CN117203278A

CN117203278A - Metal-labeled polymeric microbeads with controlled label levels

Info

Publication number: CN117203278A
Application number: CN202280027949.3A
Authority: CN
Inventors: M·A·温尼克; J·刘
Original assignee: University of Toronto
Current assignee: University of Toronto
Priority date: 2021-03-15
Filing date: 2022-03-15
Publication date: 2023-12-08

Abstract

The present disclosure relates to metal-labeled polymeric microbeads and in particular to lanthanide-labeled polymeric microbeads for use in bead-based mass spectrometry cytometry assays and multiplexing applications. The polymeric microbeads comprise a copolymer comprising a structural monomer and a metal chelating monomer.

Description

Metal-labeled polymeric microbeads with controlled label levels

Cross Reference to Related Applications

The present disclosure claims priority from U.S. patent application Ser. No. 63/161,414, filed on day 15 3 of 2021, and U.S. provisional patent application Ser. No. 63/319,608, filed on day 14 of 2022, the contents of which are incorporated herein by reference in their entireties.

Technical Field

The present disclosure relates to metal-labeled polymeric microbeads and in particular to lanthanide-labeled polymeric microbeads for use in bead-based mass spectrometry cytometry assays and multiplexing (multiplexing) applications.

Background

Mass flow cytometry (MC) is an emerging analytical technique for analyzing isotopically labeled signals on cell and microbead samples using inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS). Bead-based applications in mass cytometry require labeling microbeads with various metal ions that can be identified individually by MC.

Bead-based assays are attractive as analytical techniques because of their high sample volume efficiency for multiplex assays with high throughput capability. A wide variety of applications have been developed using bead-based assay techniques, including capture sandwich immunoassays, competitive immunoassays, serology, gene expression profiling, and genotyping ^1,2 . In contrast to classical planar or ELISA techniques, bead-based assays employ colloidal suspensions of particles as solid supports for different affinity reagents (which can target different molecules as analytes). By encoding the beads and creating a pool of beads as a classifier for the analyte, various captured analytes can be tracked by decoding and identifying individual beads throughout the experiment. In this way, multiple analyte assays can be performed simultaneously in a single assay using a set of beads functionalized with different affinity molecules. Most commercially available bead-based assays use luminescent tags as labels to bar code their beads and these classifier beads are examined by flow cytometry at high throughput. Luminex commercially available as a library of 500 differently labeled polystyrene microspheres with 3 different colors at 10 different intensity levels for bead-based assays by flow cytometry Surface functionality for attachment of bioaffinity reagents ^3-5 。

Mass flow cytometry (MC) is an emerging multiparameter analysis technique that combines the features of flow cytometry and elemental mass spectrometry to determine the characteristics of single cell or bead samples ⁵ . In mass flow cytometry, cell and bead samples are labeled with heavy metal isotopes and introduced 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 labeling, respectively. The MC can accurately detect different metal isotopes based on atomic mass, channels are not overlapped, and due to low abundance of heavy metal isotopes in biological samples, the MC avoids complex signal compensation processes related to flow cytometry. It greatly improves detection of sample characteristics compared to fluorescent-based bead assays, with enhanced resolution and sensitivity. Because a greater number of heavy metal isotopes can be used as labels, MC offers a very high probability for bead-based high throughput cytometry assays (which have much higher multiplexing capacity) ^7-8 。

To develop metal-encoded microbeads for multiplexed MC assays, abdelrahman et al introduced the idea of synthesizing libraries of multiple metal isotope-encoded microbeads that can be individually identified in MC at different concentration levels ⁹ . The theoretical maximum variability (n) of this microbead library can be calculated by the following expression:

n＝K ^M -1 (1)

here, K refers to the level of metal concentration (including a concentration of 0) in the microbeads; m is the number of different isotopes encoded in the beads. The term (-1) refers to beads with zero metal content for all isotopes that cannot be detected by MC. The selection of lanthanide isotopes as mass labels encoding PS microspheres is attractive due to their similar chemical and physical properties, low natural abundance, and their high detection sensitivity in MC ⁸ . There are 15 lanthanides whose isotopes cover at least 36 detection channels from 139amu to 176amu that are theoretically available for use as bead labels. For example, in the case of 4 concentrations of each encoded isotope, it is in principle possible toMC bead-based assays produce bead libraries with a variety of thousands of beads. To ensure the quality of the MC signal, the metal-containing microbeads employed in MC applications must be of a diameter of about And the size distribution is very narrow. Smaller microbeads tend to contain fewer metal isotopes, which may not be sufficient to be detected as a single event in the MC, while larger microbeads may not be consistently and completely consumed in the ICP torch.

The synthesis of several types of polymeric microbeads encoded with heavy metal isotopes has been reported. In the method employed by Abdelrahman et al, lanthanide-encoded Polystyrene (PS) microbeads are synthesized by multistage dispersion polymerization (DisP) in the presence of polyvinylpyrrolidone (PVP) as a steric stabilizer and Acrylic Acid (AA) as a comonomer for metal ion incorporation ⁸ . With some optimizations, abdelrahman et al can achieve a use of about 1x10 ⁸ PS microbeads encoded by lanthanide ions/beads with relatively small inter-bead variation (RCV<15%) in MC, which meets the requirements for use as a reagent in MC applications ^9,10 。

Liang et al later studied the idea of replacing comonomer AA with methacrylic acid (MAA) and achieved similar results ¹¹ . Other methods of incorporating metal ions into microbeads by absorbing styrene-soluble lanthanide chelates into PS beads in seed emulsion polymerization have also been explored, and covalent incorporation of lanthanide nanoparticles onto polymeric microbeads in dispersion polymerization has also been explored ^12,13 . However, much lower incorporation efficiencies and higher inter-bead metal content variations were observed in these later attempts as compared to the AA method developed by Abdelrahman et al.

However, the AA method is useful for synthesizing a batch of different metals with designed content for MC calibration purposesThe beads do not perform well. This strategy is not good at controlling metal incorporation in the bead synthesis, especially when a large number of different metal ions are mixed with AA in the second stage aliquots. For example, lanthanide ions with less ion radioactivity (e.g., ho ³⁺ And Lu ³⁺ ) Often compared to lanthanide ions (e.g. Ce) with greater ion radioactivity ³⁺ And Eu ³⁺ ) More efficient incorporation into microbeads ^14,15 。

This lack of controllability is a time consuming problem, especially for example when trying to synthesize multiple microbeads or libraries of microbeads encoded with various metal ions and each metal having a defined metal content at different levels.

Microbeads are useful in a variety of qualitative and quantitative applications, for example, for analyte detection. Some cytokines are believed to play a critical role in the pathology of covd-19. However, existing research tools that fully study the mechanisms of cytokine production and action have limitations ³⁴ 。

Cytokines are soluble signaling protein molecules produced by cells at picomolar to nanomolar concentrations to modulate immune responses and regulate cellular activity. They are a particularly large and diverse group of pro-and anti-inflammatory factors in the body ³⁵ . Deep characterization of cytokines in blood samples can provide key details of immune responses to diseases or infections or to vaccines, therapies, and interventions. Enzyme-linked immunosorbent assays (ELISA) are the most widely used monoplex Ab-based immunoassays for the analysis of cytokines in biological samples. However, ELISA assays require a large number of samples and are time consuming when analyzing a large number of cytokines at a time ^36,37 。

Bead-based immunoassays are a technical platform that provides information-rich multiparameter analysis in a high-throughput environment. Bead-based assays employing fluorescent dyes for signal detection are currently the gold standard for multiplexed cytokine analysis, and kits for cytokines are commercially available. In this type of assay, polymeric microbeads are labeled with 2 or 3 different fluorescent dyes at different concentrations to a well-defined intensity. Successful antigen detection is achieved by using a kit havingDye-labeled reporter antibody (Ab) recognition by different radiations ^38-44 . However, recent reports from 6 U.S. Pathology society (College of American Pathologists) cytokine surveys conducted from 2015 to 2018 describe the variability of cytokine analysis in a set of four cytokines (IL-1, IL-6, IL-8, TNF- α) ⁴⁵ . This study examined variability from laboratory to laboratory and even from method to method within the same laboratory. This report highly highlights some of the existing challenges in quantitative cytokine detection.

Disclosure of Invention

Metal-encoded polymeric microbeads that can be synthesized by multistage dispersion polymerization and that comprise structural monomers and metal-chelating monomers comprising chelating agents that chelate to metals prior to undergoing polymerization are described herein. The microbeads comprise, and are formed from, polymers including copolymers. As shown in the embodiments herein, the resulting microbeads have a narrow size distribution and defined amounts 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 may be incorporated into the entire microbead rather than merely being present on the surface. Furthermore, as described in some embodiments, the microbeads of the present disclosure may be functionalized on the surface of the microbeads and also conjugated to biomolecules, such as antibodies or other affinity reagents.

In particular, microbeads having substantially controlled metal content and methods for their preparation are provided in certain embodiments. The metal-encoded microbeads contain at least one metal element, and may contain a plurality of metal elements. The metallic element may comprise an isotope of a natural abundance of the element, or may comprise one or more enriched isotopes of the element. Lanthanide-encoded microbeads were prepared by employing polymerizable metal complexes as ligands in a two-stage dispersion polymerization, as shown in the examples. In one example, diethylenetriamine pentaacetic acid (DTPA) dianhydride is functionalized with a polymerizable monomer by reacting it with 4-Vinylbenzylamine (VBA). Then, various lanthanide metal ion complexes of this DTPA derivative were prepared. The metal-encoded microbeads are synthesized by incorporating these polymerizable metal-DTPA complexes into the dispersion polymerization of monomers such as styrene. The microbeads obtained have very small inter-bead variations in terms of their size and metal content. Similar metal incorporation efficiencies were found in bead synthesis with different metal ions complexed to the polymerizable chelating agent. The metal content in microbeads prepared using this method is linearly dependent on the metal complex feed in the bead synthesis, regardless of the metal type. Batches of microbeads encoded with three different concentrations of four lanthanides were prepared. These microbeads produced three different levels of MC signal intensity with very good baseline resolution.

As an illustrative example, and as further described herein, the surface of a collection of microbeads is surface functionalized and conjugated to antibodies and used to detect an analyte of interest (e.g., a sample biomolecule).

