synthesis as described in Table 2 using each of these different M(DTPA-VBAm2) complexes. The microbeads obtained from the synthesis of Y-1, Eu-1, Ho-1 and Lu-1 were uniform and of similar size (see Table 4). The MC signal intensities of the coding elements from these microbeads were strong and narrowly distributed, as shown in Figure 12. The metal content, evaluated by comparing the MC signal intensities from the sample microbeads and the calibration beads in the same measurement, confirmed that a large number of elements were incorporated in each microbead, as summarized in Table 4. The metal incorporation efficiencies in these bead syntheses, evaluated as described above, were all in the range of 65-72%, with a slight increase in this range for the lanthanide metal ions with larger ionic radii. (See Figure 3A).

[0197] A batch of five-element coded microbeads (5E1) was synthesized by dissolving 5 mg of each M(DTPA-VBAm2) complex (M = Y, Ce, Eu, Ho and Lu) in 15 g of ethanol as a second step aliquot for DisP reaction. The incorporation efficiency of individual metals in the bead synthesis was characterized by ICP-MS to investigate the incorporation interference between different elements when they are mixed in one reaction.

[0198] The average diameter of the 5E1 microbeads was 2.7 μm with a narrow particle size distribution (CV=1.2%). The incorporation efficiency of these five metals shown in Figure 3A indicates that 63-70% of the metal complexes introduced in the 5E1 reaction were incorporated into the beads. Different metal ions added to the 5E1 bead reaction were incorporated into the microbeads with similar efficiencies. The MC signal intensities of the coded isotopes in the 5E1 beads were compared using the seven-element coded microbeads as the calibration standard. 15 Figure 4A summarizes the median MC signal intensities of 5E1 beads, and the error bars represent the RSD of the signal intensities. 140 Ce, 151、153 EU, 165 Ho and 175 The signal intensity of Lu was in the optimal MC sensitivity range (>300 counts / bead) with a narrow signal distribution (RCV<10%). 89 Y signal intensity is MC 14 Due to the low transmission coefficient of the 89 amu channel in the 5E1 beads, there were less than 100 counts / bead. The MC signal intensity from the calibration beads was used to calculate the metal content per 5E1 bead. The results are shown in Figure 4B. Ce, Eu, Ho, and Lu in the 5E1 beads were 3.4-4.0x10 per bead. 6 The Y content was slightly higher (5.7x10 ions) as a result of there being more Y atoms in 5 mg of Y(DTPA-VBAm2) than in 5 mg of the other Ln(DTPA-VBAm2) sample. 6 ions / bead).

[0199] Control of metal ion incorporation during microbead synthesis Figure 3A summarizes the metal incorporation efficiency results in the bead synthesis batches (Y-1, Ce-1, Eu-1, Ho-1, Lu-1 and 5E1) described in Table 2 using M(DTPA-VBAm2) metal complex as the ligand. The x-axis in Figure 3 is the ionic radius of the incorporated metal ion. The incorporation efficiencies of different metals in these batches of beads overlapped in a very narrow range of 62-74%, despite the metal supply varying in these bead syntheses. The reactivity of the 4-vinylbenzylamide group appears to be independent of the metal ion bound to the chelator. As a result, the metal incorporation efficiency in the bead syntheses was consistent when M(DTPA-VBAm2) metal complex was employed as the ligand.

[0200] The relationship between the amount of M(DTPA-VBAm2) complex fed and the metal content of the resulting microbeads was investigated. In Figure 5, the metal content detected in the microbeads is plotted against the metal complex fed to the synthesis with filled symbols. To account for minor batch-to-batch variations in microbead size, the metal content in each microbead was divided by the volume of the bead, with the metal concentration in the microbead shown as the y-axis in Figure 5. The filled data points in Figure 5 show the linear dependence of the metal content in the microbeads on the amount of metal complex fed in the bead synthesis. The linear regression model fit of these data serves as a convenient guide to design the level of metal content in the metal-encoded microbeads by varying the amount of M(DTPA-VBAm2) introduced in each reaction. This situation is a significant improvement in experimental design and execution compared to the use of metal salt + acrylic acid as a means of metal-containing PS microbead preparation. Using this new method, a library of metal-encoded microbeads with various levels of metal content can be prepared that can be individually identified as classifier beads for MC bead-based bioassays.

[0201] Preparation of a series of metal-encoded classifier microbeads with different levels of metal content To prepare a library of classifier beads with maximum variation, multiple metal isotopes must be encoded into microbeads with precisely controlled metal content so that microbeads with different metal content can be individually separated by MC. A set of reaction conditions is provided that allows the synthesis of classifier beads containing three separate metal ion levels with baseline separation as described in Example 2. Intensity levels that differ by 3-fold were selected. However, other intensity levels may be selected. For example, intensity levels that differ by 2-fold or 2.5-fold may be selected.

[0202] Y(DTPA-VBAm2) (70 μmol) takes longer to dissolve in ethanol (15 g), likely due to its high polarity. Therefore, ions other than Y ions were used in these reactions. However, it is understood that Y may be a suitable metal for the microbeads of the present disclosure. For example, Y may be used with different metal chelating monomers. For example, Y may be used at different concentrations. Three samples of Ce, Eu, Ho, and Lu-containing 4-element PS microbeads were synthesized at concentration levels that differ by 3-fold. These samples are designated 4E1, 4E2, and 4E3, as shown in Table 2. The purpose was to synthesize 7-element encoded microbeads. 15 are 0.2, 0.6 and 1.8 times the MC intensity levels, respectively. 140 Ce, 151 EU, 153 EU, 165 Ho and 175 The aim was to obtain microbeads that produce MC signal intensity levels of Lu.

[0203] The microbeads obtained from the syntheses of 4E1, 4E2 and 4E3 were colloidally stable in the reaction stock and did not contain agglomerates. The average diameter of these microbeads after washing by sedimentation-redispersion ranged from 2.8 to 3.0 μm with a narrow size distribution (CV<1.5%), as shown in Table 4. As shown in Figure 3B, the incorporation efficiency of different metal ions in these three bead syntheses was consistent and close to the levels observed in the synthesis described in Figure 3A.

[0204] The MC signal intensities of the five coded lanthanide isotopes from these three batches of microbeads are shown in Figure 6 in an element-sorted panel. 140 Ce, 151 EU, 153 EU, 165 Ho and 175 The average MC signal intensity of Lu was the highest among these three batches of beads, with similar levels of 2560, 2053, 2590, 2540 and 2530 counts / bead, respectively. The signal intensity distribution of 4E3 beads was narrow, with RCV values ​​below 9%, indicating a narrow distribution of the coded lanthanide elements in the beads. 4E1 and 4E2 beads also produced sharp and narrow MC signal distributions, with intensities of about 200 and 700 counts / bead, respectively, for all coded isotopes. The histograms in Figure 6 show a clear baseline separation between these three batches of microbeads. The signal intensity of these three batches of beads increased approximately three-fold from 4E1 to 4E3 beads.

[0205] The metal content in these three microbeads was assessed by comparing the MC signal intensity to that of seven-element calibration beads. Values ​​were plotted against the metal input in the bead synthesis as open symbols shown in Figure 5. These open symbol data points followed the linear trend line observed from previous bead syntheses shown in Figure 5.

[0206] The synthesis of these three types of microbeads serves as an example to demonstrate the convenience of preparing a library of beads coded with five types of lanthanide isotopes at three different concentration levels using M(DTPA-VBAm2) as the copolymerizable metal complex in the bead synthesis. The maximum variation of this bead library, as shown in Figure 6, according to Equation 1, is 1023.

[0207] Compared to the method previously used in Liu 2020 for the synthesis of seven-element beads, the incorporation efficiency across different metals was shown to be more consistent. In Liu 2020, the incorporation efficiency varied dramatically between different metals, making controlled labeling of microbeads difficult. For example, in Liu 2020, Lu 3+ has the highest incorporation of about 50% among all the seven elements, while Ce 3+ had the least efficiency. Ce added to the reaction mixture 3+ Only about 15% of the lanthanides were incorporated into the beads. The incorporation efficiencies of the lanthanides generally showed a trend with smaller ions having greater incorporation efficiencies.