As shown herein, as an exemplary application of microbeads of the present disclosure in mass spectrometry (MC), a bead-based multiplexing assay was developed for detecting analytes including cytokines. In this assay type, classifier beads are labeled with heavy metal isotopes at different levels of metal incorporation. Each classifier bead carries a different Ab on its surface. The reporter may be a metal or metal oxide Nanoparticle (NP) with appropriate abs of other biological recognition elements bound to its surface. Samples were randomly injected into the plasma torch of an inductively coupled plasma time-of-flight mass spectrometer. The instrument is capable of achieving a single mass resolution in the range of m/z 85 to m/z 209. Thus, as shown, very high multiplexing levels are possible. In a bead-based assay by MC, cytokines were used as exemplary targets.

As described herein, a 4-fold analysis of a mixture of four cytokines is shown. 9 replicates are also illustrated. A 9-fold assay was tested on samples of Peripheral Blood Mononuclear Cells (PBMCs) comparing unstimulated samples to samples stimulated to promote cytokine secretion.

Accordingly, in one aspect, the present disclosure includes a metal-encoded microbead comprising:

a copolymer, the copolymer comprising:

structural monomers, and

a metal chelating monomer comprising a metal and a chelating agent; wherein the chelator coordinates to the metal at least at 3 sites; and is also provided with

Wherein the structural monomer does not comprise the chelating agent.

In another aspect, the disclosure includes a population of microbeads of the disclosure.

In another aspect, the disclosure includes a kit comprising a plurality of different populations of microbeads of the disclosure.

In another aspect, the present disclosure includes a method of making a metal-encoded microbead comprising:

polymerizing a structural monomer in the presence of a steric stabilizer in a nucleation stage to obtain a first mixture comprising polymerized structural monomer, unpolymerized structural monomer and the 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,

wherein the chelating agent coordinates to the metal at least at 3 sites, and wherein the metal chelating monomer is polymerizable with the structural monomer; and

Polymerizing the second mixture to form a copolymer of the microbeads;

wherein the structural monomer does not comprise the chelating agent.

In another aspect, the present disclosure includes a microbead prepared by the method of the present disclosure.

In another aspect, the present disclosure includes a method of making a metal-encoded microbead, said method comprising:

providing an aqueous dispersion of swellable seed particles and an anionic surfactant;

contacting the aqueous dispersion with a monomer comprising a structural monomer and a metal chelating monomer, wherein the metal chelating monomer comprises a metal and a chelating agent attached to at least one polymerizable end, and wherein the chelating agent coordinates to the metal at least at 3 sites;

diffusing the monomer into the seed particles to form an aqueous dispersion of swollen seed particles; and

initiating polymerization of the monomer in the aqueous dispersion of the swollen seed particles;

wherein the structural monomer does not comprise the chelating agent.

Drawings

Exemplary embodiments of the present disclosure will be further described with reference to the accompanying drawings, wherein:

FIG. 1 is a schematic view showing dissolution in D ₂ Na in O ₃ (DTPA-VBAm ₂ ) Of molecules ¹ Schematic representation of H-NMR spectra. Given Na ₃ (DTPA-VBAm ₂ ) Has a structure corresponding to the proton of the chemical shift label represented.

FIG. 2A is a metal complex Ce (DTPA-VBAm) added in the second stage ₂ ) SEM images of metal-containing microbeads Ce-1 synthesized in the presence of (c), and fig. 2B is a size diagram thereof. (d=2.9 μm, cv=1.2%).

FIG. 3 is a schematic diagram depicting five types of metal ion usage M (DTPA-VBAm ₂ ) A series of graphs of the incorporation efficiency of the complexes (m= Y, ce, eu, ho and Lu) in Y-1, ce-1, eu-1, ho-1, lu-1 and 5E1 microbeads (fig. 3A) and in 4E1, 4E2 and 4E3 microbeads (fig. 3B).

For the second phase in the aliquot at M (DTPA-VBAm ₂ ) 5-element encoded microbeads (5E 1) prepared in the presence of metal complexes (m= Y, ce, eu, ho and Lu), fig. 4A shows a graph depicting MC signal intensity, and fig. 4B shows a graph depicting metal content. a) And b) the error bars represent RSD of MC signal intensity and SD of metal content evaluated from MC signal intensity.

FIG. 5 is a graph depicting the linear dependence of metal content concentration in microbeads on the feed concentration of metal complex in second stage aliquots. The solid symbols represent data from preliminary bead synthesis (Y-1, ce-1, eu-1, ho-1, lu-1 and 5E 1). The solid line is a linear regression of these solid data points. Open symbols represent data from bead synthesis samples 4E1, 4E2, and 4E3, where we used the linear relationship observed in the preliminary bead synthesis as a guide for the bead synthesis design.

FIGS. 6A-6E are diagrams depicting the sample in phase 2 aliquots at M (DTPA-VBAm) ₂ ) A series of graphs of MC signal intensities for three different populations of PS microspheres prepared in the presence of metal complexes (m=ce, eu, ho and Lu), respectively, represent ¹⁴⁰ Ce、 ¹⁵¹ Eu、 ¹⁵³ Eu、 ¹⁶⁵ Ho sum ¹⁷⁵ MC signal intensity histogram of Lu. The x-axis in each plot represents the signal intensity of the isotope and the y-axis represents the number of beads normalized to 100. The first, second and third histograms depict the signals from the 4E1, 4E2 and 4E3 microbeads, respectively.

FIG. 7A is a diagram showing the use of M (DTPA-VBAm ₂ ) Schematic representation of antigen detection agent of encoded microspheres (Eu-1). In this protocol Eu-encoded microbeads surface-functionalized with goat anti-mouse IgG were used as reporter ¹⁷⁵ Lu-labeled mouse IgG was incubated together. And then for evidence as reporter detection ¹⁵³ Eu and ¹⁷⁵ both Lu signals were checked by MC for washed microbeads.

FIG. 7B depicts a histogram of MC measurements generated using the antigen detection agent of FIG. 7A, showing goat anti-mouse (GAM) modified Eu-1 microbeads (Eu-1/GAM) ¹⁷⁵ Lu signal intensity. In a second experiment, NAv modified Eu-1 microbeads without GAM (Eu-1/NAv) gave a weak signal, which was shown in fuchsin as a negative control.

FIG. 8A is Na ₃ (DTPA-BAm ₂ ) A kind of electronic device ¹ H-NMR spectrum, and FIG. 8B is Na ₃ (DTPA-ALAm ₂ ) A kind of electronic device ¹ H-NMR spectrum, said spectrum at D ₂ Measured in O. FIG. 8C is Na ₃ (DTPA-AmPMAm ₂ ) A kind of electronic device ¹ H-NMR spectrum. The structure of these molecules is given with protons corresponding to the chemical shift labels represented.

FIG. 9A is a diagram of Ce (DTPA-VBAm ₂ ) A kind of electronic device ¹ H-NMR (500 MHz) spectrum, FIG. 9B is Ce (DTPA-BAm) ₂ ) A kind of electronic device ¹ H-NMR (500 MHz) spectrum, FIG. 9C is Ce (DTPA-ALAm ₂ ) A kind of electronic device ¹ H-NMR (500 MHz) spectrum, and FIG. 9D is Ce (DTPA-AmPMAm ₂ ) A kind of electronic device ¹ H-NMR (500 MHz) spectrum, at D ₂ Measured in O. The formants in the figure broaden and shift because Ce (III) is a paramagnetic NMR shift reagent.

FIG. 10A is a diagram of Y (DTPA-VBAm ₂ ) A kind of electronic device ¹ H-NMR (500 MHz) spectrum, FIG. 10B is Eu (DTPA-VBAm) ₂ ) A kind of electronic device ¹ H-NMR (500 MHz) spectrum, FIG. 10C is Ho (DTPA-VBAm) ₂ ) A kind of electronic device ¹ H-NMR (500 MHz) spectrum, and FIG. 10D is Lu (DTPA-VBAm) ₂ ) A kind of electronic device ¹ H-NMR (500 MHz) spectrum, at D ₂ Measured in O. b) And c) broaden and shift the formants in c) because Eu (III) and Ho (III) are paramagnetic NMR shift reagents.

FIGS. 11A to 11D are MC in Ce-1, ce-2, ce-3 and Ce-4 microbeads, respectively ¹⁴⁰ Histogram of Ce signal intensity counts: the x-axis is ¹⁴⁰ Ce signal intensity; the y-axis is the number of beads normalized to 100.

FIG. 12A is a histogram of isotope signal intensity counts in a microbead sample of Y-1; FIG. 12B is a histogram of isotope signal intensity counts in a Eu-1 microbead sample. FIG. 12C is a histogram of isotope signal intensity counts in a sample of Ho-1 microbeads, and FIG. 12D is a histogram of isotope signal intensity counts in a sample of Lu-1 microbeads. The x-axis is isotope signal intensity; the y-axis is the number of beads normalized to 100.

FIG. 13A is a graph depicting metal ions Ce as determined by ICP-MS ³⁺ (Square) Eu ³⁺ (circle), ho ³⁺ (upward triangle) and Lu ³⁺ Graph of release profile (downward triangle) from 4E3 microbeads (solid symbols and solid lines) at 0.5% solids into pH 3.0 buffer solution (50 mM sodium acetate).

FIG. 13B is a graph depicting metal ions Ce as determined by ICP-MS ³⁺ (Square) Eu ³⁺ (circle), ho ³⁺ (upward triangle) and Lu ³⁺ Graph of release profile (downward triangle) from 4E3 microbeads (solid symbols and solid lines) at 0.5% solids into pH 7.0 buffer solution (10 mM ammonium acetate).

FIG. 13C is a graph depicting metal ions Ce as determined by ICP-MS ³⁺ (Square) Eu ³⁺ (circle), ho ³⁺ (upward triangle) and Lu ³⁺ Graph of release profile (downward triangle) from 4E3 microbeads (solid symbols and solid lines) at 0.5% solids into pH 10.5 buffer solution (200 mM sodium carbonate/sodium bicarbonate).

FIG. 13D is a graph depicting metal ions Ce as determined by ICP-MS ³⁺ (Square) Eu ³⁺ (circle), ho ³⁺ (upward triangle) and Lu ³⁺ Graph of release profile from 4E3 microbeads (solid symbols and solid lines) at 0.5% solids into 1% PVP solution (downward triangle). By way of comparison, open symbols and dashed lines represent release curves of metal ions from a batch of microbeads prepared by the AA method under the same conditions as 4e3 DTPA-beads.