[0208] As shown herein, metal complexes of structure M(DTPA-VBAm2) efficiently copolymerize with styrene in a two-step dispersion polymerization reaction in ethanol in the presence of polyvinylpyrrolidone. This reaction results in PS microbeads with diameters on the order of 2 μm and a very narrow particle size distribution (CV ≈ 1-2%). The incorporation efficiency of the metal complexes is on the order of 60-70%, with this range increasing slightly for lanthanide metal ions with larger ionic radii. This efficiency appears to be independent of the amount of metal complex introduced in the reaction, in the range of concentrations tested. This feature of the reaction makes it possible to tune the specific metal content for a sample of PS microbeads. This is a useful strategy for the preparation of both MC calibration beads and classifier beads for bead-based MC assays, as described, for example, in Example 6. Eu-labeled beads with goat anti-mouse antibodies bound to their surface were effective in the detection of Lu-labeled mouse IgG.

[0209] Example 5 Metal leakage from microbeads The stability of the microbeads prepared according to Examples 1 and 2 with respect to leaching of metal ions into aqueous media was evaluated under several experimental conditions. Sample 4E3 beads prepared by two-step DisP with M(DTPA-VBAm2) were used for this test. Samples of washed microbeads (0.5% solids) were suspended in 30 mL of three different buffers: sodium acetate (50 mM, pH 3.0), ammonium acetate (10 mM, pH 7.0), and sodium carbonate (200 mM, pH 10.5), also containing 1% PVP. The samples were stored at 4°C. At various time intervals, each sample was first vortexed and then 2 mL aliquots were taken. These aliquots were centrifuged and the supernatants were collected to measure the metal content by ICP-MS. The percentage of metal leached from the beads was measured by ICP-MS based on the metal content in the supernatant compared to the metal content in the bead dispersion. In parallel, a batch of microbeads prepared by the two-step DisP AA method was also subjected to the same experimental conditions.

[0210] The stability of a batch of M(DTPA-VBAm2)-encoded beads (4E3) against metal leaching during storage and typical application conditions was tested. These DTPA beads were stable against metal ion loss (<1.5%) upon storage in buffers with pH values ​​ranging from 3 to 10, as depicted in FIG. 13.

[0211] Leaching stability of incorporated metals Metal-encoded microbeads used in MC must be stable against metal ion leaching during storage and under typical application conditions. Samples of these microbeads at 0.5 wt% were dispersed in three aqueous buffer solutions (sodium acetate, pH 3.0; ammonium acetate, pH 7.0; sodium carbonate, pH 10.5) and stored at 4°C. In parallel, another set of microbead samples was tested in the same buffers, but these beads were stored at 4°C as described by Abdelrahman et al., 2009. 9The 4E3 beads were prepared by a two-step DisP process using acrylic acid (AA) as a ligand for incorporating metal ions as reported in. The leakage of each element from the 4E3 beads was monitored in these solutions as a function of time by ICP-MS. Metal leaching from M(DTPA-VBAm2)-encoded microbeads stored in unbuffered water in the presence of 1 wt % PVP was also monitored.

[0212] FIG. 13 shows the results of the metal leaching experiment, where filled symbols refer to DTPA beads (4E3) and open symbols refer to AA beads as a comparison. Very little detectable leakage (<0.06%) of any incorporated elements was observed from both microbeads in pH 7 buffer over a period of more than 100 days, as depicted in FIG. 13B. In the case of 4E3 DTPA beads stored in PVP solution at 4° C., negligible release (up to 0.3%) of metal elements was detected over 100 days, while AA beads released slightly more metal ions (Ce) when stored under the same conditions. 3+ Microbeads aged in acidic (pH 3) and basic (pH 10.5) buffers showed higher levels of ion loss, where elemental ion leakage reached a plateau value at 20 days with no further loss over the next 80 days. Eu from DTPA beads 3+ , Ho 3+ and Lu 3+ The loss of Ce was small (<0.2%) in pH 3 buffer, whereas it was 3+ (about 1.5%), probably Ce 3+ This is likely due to the weaker bond stability between the DTPA beads and the DTPA chelator. Overall, the DTPA beads exhibit less metal ion leakage and stronger stability under the four test conditions. [Table 4]

[0213] Example 6 Classifier Beads Surface modification and antibody conjugation M(DTPA-VBAm2)-encoded beads functionalized with a goat anti-mouse (GAM) secondary antibody (Ab) as described in Example 3 were then subjected to MC. 175 Lu-labeled Ab reporter was used to investigate the specific binding between GAM-modified Eu-1 microbeads.

[0214] Testing specific binding between classifier beads and reporters by mass cytometry For specific binding experiments, GAM-modified microbeads (Eu-1 / GAM) as classifiers were first dispersed in BSA blocking solution (50 μL, 3% BSA in PBS). After 30 min of incubation, 175 Cell staining solution (50 μL) containing Lu-labeled mouse IgG (6.25 μg) was introduced into the Eu-1 / GAM dispersion. The classifier and reporter dispersion was incubated for 2 h, followed by two cycles of centrifugation washing with cell acquisition solution (100 μL) to remove unbound reporter. The binding of reporter onto the classifier beads was examined by MC. To test nonspecific binding, NAv-modified Eu-1 beads (without GAM binding) were employed as negative control in the same binding experimental conditions and also tested by MC.

[0215] Figure 14 shows the strategy used to functionalize the surface of a microbead sample and then attach Abs to the microbead surface. The microbeads were first coated with a thin silica shell as described in Example 3, followed by Abdelrahman et al. 33Amino groups were introduced by a two-step silica sol-gel reaction as reported by. TEOS and APTES were employed in the silica coating method. NH2-modified Eu-1 microbeads with a silica shell of about 10 nm thickness and surface amino groups were obtained. The amino groups on the microbead surface were then converted to carboxyl groups (COOH) by reacting with succinic anhydride in DMSO. The carboxylate groups serve as functional groups for attaching bioaffinity drugs and help to reduce nonspecific binding of reporters to the microbead surface. NeutrAvidin (NAv) was covalently conjugated to the COOH groups on the microbead surface by EDC / NHS coupling. Finally, biotinylated GAM was attached to the NAv-modified microbead surface by strong biotin-avidin affinity.

[0216] To validate the functionality of GAM-modified Eu-1 microbeads as classifiers and as reporters 175 Lu-labeled mouse IgG was selected and incubated with Eu-1 / GAM beads. As a secondary Ab, GAM has a specific high reactivity to mouse IgG in the reporter, as shown in Figure 7(a). To test the antigen recognition, a suspension of GAM-modified microbeads (Eu-1 / GAM) was first dispersed in a bovine serum albumin-containing blocking solution (50 μL, 3% BSA in PBS). After 30 min of incubation, the reporter 175 Cell staining solution (50 μL) containing Lu-labeled mouse IgG (6.25 μg) was introduced and incubated at 23° C. for 2 h, followed by two cycles of centrifugation washing with cell acquisition solution (100 μL) to remove unbound reporter. The samples were examined by MC. As shown in FIG. 7B, a sharp chromatin staining was observed on Eu-1 / GAM microbeads with an intensity of 460 counts / bead (RCV=15%). 175 The Lu signal peak indicates strong specific binding between the GAM-modified microbeads and the reporter. In contrast, the signal obtained for the sample of Eu-1 / NAv microbeads without GAM binding gave a statistically minimized signal to zero.