FIG. 14 is a schematic of a multi-step strategy for functionalizing microbead surfaces with goat anti-mouse (GAM) through silica coating.

Fig. 15 is a schematic diagram of a microbead assay of the present disclosure.

Fig. 16 is a schematic diagram showing an exemplary bead-based multiplexed sandwich immunoassay by MC performed in a 96-well filter plate.

FIG. 17 is a dot plot of 11 types of classifier microbeads (C1-C11), wherein group A shows a mixture of 11 types of classifier microbeads (C-1-C-11) ¹⁴⁰ Ce- ¹⁴² Ce isotope plot. The oval circles separate the unimodal events of 11 types of microbeads. Panel b-k is a dot plot showing a gating strategy for identifying C-1 to C-11 microbeads individually by MC.

FIG. 18 is a histogram of reporter signal intensity on IL-4 classifier beads (C-5) in a series of quadruple assays of standard solutions at various IL-4 concentrations. (a), (b) and (c) AuNP was used as a reporter in a quadruple assay containing IL-4 at concentrations of 0, 1.2 and 20pg/mL, respectively. (d) (e) and (f) NanoGold was used as a reporter in a quadruple assay containing 0, 1.2 and 20pg/mL of IL-4 standard solution, respectively.

FIG. 19 is a standard curve of two sets of quadruple assays for (a) IL-4, (b) IL-6, (c) IFNγ and (d) TNFα . The x-axis in each plot represents analyte concentration and the y-axis represents MC signal intensity of the median of NPs attached to the corresponding classifier beads. Two different types of streptavidin conjugated reporters (AuNP and NanoGold) were studied in these quadruple assays. Results for AuNP are shown as circles (+), and NanoGold results are shown as squares (■). Will be ¹⁹⁷ Negative events with Au signal intensity ∈1 count/bead were excluded from statistical analysis for median intensity. Dose-response curves were drawn by fitting experimental results to a four-parameter logistic regression model.

FIG. 20 shows standard curves for four sets of nine replicates 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 at different concentrations of biotinylated anti-CD 163 and anti-CXCL-9 in detecting Ab mixtures. The x-axis in each plot represents analyte concentration. The y-axis in each plot represents the median of the MC signal intensities of AuNP attached to the corresponding type of classifier beads. To minimize background noise at low analyte concentrations, the concentration of biotinylated anti-CD 163 and anti-CXCL 9 in the detection Ab mixture was reduced from 2.5 to 2.0, 1.0, and 0.5 μg/mL, while the concentration of other detection abs remained constant at 2.5 μg/mL in the mixture. The results of these assays were plotted against Ab concentration, with 2.5 μg/mL plotted with filled circles (+), 2.0 μg/mL plotted with filled squares (■), 1.0 μg/mL plotted with filled triangles (∈), and 0.5 μg/mL plotted with filled diamonds (+). Will be ¹⁹⁷ Negative events with Au signal intensity ∈1 count/bead were excluded from statistical analysis for median intensity. Dose-response curves were drawn by fitting experimental results to a four-parameter logistic regression model.

FIG. 21 is a graph showing the median value of AuNP reporter attached to classifier beads in a nine-fold assay for (a) IL-1 beta, (b) IL-4, (c) IL-6, (d) IL-10, (e) IL-18, (f) IFN gamma, (g) TNF alpha, (h) CD163, and (i) CXCL-9 in stimulated and unstimulated PBMC samples at different sample dilution ratios ¹⁹⁷ Histogram of Au signal intensity. The filled bars in the figure represent the results of the measurement of stimulated samples, while the striped bars represent the results of the measurement of unstimulated samples.

FIG. 22 (a) is a schematic diagram of the structureC-1 microbead SEM image prepared by 2 stage DisP. FIG. 22 (b) is a diagram showing the use of M (DTPA-VBAm) by six types of metal ions ₂ ) Graph of the efficiency of incorporation of the complex (m= La, ce, pr, tb, ho and Tm) into C-1 microbeads. Error bars represent standard deviation of three measurements on the same solution.

FIG. 23 is a standard curve of three sets of quadruple assays for (a) IL-4, (b) IL-6, (c) IFNγ and (d) TNFα at different reporter (NP) concentrations. The x-axis in each plot represents analyte concentration. The y-axis in each plot represents the median of the MC signal intensities of AuNP attached to the corresponding classifier beads. In these quadruple assays, the concentrations of AuNP from stock solutions at 200×,400×, and 800× dilutions were studied. Their results are expressed as: 200 x dilutions are represented by circles (∈), 400 x dilutions are represented by squares (■), and 800 x dilutions are represented by triangles (). Will be ¹⁹⁷ Events with Au signal intensity ∈1 count/bead were excluded from statistical analysis for median intensity. Dose-response curves were drawn by fitting experimental results to a four-parameter logistic regression model.

Fig. 24 is a summary graph of median MC signal intensity of AuNP attached to classifier beads in a series of nine-fold assays of blank samples in the absence of analyte molecules (0 pg/mL).

FIG. 25 is a standard curve of a nine-fold assay for IL-1. Beta., IL-4, IL-6, IL-10, IL-18, IFNγ, TNFα, CD163 and CXCL-9 using the same assay conditions for the analysis of stimulated and unstimulated PBMC samples. Dose-response curves were drawn by fitting experimental results to a four-parameter logistic regression model.

Figure 26 is a graph showing cytokine concentrations in stimulated and unstimulated PBMC samples calculated based on the dose-response standard curve in figure 25. Some of the measured MC intensity values presented in fig. 21 are below the minimum of the 4P-LR modeled standard curve presented in fig. 25. The concentration was not calculated from these values.

Detailed Description

I. Definition of the definition

The definitions and embodiments described in this and other sections are intended to apply to all embodiments and aspects of the disclosure as understood by those skilled in the art as appropriate, unless otherwise indicated.

The term "and/or" as used herein means that the listed items are present or used, either alone or in combination. Indeed, this term means "at least one" or "one or more" used or present in the listed items. The term "and/or" with respect to pharmaceutically acceptable salts and/or solvates thereof means that the compounds of the present disclosure exist as a combination of individual salts and hydrates, e.g., solvates of 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 content clearly dictates otherwise. For example, embodiments that include "a compound" are understood to exhibit certain aspects with 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 different from the other component or the first component. For example, when the second combination and the first component may have the same chelating agent, the metal chelated to the second component may be different than the metal chelated to the first component. The "third" component is different from the other components, the first component, and the second component, and similarly, the further enumerated or "additional" components are different.

As used in this disclosure and in the claims, the words "comprise" (and any form of comprising) such as "comprises" and "comprises)", "having" (and any form of having "has") such as "having" and "having"), including (and any form of including such as "comprising" and "including") or "containing" are inclusive or open-ended, and do not exclude additional unrecited elements or method steps.

The term "consisting of … …" and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, and preclude the presence of other unstated features, elements, components, groups, integers, and/or steps.

The term "consisting essentially of … …" as used herein is intended to specify the presence of stated features, elements, components, groups, integers, and/or steps, and those that do not materially affect one or more of the basic and novel characteristics of the features, elements, components, groups, integers, and/or steps.

The term "suitable" as used herein means that the selection of a particular compound or condition will depend on the particular synthetic manipulation to be performed, the nature of the molecule to be converted, and/or the particular use of the compound, but such selection will be well within the skill of the person trained in the art.

In embodiments of the present disclosure, the compounds described herein may have at least one asymmetric center. Where the compounds have more than one asymmetric center, they may exist as diastereomers. It is understood that all such isomers and mixtures thereof in any ratio are encompassed within the scope of the present disclosure. It is further understood that while the stereochemistry of a compound may be as shown in any given compound listed herein, such compounds may also contain an amount (e.g., less than 20%, suitably less than 10%, more suitably less than 5%) of a compound of the disclosure having alternative stereochemistry. Any optical isomer (as an isolated, pure or partially purified optical isomer or racemic mixture thereof) is intended to be included within the scope of the present disclosure.

The compounds of the present disclosure may also exist in different tautomeric forms, and any tautomeric forms, and mixtures thereof, that are intended for formation of the compounds are included within the scope of the present disclosure.

The present specification refers to a number of chemical terms and abbreviations used by those skilled in the art. However, for clarity and consistency, definitions of selected terms are provided.

The terms "about," "substantially" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least or as high as + -5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context otherwise suggests to one of ordinary skill in the art.

The term "alkyl", as used herein, whether used alone or as part of another group, means a straight or branched chain saturated alkyl group. The possible number of carbon atoms in the mentioned alkyl groups is indicated by the prefix "Cn1-n 2". For example, the term C1-10 alkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

The term "alkylene", whether used alone or as part of another group, means a straight or branched chain saturated alkylene group; i.e. a saturated carbon chain containing substituents at both ends thereof. The possible number of carbon atoms in the alkylene groups mentioned is indicated by the prefix "Cn1-n 2". For example, the term C2-6 alkylene means an alkylene having 2, 3, 4, 5 or 6 carbon atoms.

The term "available" as in "available hydrogen atom" or "available atom" means an atom that will be known to those skilled in the art to be capable of being replaced by a substituent.

The term "amine" or "amino," whether used alone or as part of another group, as used herein refers to a group having the general formula NR 'R "wherein 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, means a saturated carbocyclic ring containing one or more rings. The possible number of carbon atoms in the cycloalkyl radicals mentioned is indicated by the numerical prefix "Cn1-n 2". For example, the term C3-10 cycloalkyl means cycloalkyl 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 ring containing at least one aromatic ring. In embodiments of the present disclosure, aryl 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 saturated or unsaturated (i.e., contain one or more double bonds). When heterocycloalkyl contains the prefix Cn1-n2, the prefix indicates the number of carbon atoms in the corresponding carbocyclic group, wherein one or more, suitably 1 to 5, ring atoms are replaced by heteroatoms 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 is a heteroatom selected from O, S and N. When heteroaryl contains the prefix Cn1-n2, the prefix indicates the number of carbon atoms in the corresponding carbocyclic group, wherein one or more, suitably 1 to 5, ring atoms are replaced by heteroatoms as defined above.

All cyclic groups (including aryl and cyclic groups) contain one or more than one ring (i.e., are polycyclic). When a cyclic group contains more than one ring, the rings may be fused, bridged, spiro, or linked by a bond.

The first ring and the second ring being "fused" means that the first ring and the second ring share two adjacent atoms therebetween.