[0217] Example 7 Ce / DTPA-encoded microbeads using methacrylamide derivative Ce(DTPA-AmPMAm2) To prepare methacrylamide-functionalized DTPA chelator, N-(3-aminopropyl)methacrylamide hydrochloride (APMAm) (2 mmol) was dissolved in anhydrous DMSO (5.0 mL) followed by the addition of triethylamine (TEA, 5.5 mmol) to deprotonate the amine hydrochloride. DTPA dianhydride (1 mmol) was then introduced to the APMAm-DMSO solution and the reaction was stirred at room temperature overnight. NaOH (1 M, 3 equiv.) in ethanol was added to the reaction to form the trisodium salt of DTPA-AmPMAm2 followed by filtration through a syringe filter (0.2 μm). The filtrate was diluted with 45 mL of acetone to precipitate the product. The precipitated DTPA salt was then collected by precipitation and dissolved in ethanol. Three cycles of dissolution-precipitation-precipitation were performed to purify the product. The product was dried overnight at room temperature under reduced pressure to remove residual solvent. 1 The structure of the product was confirmed using H-NMR.

[0218] To prepare the Ce complex of DTPA-AmPMAm2, DTPA-AmPMAm2 (0.3 mmol) and CeCl3·7H2O (0.3 mmol) were dissolved separately in 5 mL and 1 mL of DI·H2O, respectively. The Ce solution was then added to the DTPA-AmPMAm2 solution while the pH was monitored and adjusted to 6.0 with 0.01 M NaOH solution. The mixture was stirred at room temperature for 3 h. The Ce(DTPA-AmPMAm2) complex was isolated by precipitation and sedimentation with acetone. The precipitate was then dissolved in ethanol and precipitated a second time with acetone for further purification. The final product was also 1 It was characterized by H-NMR.

[0219] Polystyrene (PS) microbeads coded with Ce(DTPA-AmPMAm2) complex were prepared using a typical two-stage dispersion polymerization. PVP-55 (1.00 g), AIBN (0.25 g), TritonX305 (0.35 g) were added to a flask and completely dissolved in a mixture of styrene (6.25 g) and ethanol (18.75 g). The solution was sealed in the flask and stirred with an overhead mixer. After 30 min of nitrogen purging at room temperature, the polymerization reaction was initiated by immersing the flask in a water bath at 70°C. As a second-stage aliquot, a warm solution of Ce(DTPA-AmPMAm2) complex (54 mg, ca. 70 μmol) in ethanol (15.0 g) was injected into the reaction system 2 h after the initiation of the reaction. The reaction was stopped 24 h after initiation. A stable agglomerate-free microbead dispersion was obtained and stored at 4°C.

[0220] The particles obtained in this example were free of agglomerates, had a narrow size distribution (CV=1.3%) and a mean diameter of 2.9 μm. The Ce content in the microbeads, measured by mass cytometry, was approximately 2.7×10 7 ions / bead. As assessed by ICP-MS, 49% of the Ce added in the reaction remained in solution after the reaction was stopped. Therefore, the incorporation efficiency of the Ce(DTPA-AmPMAm2) complex in the microbeads was estimated to be 51%.

[0221] Similar methods may be used for other Ln ions. Scheme 2. Synthesis of DTPA derivative-metal complexes for two-step dispersion polymerization. The metal chelator DTPA was functionalized by reacting DTPA anhydride with amine-containing functional molecules in DMSO. Different types of metal ions were loaded onto the DTPA derivatives at pH 5–6. [ka] M=Y, Ce, Eu, Ho and Ln ions such as Lu

[0222] Example 8 Microbead-nucleic acid bioconjugation It is understood that nucleic acids or oligonucleotides can be conjugated to microbeads using methods available in the art. For example, 3'-hydroxyl groups can be conjugated to carboxylic acid (such as succinic acid) functional groups on functionalized microbeads. For example, phosphoramidite derivatives of nucleotides can be used for conjugation. Oligonucleotides can be functionalized for conjugation (e.g., to microbeads or biomolecules described herein) and are readily available commercially, for example, from Integrated DNA Technologies (IDT). For example, oligonucleotides functionalized with biotin (e.g., desthiobiotin), amines, alkyne modifiers, thiol modifiers, Acrydite, or NHS esters such as azide can be conjugated as further described herein.

[0223] Example 9 Multiplexed bead-based assay with MC experiment material The synthesis and characterization of the metal-encoded microbeads employed in this example are shown in Example 10. Buffers used in this example were purchased from Life Technologies. Antibodies (Abs), biotinylated Abs, and cytokine standards were purchased from BioLegend and R&D Systems (see Table 5). Streptavidin-conjugated gold nanoparticles (AuNPs, 10 nm, 10 OD) were purchased from Abcam. Frozen human peripheral blood mononuclear cells (PBMCs) were purchased from Immunospot (CTL, LP-188 HHU20130715). NanoGold®-streptavidin (Nanoprobe), EQ™ 4-element calibration beads (EQ4) and Cell-ID™ Pd barcoding kit were kindly provided by Fluidigm Canada. [Table 5]

[0224] Preparation of a series of metal-encoded classifier beads To develop a bead-based assay with MC, a library of microbeads labeled with various metal ions that can be individually identified by MC was prepared. A method for encoding microbeads with controlled levels of metal ions by introducing polymerizable metal complexes of structure M(DTPA-VBAm2) in a two-step dispersion polymerization (DisP) reaction is described herein. Experimental details of the formulation and processing of metal-coded classifier beads are found in Example 10. A panel of eleven bimetallic metal-coded 3 μm polystyrene (PS) microbeads was prepared employing the method of the present disclosure described in Example 10.

[0225] To prepare classifier beads capable of capturing target analytes in an immunoassay, each type of metal-encoded bead was modified with one of the Abs specific for the cytokine analyte.

[0226] Stimulation of PBMCs for cytokine secretion A frozen commercial human PBMC sample (CTL Immunospot) was thawed and added to pre-warmed RPMI serum-free medium (10 mL) containing 0.5 mL of CTL anti-agglutination washing supplement (20x). The PBMCs in the sample were spun down by centrifugation (8 min, 300 rpm). The supernatant was aspirated after centrifugation. The PBMCs were then resuspended in warm serum-containing complete RPMI (10 mL). RPMI (4.8 mL, 4.8x10 6An aliquot of this PBMC suspension in RPMI medium (100 μL) was transferred to a PS tube and stimulated with 100 μL of PBS solution containing phorbol myristate acetate (PMA, 25 nmol) and ionomycin (100 nmol). Samples containing stimulants were incubated for 5 hours at 37°C under 5% CO2. The stimulated PBMC suspension was then centrifuged to pellet the cells. The supernatant was collected as stimulated sample and stored at -80°C prior to the bead-based assay. Another aliquot of the PBMC suspension in RPMI medium was treated in parallel under the same conditions except for the addition of stimulants (PBS only). A non-stimulated sample was collected as a control in this experiment.

[0227] Bead-based sandwich immunoassay for mass cytometry. To optimize the assay conditions, several multiplexed bead-based assays were performed using a series of solutions containing mixtures of cytokines and chemokines of known concentrations as standards. These mixtures were analyzed by MC and standard curves were plotted. Figure 16 shows the assay procedure developed in this study.

[0228] A series of 4-plex assays were performed to analyze IL-4, IL-6, TNFα, and IFNγ in 10 standard samples. In these experiments, the four capture Ab-coated metal-coded classifier microbeads were first mixed in approximately equal numbers in a BSA solution (0.5% BSA in PBS). The classifier bead dispersion was then transferred to 10 wells of a 96-well filter plate (filter cutoff: 0.45 μm). Each well, containing approximately 2 million beads in the dispersion (50 μL), can analyze one standard sample. A series of 10 standard solutions (50 μL each) consisting of 0, 0.31, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000, 20000 pg / mL of the four analytes, respectively, were added to each well. The mixture of standard solution and classifier beads in each well was first agitated with a pipette and then incubated at room temperature on a microplate shaker (1200 rpm for 30 seconds, then 900 rpm for 2 hours). After incubation, the solution in the mixture was removed by vacuum filtration through the built-in filter at the bottom of the well. The classifier beads with the captured analytes on their surface were washed by two redispersion-filtration cycles with wash buffer (200 μL, 0.025% Tween® 20 in PBS) to remove any non-captured analytes that may be present. Once the wash buffer was removed from the wells by filtration, a detection Ab cocktail (100 μL, 0.5% BSA in PBS) containing four biotinylated Abs (2.5 μg / mL for each Ab) was transferred to each well. The classifier beads were redispersed with the detection Abs by agitation with a pipette. The mixture of classifier beads and detection Ab was incubated for 1 h at room temperature on a microplate shaker (900 rpm). After incubation, the mixture was filtered and washed with two redispersion-filtration cycles with wash buffer to remove unbound detection Ab on the classifier beads. The classifier beads on the filter were then redispersed with a dispersion (100 μL) of streptavidin-conjugated gold nanoparticles (AuNPs) as reporters. The reporter dispersion was prepared by diluting (e.g., 200-fold) the AuNP dispersion from the supplier with 0.5% BSA buffer.The mixture of classifier beads and AuNP reporters was incubated for 1 h on a shaker (900 rpm) and washed by two filtration-redispersion cycles with washing solution and two with PBS buffer (100 μL). To measure multiple assays in one MC experiment, the assay samples in each well (100 μL) were stained with a unique palladium barcoding solution (40 μL, 3x dilution of the stock solution in the Cell-ID™ Pd barcoding kit). The barcoding staining reaction was incubated for 30 min with stirring and quenched by two filtration-redispersion cycles with 0.5% BSA solution (200 μL) and two with water (100 μL). A total of 10 barcoded assay samples were mixed in one test tube and tested by MC using EQ4 beads as calibration standards.

[0229] In a typical multiplex assay of samples with unknown analyte concentrations, different types of metal-encoded classifier beads capable of capturing target analytes were first mixed in equal numbers in BSA solution and then transferred to a 96-well filter plate. In each well, the classifier bead dispersion (50 μL) can analyze one unknown sample by adding an aliquot (50 μL) of sample solution to the well. The assay was then incubated, washed, and stained with detection Ab and detection AuNP reporter in a similar procedure to that described in the above assay of standard samples.

[0230] Measurement equipment Mass Cytometry (MC). Microbead samples were characterized bead by bead by mass cytometry (Helios™ CyTOF® system, Fluidigm). In a typical MC measurement, barcoded immunoassay samples and EQ4 beads as internal standards were pooled in a test tube and introduced into the MC system at a rate of 30 μL / min. After signal acquisition, the MC signals were normalized with the signal from the EQ4 beads and de-barcoded to separate the results from different assays. To analyze the MC results of one assay sample, the singlets of all classifier beads included in the assay were first identified,140 Ce- 142 The results were gated on a Ce dot plot. Each type of classifier bead was then gated based on their metal-encoded signature signal. 197 Median Au signal intensity is reported as the result.

[0231] result A multiplexed bead-based immunoassay was developed at MC. Metal-coded microbeads were employed as the solid support for the immunoassay, and gold (Au) NPs as the reporter. The capture Ab of each immunoassay was bound to one of the microbead sets, each of which was composed of microbeads with uniform and distinct heavy metal isotope content. Once bound to the capture Ab, the microbeads from different sets as classifiers could be pooled together for a multiplexed assay and separated after data acquisition. The data was acquired at MC and analyzed by FlowJo™ software (see Figure 16 for experimental design). The immunoassay utilized the variability of Au NP reporters, which is proportional to the amount of analyte bound to the surface of each microbead. 197 Since the MC quantifies the amount of different heavy metal isotopes in each microbead, the pool of microbeads yields the Au signal intensity of the NP reporter for each bead set. 197 The Au signal can be separated into individual bead sets with median Au signal intensity. Due to this feature, many assays can be performed simultaneously, allowing multiplex quantification of multiple analytes in a single measurement. In addition, the concentration of an analyte in a sample can be determined by extrapolation from an internal standard.

[0232] Synthesis of metal-encoded classifier beads by dispersion polymerization. The synthesis was carried out using five types of lanthanide metal ions (La 3+ ,Pr 3+ , Tb 3+ , Ho 3+ , and Tm 3+32 types (2 5 = 32) binary coded classifier microbeads were assumed. In addition, Ce 3+ The ions can be incorporated into each bead at similar levels. 140 Ce and 142 Ce can serve as a microbead identifier for convenient gating of classifier signals of MC results. As shown, a series of eleven bimetallic encoded 3 μm polystyrene (PS) microbeads for bead-based assays were synthesized by a series of two-step dispersion polymerization (DisP) according to the bead synthesis protocol described in Example 4 herein.

[0233] Bead synthesis and characterization are described in Example 10. A scanning electron microscope image of bead sample C-1 (see Table 6) is shown in panel (a) of FIG. 22. It shows that these microbeads are uniform, with an average diameter of 3.0 μm and a CV of 1%. The median intensity levels of the MC signals of the coded isotopes ranged from 1000 to 1200 counts / bead. The table provides a summary of the beads prepared, their average diameters (and CV values) as well as the labeling patterns and signal intensities in MC detection. These 11 types of microbeads are uniform (CV approx. 1%) and share a small size with an average diameter ranging from 2.8 to 3.0 μm. For antibody binding, the beads were coated with antibodies and the Abs attached to each classifier bead are listed in the table. [Table 6]

[0234] In addition, the incorporated metal ions in these microbeads generated MC signals at similar intensity levels of 800-1200 counts / bead. All microbeads (C1-C11) generated Ce signals at similar levels in MC. 3+ Since the mixture of all beads was analyzed in one MC measurement, 140 Ce- 142One gating separated 11 types of microbead singlets from the Ce dot plot. The dot plots in Figure 17(bk) show the gating strategy to individually identify each type of classifier microbead based on their signature signal of metal encoding. Since the microbeads in this set of classifier beads were uniform with MC signal intensity at similar levels, a gating template was created in FlowJo software based on this microbead gating strategy to simplify the data analysis process of the multiplex assay.

[0235] Initial optimization of bead-based sandwich immunoassay conditions To test and optimize the reagents and conditions used in the multiplexed sandwich immunoassay method, these immunoassays were used to measure cytokine levels in a series of solutions with known cytokine concentrations as standard solutions. The effect of different assay reagents and conditions on the analytical results was evaluated by examining calibration curves from these measurements. In the first stage of assay development, a series of 4-plex assays of standard solutions were used to test a candidate reporter, which was a commercially available streptavidin-conjugated nanosized Au reporter, and optimize the reporter concentration in the assay. The assay was then expanded to a 9-plex assay to investigate the effect of detection Ab concentration on the assay performance.

[0236] In a representative experiment, a series of solutions containing known concentrations of target cytokines were prepared by diluting the standard solutions with 0.5% BSA buffer (0.5% BSA in PBS). Each of these standard solutions (50 μL) was mixed with Ab-modified classifier beads (50 μL) in a filter plate. During incubation, the Ab-modified classifier beads in the assay were allowed to capture their target analytes in the sample. After 1 h of incubation, the classifier beads were washed by filtration to remove unbound molecules in the sample. A cocktail of biotinylated detection Abs (100 μL) was then added to the assay, followed by redispersion of the classifier beads. In this step, the biotinylated Abs in the cocktail can recognize the analyte molecules captured on the classifier bead surface. The classifier beads in the assay were then washed on the filter to remove unbound detection Abs. A streptavidin-conjugated Au mass tag dispersion (100 μL) was then applied to the assay as a reporter. These mass tags could be attached to biotinylated detection Abs on classifier beads via streptavidin-biotin interactions. 47 After washing away unbound reporter particles, the assay samples were examined by MC for metal content in individual microbead events. A representative design of this bead-based sandwich immunoassay is shown in FIG.