By "bridging" a first ring with a second ring is meant that the first ring and the second ring share two non-adjacent atoms therebetween.

The first ring and the second ring are "spiro-fused" means that the first ring and the second ring share one atom therebetween.

The term "halogen" as used herein refers to a halogen atom and includes fluoro, chloro, bromo and iodo.

The term "optionally substituted" refers to a group, structure, or molecule that is unsubstituted or substituted with one or more substituents.

The term "atm" as used herein refers to atmospheric pressure.

The term "MS" as used herein refers to mass spectrometry.

The term "aq." as used herein refers to aqueous.

The term "protecting group" or "PG" or the like as used herein refers to a chemical moiety that protects or masks the reactive portion of a molecule to prevent side reactions in these reactive portions of the molecule when manipulating or reacting different portions of the molecule. After the completion of the procedure or reaction, the protecting groups are removed without degrading or decomposing the remainder of the molecule. Suitable protecting groups can be selected by those skilled in the art. Many conventional protecting groups are known in the art, for example as described in the following documents: "Protective Groups in Organic Chemistry" McOmie, J.F.W. editions, plenum Press,1973,in Greene,T.W. And Wuts, P.G.M. "," Protective Groups in Organic Synthesis ", john Wiley & Sons, 3 rd edition, 1999 and Kocienski, P.protective Groups, 3 rd edition, 2003,Georg Thieme Verlag (The America).

It will be appreciated that in the formation of the copolymer, certain polymerizable end groups of the metal chelating monomer will better match or preferentially polymerize with certain structural monomers. The reactivity of the comonomer can be determined, for example, by determination of the reaction rate using methods known in the art. For example, vinyl and methyl vinyl react with vinyl ethers, aryl vinyl reacts with other styrenes and acrylates react with other acrylates.

The term "EDTA" as used herein refers to ethylenediamine tetraacetic acid.

The term "DTPA" as used herein refers to diethylenetriamine pentaacetic acid.

The term "EGTA" as used herein refers to ethacrynic acid.

The term "EDDS" as used herein refers to ethylenediamine-N, N' -disuccinic acid.

The term "EDDHA" as used herein refers to ethylenediamine-N, N' -bis (2-hydroxyphenylacetic acid).

The term "BAPTA" as used herein refers to 1, 2-bis (o-aminophenoxy) ethane-N, N' -tetraacetic acid.

The term "TACN" as used herein refers to 1,4, 7-triazacyclononane.

The term "TACD" as used herein refers to 1,5, 9-triazacyclododecane.

The term "cyclen" as used herein refers to 1,4,7, 10-tetraazacyclododecane.

The term "cyclam" as used herein refers to 1,4,8, 11-tetraazacyclododecane.

The term "(13) aneN4" as used herein 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-diaza-octadecane.

The term "DFO" as used herein refers to deferoxamine.

The terms "TACD-type chelator", "TACN-type chelator", "cyclen-type chelator" and the like as used herein refer to chelators comprising a specified base structure (i.e. TACD, TACN, cyclen and the like), wherein the specified base structure may be further substituted at available hydrogen atoms.

The term "swellable polymer seed" as used herein refers to polymer particles capable of increasing volume. For example, the swellable polymer seed may increase in volume when contacted with a swelling agent. The swelling agent may be, for example, an anionic surfactant and/or an organic compound. Once the swellable polymer seed has swelled, compounds such as monomers (e.g., structural monomers and metal chelating monomers), steric stabilizers, and polymerization initiators may diffuse into the interior of the swollen polymer seed. Subsequent polymerization of the monomer may occur.

The term "substantially anaerobic conditions" as used herein refers to reaction conditions that are low or absent in oxygen content. For example, substantially oxygen-free conditions may refer to reaction conditions under 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 may refer to an oxygen content of between about 0ppm to about 5ppm, about 0ppm to about 3ppm, about 0ppm to about 2ppm, or about 0ppm to about 1ppm, or about 0.01ppm to about 2 ppm.

The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies, as well as binding fragments thereof. Antibodies can 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 produced by treating antibodies with pepsin. The resulting F (ab ') 2 fragments may be treated to reduce disulfide bridges, thereby producing Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, fab 'and F (ab') 2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques. Antibody fragments as used herein means binding fragments

The term "oligonucleotide" as used herein refers to a nucleic acid comprising: nucleotide or nucleoside monomer sequences consisting of naturally and non-naturally occurring bases, sugars and inter-sugar (backbone) linkages, and include single-and double-stranded molecular RNAs and DNAs. Oligonucleotides can be long (e.g., greater than 1000 monomers and up to 10K monomers), medium-sized (e.g., between 200 and 1000 nucleotides and inclusive), or short, e.g., less than 200 monomers, 100 monomers, 50 monomers, including non-naturally occurring monomers. The term "oligonucleotide" includes, for example, single stranded DNA (ssDNA), genomic DNA (gDNA), complementary DNA (cDNA reverse transcribed from RNA), messenger RNA (mRNA), "antisense oligonucleotide" and "miRNA" and oligonucleotide analogs such as "morpholino oligonucleotides", "phosphorothioate oligonucleotides" or any oligonucleotides known to those skilled in the art or analogs thereof.

The recitation of numerical ranges by endpoints herein includes 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 should also be understood that all numbers and fractions thereof are considered to be modified by the term "about".

Furthermore, the definitions and embodiments described in the specific section are intended to apply to other embodiments described herein as understood by those skilled in the art as appropriate. For example, in the following paragraphs, various aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Sub-ranges, such as each 0.1 increment therebetween, are also contemplated for the ranges described herein. For example, if the range is 0ppm to about 5ppm, 0.1ppm to about 5ppm, 0ppm to about 4.9ppm, 0.1ppm to about 4.9ppm, etc. are also contemplated.

Compounds, compositions and kits of the present disclosure

A copolymer, the copolymer comprising:

structural monomers, and

a metal chelating monomer comprising a metal and a chelating agent;

wherein the chelator coordinates to the metal at least at 3 sites; and wherein the structural monomer does not comprise the chelating agent.

The microbeads of the present disclosure may comprise a substantially uniform distribution of metal.

For example, the metal chelated to the metal chelating monomer is not limited to surface binding, but may be distributed throughout and/or within the interior portions of the microbeads of the present disclosure.

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

In some embodiments, the structural monomer does not comprise any chelating agent (e.g., does not comprise any chelating agent that coordinates to the metal at least 2 sites).

In some embodiments, the structural monomers are metal-free. For example, the structural monomer may not contain a metal by chelation, by covalent bonds (such as tellurium in the carbon backbone of the structural monomer), or optionally, by any other means. The structural monomer may not contain a transition metal or a class of transition metals. For example, the structural monomers may not include rare earth metals (such as lanthanides) and/or soft metals as described herein. In some embodiments, such as in mass flow cytometry applications, the metal chelating monomer may comprise a heavy metal (e.g., 80amu or greater heavy metal) while the structural monomer does not comprise a heavy metal (e.g., 80amu or greater heavy metal).

In some embodiments, the structural monomers are selected from the group consisting of substituted or unsubstituted styrenes, alpha-methylstyrene, acrylic acid and esters and amides thereof, methacrylic acid and esters and amides thereof, and derivatives thereof. In one embodiment, the structural monomer is selected from substituted or unsubstituted styrenes and/or combinations thereof.

In some embodiments, the metal chelating monomer has the structure of formula I prior to polymerization

Wherein the ligand is a chelator, L is a linker, X is a polymerizable end group, M is a metal, and n is 1 or an integer greater than 1, wherein the metal chelating monomer is neutral in charge prior to polymerization.

It is understood that the metal is chelated to the chelating agent of the metal chelating monomer by ionic non-covalent interactions. Thus, metals are incorporated into the microbeads of the present disclosure by non-covalent interactions.

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, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, C (O) O, or mixtures thereof. Each of alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl may independently be unsubstituted or substituted with one or more substituents that may be selected from the group consisting of: 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.

L may be attached to the chelating agent, for example, by an amide or ester group/linkage.

It will be appreciated that when the structural monomer and the metal chelating monomer have similar hydrophobicity, incorporation or mixing of one into the other is facilitated. For example, when the structural monomer is hydrophobic, the hydrophobic metal chelating monomer may have a more favorable interaction with the structural monomer, resulting in a more efficient mixing of the two monomers. Thus, in some embodiments, L may be hydrophobic.

In some embodiments, the polymerizable end group is selected from the group consisting of aryl vinyl, styrene, alpha-methyl styrene, acrylate, methacrylate, acrylamide, 2-methacrylamide, and mixtures thereof, optionally the polymerizable end group is aryl vinyl or styrene. In some embodiments, the polymerizable end group is an aryl vinyl or vinyl ester.

The chelating agent may for example be tridentate. In some embodiments, the chelator is tetradentate, pentadentate, hexadentate, heptadentate, or octadentate, optionally, the chelator is hexadentate or octadentate.

In some embodiments, the chelating agent comprises an amino polyacid moiety or derivative thereof. In some embodiments, the derivative of the amino polyacid moiety comprises an amide of the amino polyacid moiety.

For example, the amino polyacid moiety may be selected from amino polycarboxylic acids, amino polyphosphonic acids, or combinations thereof.

In some embodiments, L is a bond, the chelating agent is an amino polyacid, and the polymerizable end group is an aryl vinyl group. For example, the metal chelating monomer may be a metal-coordinated vinyliminodiacetic acid or a metal-coordinated divinylbenzene iminodiacetic acid.

In other embodiments, the amino polyacid moiety is a substituted oligomer of one or more of ethyleneimine, acrylamide, or mixtures thereof, the oligomer being substituted with two or more carboxylic acids and/or phosphonic acids. The oligomer may be a crown ether or an aza crown ether. In some embodiments, the amino polyacid moiety is a substituted oligomer of one or more of ethylene oxide, ethylene imine, propylene oxide, acrylamide, ethanolamine, propanolamine, aminophenol cyclohexane diamine, or mixtures thereof.

In yet other embodiments, the oligomer is further substituted with one or more substituents selected from the group consisting of: C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, halogen, alcohol, amine, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.

In some embodiments, the chelator is selected from DFO, EDTA, DTPA, EGTA, EDDS, EDDHA, BAPTA, H neupa, H6 phosphoa, H4CHXoctapa, H4octapa, H2 CHXddpa, H5decapa, cy-DTPA, ph-DTPA, TACN-type chelator, TACD-type chelator, cyclen-type chelator, cyclam-type chelator, (13) aneN 4-type chelator, 1, 7-diaza-12-crown-4-type chelator, 1, 10-diaza-18-crown-6-type chelator, or derivatives thereof.