[0237] After signal acquisition, the analytical results were analyzed with FlowJo software through their dot plot diagrams. Figure 17 shows the gating strategy adopted in this study. Singlet events of all classifier beads were first separated in panel (a) of Figure 17. Each classifier bead in the gated singlet events was then individually identified through a series of gating steps in the dot plot diagram shown in panel (bk) of Figure 17. 197 The Au signal was then examined and reported as the assay signal.

[0238] Selection of reporters. To find suitable metal-labeled mass tags for reporting analyte binding on classifier beads, two types of commercially available streptavidin-conjugated reporters were investigated: small diameter gold clusters (NanoGold®, d≈1.4 nm) and somewhat larger diameter gold nanoparticles (AuNPs, d≈10 nm). These were employed as reporters in a series of 4-plex assays analyzing standard solutions of 12 concentrations of 4 analytes.

[0239] The histogram shown in Figure 18 shows the signal intensity of the reporter on the IL-4-classifier in a 4-plex assay. AuNPs were employed as reporters in the assay shown in Figure 18(ac). 197 The Au signal was recorded in Figure 18(a) where no IL-4 was present in the blank control solution. The median value of all events in this plot was reported as a background noise control to reflect non-specific binding of reporter NPs to the classifier beads. 197 The intensity peak of the Au signal shifted upfield in panels (b) and (c) of FIG. 18 as the IL-4 concentration increased from 1.2 to 20 pg / mL. At 1.2 pg / mL, there were nearly 0 counts per bead. 197 Some events with Au signal intensity were recorded in Figure 18(b). These "0" events may be instrument noise due to artifacts during the signal acquisition process or due to some classifier beads carrying a small number of reporters that were below the MC detection limit. To remove uncertainties and simplify the data analysis process, the median intensity of positive events (reporter signal intensity > 1 count / bead) in positive samples was reported and plotted in this study.

[0240] In the histograms shown in panels (d), (e), and (f) of FIG. 18, standard solutions containing IL-4 at concentrations of 0, 1.2, and 20 pg / mL, respectively, were analyzed employing NanoGold® as a reporter.197 A much weaker intensity of the reporter signal for Au was observed compared to the results of the analysis using AuNPs as reporter: Many more events with a signal intensity of about 0 were shown in the histogram shown in panel (e) of Figure 18 compared to panel (b).

[0241] A typical log-log calibration curve for a bead-based assay begins with a low, flat region at low analyte concentrations. The curve then rises with increasing analyte concentration, followed by a plateau at higher concentrations. 41、43、48 In this example, the detection range of the assay was estimated based on the slope region of the calibration curve, within which the analyte concentration is typically measurable.

[0242] Figure 19 is a summary of the median MC signal intensities of different types of NPs attached to classifier beads at different analyte concentrations. In the low concentration region (up to about 1 pg / mL), the signal intensities of both types of reporters were low (about 10 counts / bead) and insensitive to increasing analyte concentrations. At higher analyte concentrations, ranging from 1 to 1000 pg / mL, the signal intensities of these reporters increased significantly with increasing analyte concentrations. The signal intensity of AuNPs approached a plateau when the analyte concentration exceeded 1000 pg / mL, while the curves of the assays using NanoGold® as reporters showed a similar trend, but with much lower intensities compared to those using AuNPs as reporters. Since much higher intensities were obtained at lower analyte concentrations in these assays, AuNPs were selected as the reporter candidate for the next bead-based assay in this study to optimize the lower detection limit.

[0243] Optimization of reporter concentration. The effect of NP concentration on the assay signal intensity level was next investigated. In this study, the stock AuNP dispersion was diluted 200, 400 and 800 times with 0.5% BSA buffer. These dilutions were then used as reporter solutions in three sets of 4-plex assays to analyze the standard solutions. Figure 23 shows the calibration curves of the 4-plex assays performed under different AuNP concentrations. The signal intensities of AuNPs at all three dilutions showed minimal differences at analyte concentrations above 20 pg / mL. The detection limits of the assays stained with 200x diluted AuNP dispersions were as low as 0.3 pg / mL for IL-4, IL-6 and TNFα, while the detection limit for IFNγ was slightly higher at 1.2 pg / mL. The 200x AuNP dilution was selected for use in the bead-based assays in this study.

[0244] Optimization of detection Ab concentration (in 9-plex assays). After using a series of 4-plex assays as proof-of-concept experiments, the multiplexing capacity of the assay was extended to nine analytes by adding the analysis of IL-1β, IL-10, IL-18, CD163, and CXCL-9 to the panel. The experimental conditions developed for the 4-plex assays were adapted to the analysis of a series of standard solutions containing nine analytes. In the first set of 9-plex assays, the concentration of each type of biotinylated Ab in the detection Ab cocktail was 2.5 μg / mL. The results are plotted as filled circles (●) in Figure 20. The median intensity levels of AuNP signals on all types of classifier beads at low analyte concentrations (≦1.22 pg / mL) were observed to be higher (approximately 100 counts / bead) compared to the 4-plex assays. Without intending to be bound by theory, these relatively higher levels of signal intensity at low analyte concentrations may be due to background noise in the assay, likely the result of non-specific binding.

[0245] To minimize non-specific binding in the 9-plex assays, three sets of 9-plex assays were performed in which the concentrations of biotinylated anti-CD163 and anti-CXCL-9 were reduced to 2.0, 1.0, and 0.5 μg / mL, while the concentrations of other detection Abs were maintained at 2.5 μg / mL in the detection Ab cocktail. The concentrations of detection anti-CD163 and anti-CXCL-9 were reduced in these assays, in part because possible cross-reactivity and interference of detection Abs against CXCL-9 and CD163 with interleukins (ILs) at high Ab concentrations may result in more non-specific binding. The calibration curves of these 9-plex assays are plotted in FIG. 20. For assays of standard solutions containing low concentrations of analyte (≦1.22 pg / mL), the median signal intensities in assays using low concentrations of biotinylated anti-CD163 and anti-CXCL-9 Abs (0.5 and 1.0 μg / mL) were significantly lower than those obtained using higher concentrations of detection Abs (2.0 and 2.5 μg / mL). For assays of standard solutions with high analyte concentrations (≧1250 pg / mL), the signal intensity levels and assay sensitivity were similar under all experimental conditions. Overall, the detection sensitivity at low analyte concentrations and the detection range for the analytes were improved when the concentrations of biotinylated anti-CD163 and anti-CXCL-9 in the detection Ab cocktail were reduced to 0.5 and 1.0 μg / mL. As shown in FIG. 24, the background noise in these 9-plex assays of blank solutions was also significantly reduced when the concentrations of biotinylated anti-CD163 and anti-CXCL-9 Abs were reduced from 2.0 to 0.5 μg / mL. Based on these results, it appears that nonspecific binding of biotinylated anti-CD163 and anti-CXCL-9 to classifier beads can be minimized by reducing their detection Ab concentrations in the assay.

[0246] Bead-based sandwich immunoassay for cytokines in biological samples To investigate the performance of the 9-plex assay on biological samples, a commercially available PBMC sample (from a healthy donor) was used. One sample of PBMC suspended in cell culture medium (RPMI) was stimulated with PMA / ionomycin and analyzed for extracellular release of cytokines in the supernatant of the stimulated PBMC suspension by the 9-plex assay using the optimized assay conditions described above. PMA has a structure similar to diacylglycerol and can diffuse through the cell membrane into the cytoplasm. In the cytoplasm, PMA activates protein kinase C. When used in combination with ionomycin, a calcium ionophore that induces calcium release, moderate levels of cytokines are released from the cells. Unstimulated samples were collected from the supernatant of unstimulated PBMC suspension and analyzed as a control in this experiment. Prior to the assay, stimulated and unstimulated samples were diluted 2x, 4x, 16x, 64x and 256x in the assay to vary the analyte concentration during measurement so that some of the assays produced MC intensity values ​​within the range of the calibration curve. In these assays, AuNPs attached to classifier beads 197 Median Au signal intensities are shown in Figure 21. In a typical assay, sample solution (50 μL) was added to classifier dispersion (50 μL) either undiluted or diluted at 1:2, 1:8, 1:32 or 1:128 ratios. The detection Ab concentrations of anti-CD163 and anti-CXCL-9 in this assay were 0.5 μg / mL and 1.0 μg / mL, while the concentration of other detection Abs was 2.5 μg / mL in the cocktail. The signal was significantly higher than that from unstimulated samples. 197 Au signals were detected from the IL-4, IFNγ and TNFα classifier beads in assays of stimulated samples at all dilutions. This result is consistent with the fact that the stimulation process can lead to increased release of IL-4, IFNγ and TNFα by PBMCs and is consistent with the results of Ai et al. 18 This is generally consistent with findings reported by others regarding use stimulated by a similar process.