For example, the TACN-type chelating agent may 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-bridged TETA, diAmSar, or derivatives thereof.

In other embodiments, the (13) aneN 4-type chelating agent is selected from TRITA or derivatives thereof.

In yet other embodiments, the 1, 10-diaza-18-crown-6-type chelator is selected from MACROPA or a derivative thereof.

In some embodiments, the chelator is selected from DTPA, cy-DTPA, ph-DTPA or derivatives thereof.

For example, derivatives of DTPA may include DTPA having two adjacent carbon atoms joined together with atoms therebetween to form a 5-or 6-membered ring (optionally, a cycloalkyl ring, an aryl or heteroaryl ring).

Prior to polymerization, the monomers described herein are unreacted, i.e., the monomers contain polymerizable end groups that can participate in the polymerization. In some embodiments, the metal chelating monomer is prior to polymerization

Wherein L and X are as described herein.

In other embodiments, prior to polymerization, the metal chelating monomer is selected from the group consisting of:

/>

or a mixture thereof.

For example, the mixture may include one or more of the metal chelating monomers having different metals.

In other embodiments, the chelator comprises porphyrin or phthalocyanine.

For example, the chelating agent may be a substituted or unsubstituted porphyrin.

In some embodiments, the porphyrin and phthalocyanine are each independently substituted with a 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.

When the chelating agent comprises or is porphyrin or phthalocyanine, the metal may be a soft metal. For example, the soft metal may be cadmium, cobalt, copper, iron, zinc, nickel, tin, osmium, palladium, platinum, gold, thallium, mercury, or lead, including isotopes thereof, and mixtures thereof. In some embodiments, the soft metal has an atomic mass of 80amu or more.

In some embodiments, the metal chelating monomer prior to polymerization is selected from

Or mixtures thereof, and wherein n is an integer from 1 to 4.

For example, L may be aniline.

In some embodiments, n is 2 or at least 2.

In other embodiments, the metal chelating monomer is selected from

Or a mixture thereof.

The microbeads of the present disclosure may further comprise a steric stabilizer. The steric stabilizer may be PVP, polyvinyl alcohol, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polyacrylic acid, a water soluble homopolymer of an acrylate, a water soluble homopolymer of a methacrylate, a water soluble homopolymer of acrylamide, a homopolymer of methacrylamide, a water soluble copolymer steric stabilizer or a mixture thereof.

In some embodiments, the water-soluble copolymer steric stabilizer is selected from the group consisting of acrylate, methacrylate, acrylamide or methacrylamide copolymers with methyl acrylate and/or ethyl acrylate, or mixtures thereof.

The copolymer may also be crosslinked, as is achieved, for example, when the metal chelating monomer and/or the structural monomer has two or more polymerizable groups.

For example, the microbeads of the present disclosure may comprise a variety of metals. For example, each metal may be incorporated by the same type or different types of metal chelating monomers. The metal may be a variety of metals.

In some embodiments, the plurality of metals includes one or more enriched isotopes.

In some embodiments, the plurality of metals comprises at least 2 metals, at least 3 metals, or at least 4 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 the other metal in the plurality of metals. For example, this can be determined by mass flow cytometry on a population of microbeads or individual microbeads.

The metal may be, for example, a transition metal (i.e., a metal from groups 3-12 of the periodic table, or from the lanthanide or actinide series). The metal may be, for example, indium, bismuth or a rare earth metal. The rare earth metal may 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 may 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, different isotopes thereof, 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, different isotopes thereof, and mixtures thereof.

In still other embodiments, the rare earth metal is selected from 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, 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 microbeads. It may also be compartmentalized, for example, in the interior of the microbeads.

In some embodiments, the microbeads have a glass transition temperature (as measured, for example, by Differential Scanning Calorimetry (DSC)) of about 60 ℃ or greater than 60 ℃, optionally, about 70 ℃ or greater than 70 ℃, about 80 ℃ or greater than 80 ℃, about 90 ℃ or greater than 90 ℃, about 100 ℃ or greater than 100 ℃, about 115 ℃ or greater than 115 ℃, about 125 ℃ or greater than 125 ℃ or about 135 ℃ or greater than 135 ℃. For example, DSC results are obtained at a scan rate of about 10 ℃/min to about 20 ℃/min during the second or third thermal scan.

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 dimensions suitable for mass flow cytometry.

In yet other embodiments, 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 of metal or less than 1% leakage of metal when stored in the buffer and/or physiological medium.

In some embodiments, the surface of the microbead comprises functionalization for attachment to a biomolecule. Functionalization can be introduced by adding a coating.

In some embodiments, the attachment is covalent attachment. For example, as shown in the examples, the microbeads may be coated with silica and functionalized with reactive functional groups, such as carboxylic acid groups. Biomolecules may be added to the microbeads.

Biomolecules can be classified as proteins, oligonucleotides, lipids, carbohydrates or small molecules. Alternatively or additionally, biomolecules may be classified by their function. The biomolecules are not particularly limited and different functionalization may be used to conjugate the biomolecules to the microbeads. For example, the oligonucleotide can be a single stranded DNA molecule, optionally, a cDNA that hybridizes under stringent conditions to a target nucleic acid analyte (e.g., a sample nucleic acid biomolecule), or the oligonucleotide can be an aptamer. For example, the biomolecule may be an oligonucleotide that specifically hybridizes to a target oligonucleotide (e.g., to a sample oligonucleotide), such as a target mRNA endogenous to the sample. Hybridization may be hybridization of sequences of more than 8, more than 10, more than 15 or more than 20 nucleotides.

In certain aspects, biomolecules may be classified by their function. For example, the biomolecule may be an affinity reagent, an antigen (e.g., an analyte specifically bound by the affinity reagent), or an enzyme substrate. The affinity reagent may be an antibody (e.g., or fragment thereof), an aptamer, a receptor (e.g., or portion thereof), or any other biomolecule that specifically binds to a target (e.g., avidin, such as streptavidin, that specifically binds biotin). For example, beads may be associated with antibodies, which may be used to detect the presence of their target antigens, such as cytokines, viral proteins, cancer biomarkers, etc., in a sample. In certain methods and kits, the microbeads can be functionalized with avidin for attachment of another biomolecule functionalized with biotin (e.g., to allow the beads to be adapted for any of a variety of different assays). An antigen may be a protein (or peptide sequence thereof) comprising an epitope that is specifically bound by an affinity reagent, such as an antibody. For example, the beads may be attached to a viral antigen (such as a viral protein sequence) and may be used to detect the presence of antibodies in a sample that specifically bind to the viral antigen, as further described herein. The enzyme substrate may 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 may be a protein (e.g., or a peptide sequence thereof) that is a substrate for an enzyme, such as a protease, phosphatase, kinase, methyltransferase, demethylase. Non-protein substrates include, for example, double-stranded oligonucleotides comprising restriction sequences cleavable by restriction enzymes or sites for DNA repair (e.g., nicks), oligonucleotide sequences comprising sequences targeted by DNA methyltransferases, or any non-protein substrate known to those of skill in the art. For example, the beads may be attached to a substrate and exposed to a sample comprising an enzyme that modifies the substrate, and the modification of the substrate (or lack thereof) may be detected (e.g., as further described herein).

In some embodiments, the affinity reagent is or includes an antibody or binding fragment thereof. The antibody may be, for example, a biotinylated antibody or binding fragment, and may be added directly or indirectly to the microbeads.

For example, as demonstrated in the examples, neutravidin (NeutrAvidin, NAv) was covalently conjugated to COOH groups on the surface of silica coated microbeads by EDC/NHS coupling. Biotinylated antibodies were attached to NAv modified microbead surfaces with strong biotin-avidin affinity.

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

In some embodiments, the affinity reagent (optionally, antibodies) is specific for cytokines, optionally, chemokines, interferons, lymphokines, monokines, interleukins (such as IL-1-36), tumor necrosis factors, and colony stimulating factors.

Antibodies may also be specific for pathogenic proteins such as viral, bacterial or fungal pathogens. Such microbeads may be used to detect the presence of such pathogens or products thereof in a sample, such as an environmental or patient sample.

In other embodiments, the antigen is a viral antigen. Such microbeads comprising viral antigens may be used to detect the presence of viral antibodies in a patient sample. Similarly, antigens such as antigens from other pathogens.

In yet other embodiments, the copolymer of the microbeads further comprises a third monomer, which is present at least on the surface of the microbeads and comprises at least one reactive functional group. In some embodiments, the microbeads are functionalized by at least one reactive functional group contained in a third monomer, for example by three-stage dispersion polymerization. In some embodiments, the third monomer is a substituted or unsubstituted acrylic acid or methacrylic acid. Examples of three-stage dispersion polymerization are described in Abdelrahman et al, JACS,2009,15276, the contents of which are hereby incorporated by reference.

For example, the surface of the microbeads may be functionalized with reactive functional groups.

In some embodiments, the reactive functional groups are on a coating of silica.

In other embodiments, the reactive functional group is selected from an amine, thiol, alcohol, aldehyde, carboxylic acid, epoxide, vinyl, alkyne, maleimide, or click chemistry moiety (such as, for example, dibenzocyclooctyne (DBCO), azide, trans-cyclooctene (TCO), or tetrazine or derivative) or mixtures thereof.

In still other embodiments, the functionalization comprises a coating of silica on the surface of the microbead, optionally, the functionalization further comprises functionalizing the coating of silica.

In some embodiments, the surface of the microbeads is functionalized with reactive functional groups.

In other embodiments, the reactive functional groups are on a coating of silica.

For example, the reactive functional group may be selected from amines, thiols, alcohols, aldehydes, carboxylic acids, epoxides, vinyl groups, alkynes, maleimides, or mixtures thereof.

It will be appreciated that functionalization of the silica coating can be performed using methods known in the art. For example, a method of functionalizing microbeads by means of a silica coating is described in US2019/0091647, the content of which is hereby incorporated by reference.

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 mixtures thereof.

In yet other embodiments, the surface of the microbead is conjugated to a biomolecule.

In some embodiments, the metal provides a bar code 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 rather than multiple metal atoms.

In some embodiments, the microbeads comprise polymer seeds that do not comprise metal chelating monomers. In some embodiments, the polymer seed has an interior space formed by swelling the polymer seed, and the copolymer is present at least within the interior space of the polymer seed.

In some embodiments, the polymer seed comprises a structural monomer; optionally, wherein the structural monomers of the polymer seed are structurally identical to the structural monomers of the copolymer.

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%.

In some embodiments, each microbead comprises a plurality of metals, the average amount of each metal of the plurality of metals in the entire population of microbeads being within about 10% or 10% of the average amount of another metal of the plurality of metals.

In other embodiments, the plurality of metals includes one or more enriched isotopes.

In some embodiments, the amount of each metal of one microbead in the population of microbeads is within about 20% or 20% of the amount of the same metal of another microbead in the population of microbeads, alternatively within about 10% or 10%, alternatively within about 5% or 5%.

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 still 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 the population of microbeads comprise the same metal in substantially the same amount.

In some embodiments, the same metal is a plurality of metals and the microbeads comprise each of the plurality of metals in substantially the same amount.

Another aspect is a composition comprising a microbead or a plurality of microbeads. The composition may be, for example, a buffer solution, e.g., buffered to a pH of about 7. The buffer solution may comprise ammonium acetate and similar buffers. The composition may further comprise PVP.

In one embodiment, the composition is an aqueous colloidal suspension.

The composition may comprise one or more components selected from the group consisting of stabilizers, preservatives, buffers, and mixtures thereof.

In another aspect, the present disclosure includes a kit comprising the microbeads of the present disclosure, a population of a plurality of different microbeads, and/or a composition.

In some embodiments, the population of each microbead is distinguishable from the population of another microbead based on the metal or metals of the microbeads.

In some embodiments, the microbeads in at least one population of microbeads (e.g., each population) comprise a metal or metals that are different from the metal or metals of the microbeads in another population of microbeads.

In some embodiments, the microbeads in at least one population of microbeads (e.g., each population) and the microbeads in another population of microbeads comprise different ratios of multiple metals.

In some embodiments, the microbeads in each population of microbeads are conjugated to different biomolecules. For example, the microbeads may serve as a barcoding agent for biomolecules attached to the microbeads by the nature of the metal or metals in the microbeads.

In some embodiments, the structural monomer and the metal chelating monomer may have been copolymerized according to any of the methods described herein. Notably, the structural monomers and/or metal chelating monomers used in the methods described herein can have any of the aspects described in this section.

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 or be used in mass spectrometry assays, as further described herein. Aspects of the disclosure also include additional methods, such as mass spectrometry, as further described herein.

Methods of the present disclosure

polymerizing the second mixture to form a copolymer of the microbeads;

wherein the structural monomer does not comprise the chelating agent.

It will be appreciated that for efficient polymerization, the structural monomers and metal chelating monomers should be soluble in the reaction medium. Furthermore, it is understood that when the monomer is substituted, for example with substituents that may interfere with polymerization (e.g., halogens, amines, alcohols), the substituents may be temporarily protected prior to and/or during polymerization using protecting groups known in the art. After polymerization, the protecting groups may be selectively removed and the substituents selectively deprotected using methods known in the art.

In some embodiments, the metal is a plurality of metals.

In other embodiments, the structural monomer is polymerized in the nucleation stage to a degree of completion of about 5% to about 20% based on the structural monomer.

In yet other embodiments, the polymerization of the second mixture occurs to about 75% to about 100% completion, about 80% to about 99% completion, about 85% to about 95% completion, about 85% to about 93% completion, based on the structural monomers.

In some embodiments, the 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.

In some embodiments, the method further comprises functionalizing the microbeads.

In some embodiments, the functionalization of the microbeads includes

Mixing the polymerized second mixture with a third monomer to obtain a third mixture, the third monomer comprising a reactive functional group; and

polymerizing the third mixture.

For example, the reactive functional group may be selected from amines, thiols, alcohols, aldehydes, carboxylic acids, epoxides, vinyl groups, alkynes, maleimides, or mixtures thereof. It is understood that certain reactive functional groups may interfere with the polymerization process and may be protected prior to and/or during polymerization using protecting groups known in the art. For example, amines and thiols can be protected by protecting groups. For example, monomers substituted with reactive functional groups may be used in a protected form such that the reactive functional groups will not interfere with the polymerization process. Optionally, the protected reactive functional groups may be deprotected using methods known in the art.

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

In other embodiments, functionalization of the microbeads includes coating the microbeads with silica.

In other embodiments, the functionalization of the microbeads further includes a coating of functionalized silica.

For example, functionalization of a coating of silica can include reacting the coating of silica with an organosilane. For example, the organosilane may be selected from chlorosilanes, alkoxysilanes, derivatives thereof, and mixtures thereof.

In some embodiments, reacting the coating of silica with the organosilane is performed in the presence of a catalyst. For example, the catalyst may 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 group is selected from amines, thiols, alcohols, aldehydes, carboxylic acids, epoxides, vinyl, alkynes, maleimides, or mixtures thereof.

In some embodiments, the organosilane is APTES.

In other embodiments, the method further comprises conjugating the microbeads to a biomolecule.

In some embodiments, the biomolecule is as defined herein.

In some embodiments, the microbeads have diameters 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.

In some embodiments, the microbeads are microbeads of the present disclosure.

contacting the aqueous dispersion with a monomer comprising a structural monomer and a metal chelating monomer, wherein the metal chelating monomer comprises a metal and a chelating agent attached to at least one polymerizable end, and wherein the chelating agent coordinates to the metal at least at 3 sites; and

wherein the structural monomer does not comprise the chelating agent.

Providing an aqueous dispersion comprising swellable polymer seeds, an organic compound, an anionic surfactant, and optionally an organic solvent in which the organic compound is soluble;

allowing the organic compound to diffuse into the swellable polymer seed;

contacting the aqueous dispersion with a mixture comprising a structural monomer and a metal chelating monomer; optionally, the mixture further comprises a steric stabilizer and/or a polymerization initiator; and

polymerizing the mixture to obtain a copolymer of the microbeads;

wherein the organic compound has a molecular weight of less than 5000Da and less than 10 at 25 DEG C ^-2 g/L water solubility.

preparing swellable polymer seeds by emulsion polymerization, wherein anionic surfactants are used as emulsifiers under substantially oxygen-free conditions;

contacting the swellable polymer seed with an aqueous dispersion comprising an organic compound, an anionic surfactant, and optionally an organic solvent in which the organic compound is soluble;

diffusing the organic compound into the swellable polymer seed;

polymerizing the mixture to obtain a copolymer of the microbeads;

In some embodiments, the structural monomer is selected from the group consisting of: acrylic, methacrylic and vinyl monomers selected from the group consisting of styrene, divinylbenzene (DVB), ethylvinylbenzene, vinylpyridine, amino-styrene, methyl-styrene, dimethylstyrene, ethylstyrene, ethyl-methyl-styrene, 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 an aqueous dispersion of swellable seed particles includes preparing monodisperse swellable seed particles by emulsion polymerization.

In some embodiments, the aqueous dispersion of swellable seed particles further comprises an organic compound having a molecular weight of less than 5000 daltons and less than 10 at 25 ℃ and optionally an organic solvent in which the organic compound is soluble ^-2 g/L water solubility.

In some embodiments, the swellable seed particles are monodisperse swellable seed oligomer particles.

For example, the anionic surfactant may be an alkyl sulfate or alkyl sulfonate. In some embodiments, the anionic surfactant is C _8-16 Alkyl sulfate or sulfonate or salts thereof. For example, the anionic surfactant may be decyl sulfate, dodecyl sulfateDecyl sulfonate, dodecyl sulfonate or salts thereof. In some embodiments, the anionic surfactant is sodium dodecyl sulfate or sodium decyl sulfate.

In some embodiments, the 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 the group consisting of: alcohols, ethers, ketones, dialkyl sulfoxides (e.g., DMSO), dialkyl formamides (e.g., DMF), or mixtures thereof.

Synthesis of monomers of the present disclosure

The monomers of the present disclosure can be prepared by various synthetic processes. The selection of particular structural features and/or substituents may affect the selection of one process relative to another. The selection of a particular process to prepare a given monomer is within the knowledge of one skilled in the art. Some of the 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 readily prepared from available precursors using direct transformations well known in the art. In the following schemes showing the preparation of the second monomer of the present application, all variables are as defined in the specification unless otherwise indicated.

The compounds of formula (I) may be prepared, for example, according to the procedure set forth in the schemes below. In the structural formulae shown below, variables are as defined in formula (I) unless otherwise indicated. Those skilled in the art will appreciate that many of the reactions depicted in the schemes below will be sensitive to oxygen and/or water and will be aware of the reactions being carried out under anhydrous inert atmospheres, if desired. The reaction temperature and time will be presented for illustrative purposes only and may be varied to optimize the yield (noun) rate as will be appreciated by those skilled in the art.

Thus, in one embodiment, the compound having formula I is prepared as shown in scheme a. A chelator or ligand of formula a is attached to one or more linkers of formula B having a polymerizable end group to form a metal-chelating-capable monomer of formula C. Metal D may then be chelated to the monomer having formula C to form a metal chelate monomer having formula I.

Scheme A

In some embodiments, the ligand having formula a may comprise one or more carboxylic acid groups. Thus, the steps in scheme a may be completed according to scheme B. The ligand of formula E may be attached to one or more linkers of formula B having a polymerizable group by amide bond formation or esterification to obtain a metal-chelating-capable monomer of formula F. It will be appreciated that amide bond formation may be carried out using methods known in the art, for example by the formation of activated esters.

Scheme B

It will be appreciated that the metal chelation step b may be carried out using methods known in the art. For example, in some embodiments, the metal-chelating monomer having formula C or F and the metal having formula I may each be dissolved in a suitable solvent. Suitable solvents may be selected by those skilled in the art and may include water. The metal having formula D may be a salt of a metal, such as a halide salt of a metal. The solution capable of metal chelating monomer and the solution of metal may be mixed together. For example, the pH of the resulting mixture may be monitored and adjusted to an appropriate pH. In some embodiments, suitable pH includes a pH of about 5.5 to about 7.5, or about 6. The resulting mixture may be stirred until a metal chelating monomer having formula I is formed.