[0247] Using the same assay conditions, a series of standard solutions containing nine target cytokines with known concentrations were measured. A series of calibration curves were then generated by fitting the assay results of the standard solutions with a four-parameter logistic regression (4P-LR) model (see FIG. 25). These calibration curves were used to evaluate the concentrations of these cytokines in unstimulated and stimulated samples. However, some assay results in FIG. 21 were smaller than the minimum value of the calibration curve shown in FIG. 25. Thus, concentrations were not derived from these values. FIG. 26 summarizes the preliminary findings of cytokine concentrations by fitting the analytical results shown in FIG. 21 to the calibration curve shown in FIG. 25. Since the dilution factor was taken into account in the calculation of cytokine concentrations shown in FIG. 26, theoretically, similar concentration values ​​for the same measured samples should be observed from different dilutions. For the measurement of IL-4 concentrations in stimulated samples, samples diluted at 1:1, 1:2, 1:8, 1:32 and 1:128 ratios reported IL-4 concentrations of 600, 430, 460, 640 and 1110 pg / mL in the stock solutions of stimulated samples, respectively, while for unstimulated samples, none of the assays could generate sufficient signal intensity to assess IL-4 concentrations, probably due to the low IL-4 concentrations in unstimulated samples. For the measurement of IFNγ concentrations in stimulated samples, all the assays at different dilutions reported extremely high signal intensities, which were higher than the intensities of most concentrated standard solutions.

[0248] conclusion A series of eleven types of lanthanide-encoded microbeads were synthesized by two-step DisP. The metal content of six metals in these microbeads was precisely controlled by varying the amount of metal complex fed in the second step of DisP to generate microbeads that generated MC signals with a median intensity of approximately 1000 counts / bead. These microbeads were characterized by size (CV 直径 <2%) and uniform in metal content (RCV<15%), which makes them good candidates for classifier beads in bead-based assays.

[0249] Cytokine levels were analyzed with this bead-based assay with MC. To develop a multiplexed bead-based sandwich immunoassay method for cytokines and chemokines with MC, the surface of metal-encoded microbeads was modified with various types of Abs and employed as classifier beads to capture target analytes. To develop the reporter system and optimize the assay conditions, a series of bead-based assays were first performed to analyze a series of standard solutions containing up to nine types of cytokines and chemokines with known analyte concentrations. In this experiment, the MC signal intensity of the reporter NPs responded to the concentration difference of the standard solutions. These assays showed high sensitivity at low analyte concentrations. However, non-specific binding of some detection Abs resulted in increased background noise problems. Background noise may impair the detection limit of these assays. In addition, the supernatants of two kinds of PBMC samples were analyzed with the 9-plex assay developed in this study for cytokines and chemokines. The analysis results showed that the supernatants of PMA / ionomycin-stimulated samples contained higher levels of IL-4, IFNγ, and TNFα compared to non-stimulated samples. This finding was generally consistent with results reported in the literature for cytokine detection measured by other assays. The results presented in this study indicate that the bead-based sandwich immunoassay method with MC can be used for the simultaneous detection of different analytes.

[0250] Example 10: Additional Experimental Details and Discussion of Example 9 material Styrene (St, Sigma-Aldrich, ≥99%), polyvinylpyrrolidone (PVP, Mw approx. 55 kDa), 2,2-azobis(2-methylpropionitrile) (AIBN, 98%), Triton X305 (TX305, 70% aqueous solution), diethylenetriaminepentaacetic acid dianhydride (DTPA acid dianhydride, 98%), phorbol 12-myristate 13-acetate (PMA), ionomycin, hydrogen peroxide solution (H2O2, 30% in H2O), sulfuric acid (trace metals grade), and metal salts with purity ≥ 99.99% (trace metals standard) including lanthanum(III) chloride heptahydrate (LaCl3·7H2O), cerium(III) chloride heptahydrate (CeCl3·7H2O,), praseodymium(III) acetate hydrate (Pr(OAc)3·xH2O), terbium(III) chloride hexahydrate (TbCl3·6H2O), holmium(III) chloride hexahydrate (HoCl3·6H2O), and thulium(III) chloride hexahydrate (TmCl3·6H2O) were purchased from Millipore-Sigma. 4-Vinylbenzylamine (VBA, ≥ 92%) was purchased from TCI America. Absolute ethanol (EtOH) was prepared from commercial alcohol (Mississauga, Ontario). Single element standard solutions for inductively coupled plasma mass spectrometry (ICP-MS) calibration were purchased from PerkinElmer (Pure Plus).

[0251] Preparation of metal-encoded classifier beads Metal complexes of DTPA bisvinylbenzylamide (DTPA-VBAm2) were employed as chelating agents to incorporate different types of metal ions into polystyrene (PS) microbeads. The synthesis and metal loading procedures of the polymerizable DTPA-VBAm2 metal chelator are described in Example 4. To encode La, Ce, Pr, Tb and Tm into the microbeads, La(DTPA-VBAm2), Ce(DTPA-VBAm2), Pr(DTPA-VBAm2), Tb(DTPA-VBAm2) and Tm(DTPA-VBAm2) were prepared in aqueous solution by mixing DTPA-VBAm2 with LaCl3, CeCl3, Pr(OAc)3, TbCl3 and TmCl3, respectively. These metal complexes were 1 Characterized by 1 H NMR.

[0252] Microbeads as classifier beads for bead-based assays were synthesized by a series of two-step dispersion polymerization (DisP) as described in Table 7. In a typical bead synthesis, the first step of polymerization of styrene (6.25 g) in absolute ethanol (18.75 g) was initiated with AIBN (0.25 g) at 70 °C in the presence of PVP (1 g) and TX305 (0.35 g) as stabilizers. The reaction was protected with N2 purge (3 mL / min) controlled by a gas mass controller (OMEGA). Two hours after the start of the reaction, an aliquot (15 g) of warm ethanol containing different types of metal complexes of DTPA-VBAm2 was added to the DisP reaction system to incorporate metal ions into the microbeads. The feeding amount of M (DTPA-VBAm2) in the second step aliquot was optimized to generate metal ion-encoded microbeads that generated a designed level of signal intensity in MC (see Table 8). The reaction was stopped 24 hours after initiation and cooled to room temperature. The microbeads in the reaction dispersion were purified by two precipitation-redispersion cycles with absolute ethanol and four with water. The purified microbead dispersion was used for scanning electron microscope (SEM) imaging, MC measurement and further surface modification.

[0253] The diameter and diameter distribution of the microbeads were characterized from their SEM images using a Hitachi S-5200 microscope. Typically, 2 μL of the diluted bead dispersion was dropped onto a 300 mesh formvar / carbon-coated copper grid and allowed to dry. The diameter of the microbeads was measured manually from multiple SEM images using ImageJ software. The mean diameter, standard deviation (SD) and coefficient of variation (CV) were calculated based on at least 300 measurements.

[0254] In the first two trials, the feeding amounts of metal complexes to the synthesis mixture were investigated to obtain microbeads that could generate MC signals with the desired range of intensities. These microbeads are denoted as Trial 1 and Trial 2. Based on the feeding amount formulations developed in the trial experiments, eleven samples were then prepared as classifier beads. These classifier beads are denoted as C-1, C-2, ...C-11, respectively, and are listed in the table.

[0255] Acid digestion of microbeads After DisP was stopped, an aliquot of the C-1 reaction dispersion was digested with H2SO4 / H2O2 at 250 °C and analyzed by inductively coupled plasma mass spectrometry (ICP-MS) for the total metal content in the reaction system. For the digestion of the microbeads in the reaction dispersion, the microbead dispersion (100 μL, 0.5-2% solid content) and 500 μl of sulfuric acid were mixed in a 20 mL glass vial and heated to 250 °C on a hot plate and held for 40 min with magnetic stirring, followed by the addition of 30% H2O2 solution (50 μL). For ICP-MS analysis, the digestion solution was then diluted with 2% HNO3. To quantify the free metal content in the reaction dispersion, the reaction stock dispersion was filtered through a syringe filter (0.2 μm, nylon) to remove the microbeads, and the filtrate was collected for ICP-MS analysis. The efficiency of metal incorporation during the C-1 reaction was estimated by comparing the total metal content with the free metal content in the reaction mixture, as described above in Example 4.

[0256] Measurement equipment Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). An ICP-MS (iCAP-Q, Thermo Scientific) system was used to quantify the metal content in the samples. Elemental standard solutions were serially diluted with 2% HNO3 to a series of concentrations of 40, 20, 10, 1 and 0.1 ppb as calibration solutions. The metal content in each solution sample was determined based on the calibration fitting curve with a detection limit of less than 10 ppt.

[0257] Additional Results and Discussion Synthesis of metal-encoded classifier beads by dispersion polymerization. In this study, metal-encoded microbeads were prepared by a two-step DisP to incorporate metal ions into PS microbeads using a polymerizable metal complex, M(DTPA-VBAm2). The synthesis of the DTPA-VBAm2 chelator was carried out by reacting DTPA dianhydride with 4-vinylbenzylamine in anhydrous DMSO. La 3+ , Ce 3+ ,Pr 3+ , Tb 3+ , Ho 3+ and Tm 3+ was loaded onto the DTPA-VBAm2 chelator in aqueous solution at pH 5-6. The products of these syntheses were 1 The chelator synthesis and metal loading procedures are described in detail in Example 4.

[0258] To develop a formulation for classifier bead synthesis, the steps were: optimizing the amount of M(DTPA-VBAm2) delivered in the second stage aliquot to produce a classifier bead with intensity levels of 800-1000 counts / bead for each of these isotope channels, in the MC. 139 La, 140 Ce, 141 Pr, 159 Tb, 165 Ho and 1 69The aim of the study was to prepare microbeads that generate signals of Tm. Based on the linear relationship between metal dosage and metal content discussed in Example 4 herein, several trial syntheses were carried out to design dosages of six metal complexes. Based on the dosage formulations developed in the trial experiments, C-1 sample was prepared as a series of classifier beads coded with six types of metal ions.

[0259] In addition, the metal incorporation efficiency of metal ions into C-1 beads was evaluated by ICP-MS. The results shown in panel (b) of FIG. 22 indicate that all six types of metal ions were efficiently incorporated with efficiencies ranging from 63 to 74%. The incorporation efficiency of each metal was evaluated by comparing the post-synthesis metal content with the total metal content in the reaction system. A slight increase in efficiency was observed for lanthanide metal ions with larger ionic radii, which is consistent with the findings described in Example 4 above. Overall, C-1 beads are suitable as candidate classifier beads.

[0260] The size of microbeads prepared by DisP varies from batch to batch, possibly due to the sensitivity of particle nucleation to reaction conditions. To optimize and prepare microbeads with small batch-to-batch particle size variation, several control factors were investigated in the experimental conditions, and it was found that the particle size reproducibility could be improved by keeping the N2 purging and reaction heating procedures constant from batch to batch. As summarized in Table 6, 11 types of microbeads (C-1 to C-11) were prepared that were uniform (CV<2%) and shared a small size with an average diameter ranging from 2.8 to 3.0 μm.

[0261] After formulation optimization, additional types of classifier beads were prepared by adopting the feeding amounts of metal complexes developed for the C-1 synthesis. In the first set of samples C-2-C-6, two types of metal ions, Ce(DTPA-VBAm2)+a second type of metal complex, were added in the bead synthesis. The amount of each metal complex in these bimetallic bead syntheses was the same as in the synthesis of C-1 beads. The same principle was then also applied to several trimetallic encoded microbeads (C-7-C11). [Table 7] [Table 8]

[0262] While the present disclosure has been described with reference to what are presently considered to be the preferred embodiments, it is to be understood that the present disclosure is not limited to the disclosed embodiments, but rather, the present disclosure is intended to cover various modifications and equivalent combinations within the spirit and scope of the appended claims. All publications, patents, and patent applications are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference in its entirety. In particular, the sequences associated with each accession number provided herein, including, for example, the accession numbers and / or biomarker sequences (e.g., proteins and / or nucleic acids) provided in the tables or elsewhere, are incorporated by reference in their entirety. The claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.

[0263] Citations of References Referenced herein [Table 9] TIFF2024511743000028.tif197132TIFF2024511743000029.tif200132TIFF2024511743000030.tif199132TIFF2024511743000031.tif196132TIFF2024511743000032.tif44132

Claims

1. Structural monomers, and Metal chelating monomers containing a metal and a chelating agent A copolymer comprising A metal-encoded microbead comprising: said chelating agent coordinates said metal at at least three sites; said structural monomer does not include said chelating agent; the metal is a plurality of metals; Metal-encoded microbeads, which are one or more of the following: the plurality of metals includes one or more enriched isotopes; the plurality of metals comprises at least two metals, at least three metals, or at least four metals; the amount of each metal in the plurality of metals is within about 20% or about 10% of the amount of another metal in the plurality of metals; and The metal is dispersed throughout the microbeads.

2. 2. The microbead of claim 1, wherein the structural monomer is selected from substituted or unsubstituted styrene, α-methylstyrene, acrylic acid and its esters and amides, methacrylic acid and its esters and amides, and derivatives thereof, optionally wherein the structural monomer is styrene.

3. The metal chelating monomer has the formula I: 【Chemistry 1】 wherein Ligand is said chelating agent, L is a linker, X is a polymerizable end group, M is a metal, and n is an integer equal to or greater than 1, and said metal chelating monomer is charge neutral prior to polymerization; and / or L is selected from a bond, a C3-C8 alkylamine, a C3-C8 alkylene, a C3-C8 cycloalkyl, a C3-C8 heterocycloalkyl, a 5- or 6-membered aryl or heteroaryl, an alkylaryl, an alkylheteroaryl, a C3-C8 cycloalkylaryl, a C3-C8 cycloalkylheteroaryl, C(O), —C(O)O, or a mixture thereof, wherein each of said alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl is independently unsubstituted or substituted with one or more substituents selected from a C1-C6 alkyl, a C1-C6 alkenyl, a C3-C8 cycloalkyl, a C3-C8 heterocycloalkyl, an amide, an ester, an aryl, a heteroaryl, an alkylaryl, an alkylheteroaryl, a C3-C8 cycloalkylaryl, a C3-C8 cycloalkylheteroaryl, CN, or a mixture thereof; 3. The microbead according to claim 1 or 2, wherein optionally the polymerizable end group is selected from allyl vinyl, styrene, α-methyl styrene, acrylate esters, methacrylate esters, acrylamide, 2-methyl acrylamide, and mixtures thereof, and optionally the polymerizable end group is allyl vinyl or styrene.

4. 4. The microbeads of claim 3, which are one or more of the following: L is attached to the chelator via an amide or ester, and the chelating agent is tetradentate, pentadentate, hexadentate, heptadentate, or octadentate, optionally the chelating agent is hexadentate or octadentate; Optionally, the chelating agent comprises an aminopolyacid moiety, or a derivative thereof; optionally, the aminopolyacid moiety is selected from an aminopolycarboxylic acid, an aminopolyphosphonic acid, or a combination thereof; optionally, the aminopolyacid moiety is one or more substituted oligomers of ethyleneimine, propyleneamine, or mixtures thereof, the oligomer being substituted with two or more carboxylic and / or phosphonic acids; optionally, the oligomer is a crown ether or an azacrown ether.