Throughout the processes described herein, it will be appreciated that where appropriate, suitable protecting groups are added to and subsequently removed from the 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", T.W.Green, P.G.M.Wuts, wiley-Interscience, new York, (1999). It will also be appreciated that the conversion of a group or substituent to another group or substituent can be performed by chemical manipulation on any intermediate or final product on a synthetic pathway toward the final product, with the type of conversion possible being limited only by the inherent incompatibility of the other functional groups carried by the molecule at this stage with the conditions or reagents employed in the conversion. Such inherent incompatibilities and the manner in which such inherent incompatibilities are avoided by performing appropriate transformations and appropriate sequences of synthetic steps will be readily understood by those skilled in the art. Examples of transformations are given herein, and it is understood that the transformations described are limited only to general groups or substituents that exemplify the transformations. Other suitable transformations are described and referenced in "Comprehensive Organic Transformations-A Guide to Functional Group Preparations" R.C. Larock, VHC Publishers, inc. (1989). Other references and descriptions of suitable reactions are described in textbooks of organic chemistry, e.g. "Advanced Organic Chemistry", march, 4 th edition, mcGraw Hill (1992) or "Organic Synthesis", smith, mcGraw Hill (1994). Techniques for purifying intermediates and end products include, for example, direct and reverse phase chromatography on columns or rotating plates, recrystallization, distillation, and liquid-liquid or solid-liquid extraction, as will be readily appreciated by those skilled in the art.

V. Mass Spectrometry methods and kits

The microbeads and kits described above may include additional aspects for use in mass spectrometry assays. Methods and kits for mass spectrometry are described below, which may include or use any suitable microbead described elsewhere herein. Suitable assays include assaying for activity of a target or sample biomolecule, such as a target oligonucleotide, a protein (e.g., a cytokine, a cancer biomarker, or an antibody to a specific antigen), or an enzyme (e.g., an enzyme such as a kinase, phosphatase, protease, or any other enzyme of interest that modifies a biomolecule attached to a microbead). The assay may use hybridization and/or sandwich ELISA formats to detect sample biomolecules. Suitable assays also include analysis of cells (e.g., using microbeads as a standard for calibration, normalization, or quantification in mass cytometry assays).

Thus, the microbeads may exhibit low fluorescence (e.g., may have a fluorescence quantum yield in the visible and/or UV range of less than 0.2, less than 0.1, less than 0.05, or less than 0.02), but will still be suitable for analysis by mass spectrometry (e.g., atomic mass spectrometry, such as ICP-MS) or another form of elemental analysis (e.g., ICP-OES, x-ray dispersion spectrometry).

The assay methods and kits described herein can be used to analyze 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., comprising suspended sample biomolecules). The sample may be a cell line, a cell culture suspension, or may be harvested 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., biopsy of solid tissue). The sample may comprise whole cells or may be homogenized from a sample containing whole cells (e.g., as a lysate thereof). In certain aspects, the sample can be a purified sample biomolecule characterized by an assay described herein or using a kit described herein. For example, an assay or kit may be screened for potential sample biomolecules that are drug candidates. Notably, additional steps can be performed in any of the methods described herein to remove unbound sample biomolecules from the microbeads, unbound reporter from the microbeads, or unbound mass-labeled antibodies from the cells.

In imaging mass spectrometry (IMC), a sample may be a tissue section labeled with a mass tag (e.g., a mass-labeled antibody) for analysis by imaging mass spectrometry (e.g., LA-ICP-MS or SIMS). In suspended mass flow cytometry, suspended particles (such as cells and/or beads) can be introduced to a mass flow cytometer (e.g., ICP-MS system) for analysis.

The mass spectrum of the present application may be an atomic mass spectrum. The mass spectrometer of the present application can detect multiple mass channels simultaneously. The different mass channels may correspond to metals or isotopes thereof from different mass tags. Such simultaneous mass spectrometers may be, for example, time-of-flight mass spectrometers (TOF-MS) or magnetic sector mass spectrometers. The mass spectrometer may atomize the sample for atomic mass spectrometry. For example, ICP, laser ablation ICP, secondary ions (e.g., for Secondary Ion Mass Spectrometry (SIMS)), or any suitable ionization source may be used to atomize the sample. Mass flow cytometry can specifically detect metals from the mass tags and/or microbeads described herein, and can, for example, include ion optics for filtering out lighter atoms (e.g., endogenous elements such as C, N, O and light metals, and optionally additional plasma gas elements such as argon and argon dimers if the mass spectrometer is an ICP system). Exemplary mass flow cytometers are further described in U.S. patent publication nos. 20050218319 and 20160049283, which are incorporated herein by reference.

An exemplary assay protocol is shown in fig. 15. The copolymer microbeads of the present disclosure may comprise a barcode. The bar code may be a plurality of metals or enriched metal isotopes chelated by the metal chelating monomer. The barcode may be an assay barcode (i.e., the sample biomolecules to which the identification microbeads are functionalized for binding). Alternatively or additionally, the microbeads may contain a sample barcode for identifying the sample with which the microbeads were or are to be mixed. The microbeads may be attached to (e.g., covalently bound to) biomolecules (e.g., capture biomolecules) that specifically bind to the sample biomolecules. The biomolecules may for example comprise affinity reagents (such as antibodies) that specifically bind to sample antigen biomolecules of interest (such as viral particles, cytokines, cancer biomarkers). The biomolecules may for example comprise oligonucleotides (such as ssDNA oligonucleotides) 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 biomolecules of each bead to bind to their respective sample biomolecules, as shown in fig. 15. Furthermore, as shown in fig. 15, the reporter may be bound 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 quality label. Thus, the methods of the present disclosure can include binding a sample biomolecule to a biomolecule (e.g., a capture biomolecule) attached to the barcoded microbead, and can further include binding a mass label reporter to the sample biomolecule. The bar code and mass label can then be detected by atomic mass spectrometry as described herein. Also, certain kits described herein can include optionally, barcoded microbeads attached to biomolecules that specifically bind to the sample biomolecules, and the kit can further associate a mass label with the reporter of the sample biomolecules.

In some embodiments, the kit comprises a population of microbeads or a population of a plurality of different microbeads, as described in any of the other embodiments herein. In certain aspects, the population of each microbead can be distinguishable from the population of another microbead (e.g., by atomic mass spectrometry) based on the metal or metals of the microbeads. For example, the microbeads in at least one population of microbeads (e.g., each population) may comprise different metals or multiple metals from the microbeads in another population of microbeads, or may comprise different ratios of multiple metals from the microbeads in another population of microbeads.

In some embodiments, the microbeads in each population of microbeads in the kit are conjugated to different biomolecules. In some embodiments, the kit may further comprise a reporter comprising a mass label, such as a reporter capable of specifically binding to a sample biomolecule that is specifically bound by at least one of the different biomolecules. Different biomolecules attached to the microbeads of the kit may specifically bind different sample biomolecules. The sample biomolecule may be any biomolecule described herein as being present in a sample. Such sample biomolecules may be proteins (e.g., or peptides thereof), oligonucleotides, lipids, carbohydrates, or small molecules. 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 comprise one or more modified residues.

The sample biomolecule may comprise an oligonucleotide (e.g., RNA), and at least one of the different biomolecules 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 a plurality of mass-labeled oligonucleotides to the sample biomolecule. Alternatively, a reporter comprising a mass label may be hybridized indirectly to a 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, wherein the antibody is capable of binding to the antigen and the oligonucleotide conjugated to the antibody is directly or indirectly bound (e.g., by hybridization) to a mass tag oligonucleotide of the reporter. Such indirect hybridization may allow for a variety of mass tags to be associated with the sample biomolecules, such as by hairpin linkage, branched in situ hybridization, or any other suitable method. Thus, in some embodiments, a reporter as described herein may (or may not) comprise a system of separate biomolecules that together associate a mass label with a sample biomolecule.

In some embodiments, at least one of the different biomolecules attached to the microbeads is an antibody (e.g., or fragment thereof, such as a nanobody). For example, the sample biomolecule is a viral particle, wherein at least one of the different biomolecules is a first antibody that specifically binds to the viral particle, and wherein the reporter comprises a second antibody that specifically binds to the viral particle.

In some embodiments, the at least one sample biomolecule is a cytokine, e.g., wherein at least one of the different biomolecules attached to the microbead is a first antibody that specifically binds the cytokine, and wherein the reporter comprises a second antibody that specifically binds the cytokine.

In some embodiments, the cytokine is selected from the group consisting of IL-18, IL-1F4, TNF alpha, IL-6, IFN gamma, IL-4, CD163, CXCL-9/MIG, IL-10, IL-1 beta, and combinations thereof.

In some embodiments, at least one sample biomolecule is a cancer biomarker (e.g., a prostate specific antigen), wherein at least one of the different biomolecules is a first antibody that specifically binds the cancer biomarker, and wherein the reporter comprises a second antibody that specifically binds the cancer biomarker.

In some embodiments, at least one of the different biomolecules comprises a viral antigen, wherein the sample biomolecule is an antibody that specifically binds to the viral antigen, and wherein the reporter comprises a second antibody that binds to the sample biomolecule.

The methods and kits of the present disclosure can include a plurality of different reporters, wherein each of the different reporters is capable of binding to a sample biomolecule that is specifically bound by a different biomolecule. The plurality of different reporters can each comprise the same mass label (detected in the same mass channel) or different mass labels (detected in different mass channels). The mass label can include metal nanoparticles, such as metal nanocrystals (e.g., gold nanoparticles), quantum dots, polymer nanoparticles, and the like. The nanoparticles may be or less than 100nm, less than 50nm, less than 20nm, less than 10nm in diameter, such as between 2nm and 100nm or between 5 and 50 nm. The mass label can include a metal chelating polymer, such as a linear or branched polymer comprising metal binding (e.g., metal chelating) side groups. Other mass labels may be suitable, such as an organic tellurium polymer mass label in which a tellurium atom is covalently bound to a carbon atom of the polymer, as well as within the scope of the present disclosure. The mass label may have one or more atoms of a metallic element or enriched isotope thereof. The mass label can be conjugated to a biomolecule (e.g., a reporter-binding sample biomolecule) by any suitable conjugation means described herein or known to those of skill in the art.