5. the oligomer is further substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof; or The chelating agent is selected from DFO, EDTA, DTPA, EGTA, EDDS, EDDHA, BAPTA, H4neunpa, H6phospa, H4CHXoctapa, H4octapa, H2CHXdedpa, H5decapa, Cy-DTPA, Ph-DTPA, TACN type chelating agent, TACD type chelating agent, cyclen type chelating agent, cyclam type chelating agent, (13)aneN4 type chelating agent, 1,7-diaza-12-crown-4 type chelating agent, 1,10-diaza-18-crown-6 type chelating agent, or derivatives thereof; The microbeads according to claim 4.

6. 6. The microbead of claim 5, wherein the TACN type chelator is selected from NOTA, NOPO, TRAP, or derivatives thereof, the cyclen type chelator is selected from DOTA or derivatives thereof, the cyclam type chelator is selected from TETA, cross-linked TETA, DiAmSar, or derivatives thereof, the (13)aneN4 type chelator is selected from TRITA or derivatives thereof, the 1,10-diaza-18-crown-6 type chelator is selected from MACROPA or derivatives thereof, and / or the chelator is selected from DTPA, Cy-DTPA, Ph-DTPA, or derivatives thereof.

7. The metal chelate monomer is 【Chemistry 2】 【change】 and optionally L is as defined in claim 3, Optionally, the metal chelating monomer is 【Chemistry 3】 4. The microbead of claim 3, wherein the microbead is selected from the group consisting of:

8. the chelator comprises a porphyrin or a phthalocyanine, optionally the chelator is a substituted or unsubstituted porphyrin; and Optionally, prior to polymerization, the metal chelating monomer comprises 【Chemistry 4】 or mixtures thereof, and n is an integer from 1 to 4, optionally n is at least 2; 5. The microbead of claim 4, optionally wherein L is aniline.

9. The method of claim 8, wherein the metal comprises indium, bismuth, or a rare earth metal, optionally wherein the rare earth metal is selected from a lanthanide metal, yttrium, or a mixture thereof; Optionally, the metal comprises a rare earth metal selected from Y, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, individual isotopes thereof, or mixtures thereof; and Optionally, the rare earth metal is 89Y, 139La, 136Ce, 138Ce, 140Ce, 142Ce, 141Pr, 142Nd, 143Nd, 145Nd, 146Nd, 148Nd, 145Pm, 144Sm, 149Sm, 150Sm, 152Sm, 154Sm, 151Eu, 153Eu, 154Gd, 155Gd, 156Gd, 157Gd, 158Gd, 160Gd, 152Gd, 15 2. The microbead of claim 1, selected from 9Tb, 156Dy, 158Dy, 160Dy, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 162Er, 164Er, 166Er, 167Er, 168Er, 170Er, 169Tm, 168Yb, 170Yb, 171Yb, 172Yb, 173Yb, 174Yb, 176Yb, 175Lu, or mixtures thereof.

10. One or more of the following: the microbeads have a glass transition temperature of about or greater than 60°C, optionally about or greater than 70°C, about or greater than 80°C, about or greater than 90°C, about or greater than 100°C, about or greater than 115°C, about or greater than 125°C, or about or greater than 135°C; The microbeads have a diameter of about 0.6 μm to about 20 μm, about 1 μm to about 15 μm, about 2 μm to about 10 μm, about 2 μm to about 6 μm, and the microbeads are colloidally stable in water, or the surface of the microbead comprises functionalization for attachment to biomolecules; Optionally one or more of the following: The attachment may be a covalent or non-covalent attachment; the surface of the microbeads is functionalized with avidin, streptavidin, neutravidin, or a mixture thereof; and the surface of the microbead is conjugated to the biomolecule; Optionally, the biomolecule is selected from a protein, an oligonucleotide, a small molecule, a lipid, a carbohydrate, or a mixture thereof; Optionally, said biomolecule is an affinity reagent, optionally said affinity reagent is an antibody, optionally said antibody is specific for a cytokine, optionally a chemokine, an interferon, a lymphokine, a monokine, an interleukin such as IL-1-36, a tumor necrosis factor, and a colony stimulating factor, and optionally said antigen is a viral antigen, and Optionally, the functionalization comprises a coating of silicon dioxide on the surface of the microbead, and optionally, the functionalization further comprises functionalizing the coating of silicon dioxide. The microbeads according to any one of claims 1 to 3.

11. The microbead of claim 10 , wherein the metal provides a barcode that identifies the biomolecule.

12. A population of microbeads as defined in any one of claims 1 to 3, optionally one or more of the following: the population has a particle size distribution with a coefficient of variation (CV) of about 10% or less than 10%, optionally the coefficient of variation is less than 5%; each microbead comprises a plurality of metals, the average amount of each metal of the plurality of metals across the population of microbeads being at or within about 10% of the average amount of another metal of the plurality of metals; Optionally, the plurality of metals includes one or more enriched isotopes; the amount of each metal in the population of microbeads has a distribution with a coefficient of variation of about or less than 20%, or about or less than 10%, the amount of each metal in one microbead of said population of microbeads is within about 20% or 20%, or within about 10% or 10%, or within about 5% or 5% of the amount of the same metal in another microbead of said population of microbeads; and The microbeads of the population of microbeads comprise a same metal in substantially the same amount, optionally the same metal is a plurality of metals, and the microbeads comprise each metal of the plurality of metals in substantially the same amount.

13. A kit comprising a plurality of distinct populations of microbeads as defined in claim 12, optionally one or more of the following: Each population of microbeads is distinguishable from another population of microbeads based on the metal or metals of the microbeads; and The microbeads of at least one population of microbeads comprise a metal or metals that is different from the metal or metals of the microbeads of another population of microbeads, or the microbeads of at least one population of microbeads comprise metals in a different ratio than the microbeads of another population of microbeads.

14. The kit of claim 13 , wherein the microbeads of each population of microbeads are conjugated to a different biomolecule.

15. polymerizing the structural monomers in the presence of a steric stabilizer in a nucleation stage to obtain a first mixture comprising polymerized structural monomers, unpolymerized structural monomers, and a steric stabilizer; combining said first mixture with a metal chelating monomer comprising a metal and a chelating agent attached to at least one polymerizable end group to obtain a second mixture; combining said chelating agent with at least three sites to coordinate a metal and said metal chelating monomer is polymerizable with the structural monomer; and polymerizing the second mixture to form a copolymer of the microbeads; The structural monomer is polymerized without the chelating agent.

23. A method for preparing metal-encoded microbeads comprising:

16. One or more of the following: the metal is a plurality of metals; said structural monomers are polymerized in said nucleation step to about 5% to about 20% completion based on said structural monomers; and Polymerization of said second mixture occurs to from 75% to about 100% completion, from about 80% to about 99% completion, from about 85% to about 95% completion, from about 85% to about 93% completion based on said structural monomers; or further comprising the step of functionalizing the microbeads; Optionally, the functionalization of the microbeads comprises: mixing the polymerized second mixture with a third monomer to obtain a third mixture, the third monomer comprising a reactive functional group; Optionally, the reactive functional groups are selected from alcohols, aldehydes, carboxylic acids, epoxides, vinyls, alkynes, maleimides, or mixtures thereof; and polymerizing said third mixture. Including, 14. The method of claim 13, optionally one or more of the following: Functionalizing the microbeads includes coating the microbeads with silicon dioxide; The functionalization of the microbeads further comprises functionalizing the coating of silicon dioxide; and Further comprising conjugating said microbeads to a biomolecule.

17. 17. Microbeads prepared by the method of claim 15 or 16.

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