In some embodiments, the biomolecule attached to the microbead is an enzyme substrate for the sample biomolecule. The methods or kits of the present disclosure may further comprise a reporter that specifically binds (e.g., is capable of specifically binding) the enzyme substrate when the enzyme substrate has been modified by the sample biomolecule or specifically binds (e.g., is capable of specifically binding) the enzyme substrate when the enzyme substrate has not been modified by the sample biomolecule. For example, the enzyme substrate may comprise a kinase substrate, the sample biomolecule (e.g., target analyte) may be a kinase that phosphorylates the kinase substrate, and the reporter may be a phosphorylation-specific antibody that binds to the phosphorylated substrate. In another example, the enzyme substrate includes a mass tag that is removed when the enzyme substrate is modified by a sample biomolecule, such as a peptide sequence that is cleaved by the sample biomolecule as a protease. In some embodiments, therefore, the absence of a signal of the mass label may be indicative of the presence of a sample biomolecular enzyme. Examples of enzyme assays are described in U.S. patent publication No. 20070190588, which is incorporated herein by reference.

In some aspects of the kits and methods of the present disclosure, microbeads from multiple different populations are mixed (in a first microbead mixture). Aspects may further include a second mixture of microbeads comprising the same biological molecule as the first mixture of microbeads, wherein the microbeads of the first mixture comprise a sample barcode that is different from the microbeads of the second mixture. The sample bar code is, for example, on the interior of the microbeads (e.g., may be a metal subset sequestered by metal-chelating monomers of the microbeads of the first and second mixtures). Alternatively, wherein the sample bar code may be on the surface of microbeads of the first and second mixtures. The method or kit may include multiple sample barcodes in separate partitions that are functionalized to bind to the surface of microbeads from multiple different populations (e.g., microbeads wherein the sample barcodes of each partition are applied to different mixtures). The sample barcoded microbeads may be mixed together (e.g., after mixing with their respective samples, but optionally, prior to any mixing with the reporter) and then analyzed by mass spectrometry. Mass spectra from the sample bar code of each microbead can thus be used to identify the sample from which it is derived.

In certain aspects, the kit or method may further comprise a set of mass-labeled antibodies mixed with each other, wherein at least some of the antibodies of the set are specific for a cell surface marker (e.g., for binding to a cell surface protein in a cell sample). The plurality of antibodies can be mixed with microbeads of the kit, e.g., in a buffer solution or in lyophilized form (e.g., less than 5% or less than 1% moisture by volume). The plurality (e.g., each) of mass-labeled antibodies comprises the same metal as the metal 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, a cell as described herein may include one or more metals (or enriched isotopes thereof) that the non-naturally occurring cell typically comprises, such as an iridium intercalator that binds to DNA of the cell. Microbeads can be analyzed with cells as a quality standard and/or for detecting biomolecules (e.g., cytokines, antibodies, cancer biomarkers) in a sample solution (e.g., cell culture supernatant or serum).

In some embodiments, a kit comprising one or more populations of microbeads as described herein further comprises a steric stabilizer (e.g., mixed with the microbeads of the kit). As described herein, the steric stabilizer may be polyvinylpyrrolidone (PVP) in an amount such as greater than 0.05%, 0.1%, 0.2%, 0.5% or 1% by weight; such as about 0.05%, about 10% by weight or between them; about 0.1%, about 5%, or therebetween; about 0.2%, about 2%, or therebetween. Alternatively or additionally, the microbeads of the kit may be in a buffer solution at a pH of about 4, about 10, or between them; a pH of about 5, about 9, or between them; a pH of about 6, about 8, or between them; a pH of greater than about 3; a pH of greater than about 4; or a pH of less than about 10.

In some embodiments, the microbeads of the kits of the present disclosure are lyophilized. For example, the microbeads of the kit have a moisture of 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 between about 0.05% and about 5% by weight.

In some embodiments, microbeads may be fused to a solid support, such as for calibration (e.g., as particles), normalization, or quantification in an imaging mass spectrum (or imaging mass spectrometry) as further described herein. The solid support may 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 (e.g., for application to a microscope slide in any of the methods of the invention). The solid support may further comprise a biological sample.

In some embodiments, the solid support comprises at least 2, such as 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, such as at least 10000 fusion microbeads. The microbeads (reference particles) may all be the same, or the microbeads may differ in metal or their amounts. When different microbeads are used, there will typically be multiple microbead populations each.

The microbeads may be dispersed on the solid support such that substantially all of the microbeads are individually (i.e., discretely) located on the solid support such that each fusion microbead may be individually identified and sampled. It will be appreciated by those skilled in the art that the solid support may further comprise some fused microbeads that have been coagulated on the sample carrier, and thus these coagulates may not be suitable for sampling for calibration and normalization of signal intensity. For example, up to about 2%, such as 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 coalesce on the solid support. In other words, at least about 80% of the microbeads, such as at least about 85%, at least about 90%, at least about 92%, or at least about 95%, may be individually separated. The optical interrogation can identify which locations of the solid support have discrete microbeads and can direct acquisition by an imaging mass spectrometer.

The step of fusing the at least one microbead to the solid support may comprise heating the solid support. In some embodiments, the step of fusing the at least one microbead with the sample carrier or solid support comprises heating the sample carrier or solid support at a temperature above the glass transition temperature of the microbead and subsequently cooling the sample carrier or solid support below the glass transition temperature of the microbead. In other words, fusing at least one microbead to a sample carrier or solid support can occur by vitrification. In some embodiments, the sample carrier or solid support is heated to a maximum of up to 300 ℃, e.g., up to 275 ℃, up to 250 ℃, up to 225 ℃, or up to 200 ℃.

The kit of any of the embodiments described herein may further comprise one or more of the following: buffers (e.g., PBS, erythrocyte lysis buffer, wash buffer, staining buffer, and/or buffers for recombinant lyophilized reagents such as lyophilized microbeads, lyophilized reporters, lyophilized antibodies), anticoagulants (e.g., for processing blood samples), fixative reagents, permeabilization reagents, or any reagents for performing the methods or embodiments described herein.

The methods of the present disclosure include analyzing any microbead described herein by mass spectrometry. For example, a microbead (e.g., one or more populations of microbeads) can be an elemental standard for calibrating a mass spectrometer and/or for normalization (e.g., normalization of mass spectra obtained from mass labels) or quantification (e.g., quantification of the amount of antibody in a cell or pixel) as further described herein.

In some embodiments, the mass spectrometry method comprises: mixing a population of different microbeads of the present disclosure with a sample, wherein the microbeads in each population of microbeads bind to different biomolecules, and wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or metals of the microbeads; binding different sample biomolecules of the sample to different biomolecules of the population of different microbeads; binding the reporter directly or indirectly to each of the different sample biomolecules, wherein the reporter bound to each of the different sample biomolecules comprises a mass label; and the metal and mass labels of the individual microbeads were detected by mass spectrometry.

Such a method may further comprise attaching different biomolecules to the microbeads in the population of different microbeads prior to the step of mixing the population of different microbeads with the sample. One or more of the different sample biomolecules may include an oligonucleotide, an antibody (another affinity reagent), a cytokine, a cancer biomarker (such as one or more prostate specific antigens), and the like.

In some embodiments, the mass spectrometry method uses the kit of any of the embodiments described herein, and further comprises the steps of: mixing a population of different microbeads with the sample, wherein the microbeads in each population of microbeads bind to different biomolecules, and wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or metals of the microbeads; binding different sample biomolecules of the sample to different biomolecules of the population of different microbeads; binding the reporter directly or indirectly to each of the different sample biomolecules, wherein the reporter bound to each of the different sample biomolecules comprises a mass label; and detecting the metal and mass labels of each microbead by mass spectrometry.

In some embodiments, a kit or method for mass spectrometry of a cell sample using the microbeads of any of the embodiments described herein comprises the steps of: providing a plurality of mass-labeled antibodies, wherein each of the mass-labeled antibodies is conjugated to a polymeric mass label that chelates a plurality of atoms of the metal or enriched isotope thereof; contacting the sample with a plurality of mass-labeled antibodies; mass-labeled antibodies bound to the sample and microbeads were detected by mass spectrometry. The microbeads and cell samples may be combined prior to analysis by mass spectrometry, or may be analyzed in a separate sample run. Microbeads may be used to determine one or more sample biomolecules. Alternatively or additionally, the microbeads may be elemental standard for calibration, normalization, and/or quantification as further described herein.

The microbeads may be a single population of one or more metals as described herein, of uniform size and/or amount. Alternatively, the microbeads may be different populations each characterized by a different set of metals, combination of metals, and/or amount of metals. For example, a population of microbeads together have metals from more than 10 different elements (e.g., more than 30 different isotopic masses). For example, the microbeads of the present application may together 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). Microbeads may be added with cells in a suspension mass flow cytometry workflow or may be provided on the same carrier as a tissue section or cell smear in an imaging mass flow cytometry workflow. Microbeads may be used in assays as described herein, obtained from a mass channel that may be standard (e.g., provide a signal in most or all of the mass channels in which mass tags detected by mass spectrometry are detected). Mass spectra of different amounts of microbeads having the same metal (e.g., or enriched isotopes thereof) can be used to generate curves (e.g., signal intensity for known amounts of metal) for use in calibration, normalization, or quantification as further described herein.

The microbeads may be used to calibrate a mass spectrometer used in the detection step of any of the methods described herein. Calibration may be with respect to 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 peaks from a particular isotope. Mass resolutions higher than certain values may indicate eligibility. The quality calibration may include the following auto-tuning: examining the values of one or more mass channels (e.g., metals from the microbead standard) and then calculating the TOF values of additional mass channels and/or may include comparing the correct ions to the detection channels such that the full signal of each ion is collected. The dual count calibration may determine a dual count coefficient (for correlating pulse count and intensity (the dual count coefficient converts a similar signal to an ion count signal). When, for example, ion concentration increases during a cell or bead event and pulses overlap, this correlation may be important.

The microbeads may be used as a standard for normalizing mass spectrometry signals obtained from a mass label (e.g., a mass label of a sample as described herein) based on mass spectrometry signals obtained from the microbeads (e.g., obtained from microbeads comprising one or more of the same metals as at least one of the mass labels, or a similar mass spectrum, or a standard curve generated from a plurality of microbead populations comprising different amounts of metals). Each of the tags may provide signals in one or more mass channels. Such normalization of mass label signals may be used for individual cells or assay microbead events (e.g., in suspension mass flow cytometry) or individual cells, assay microbeads, or pixels (e.g., in imaging mass flow cytometry). For example, individual cells of a cell slide may be detected by IMC, or cells of a solid tissue section may be algorithmically segmented based on cell membrane staining, and mass tag signals in individual cells may be normalized as described hereinOr quantitative. The mass label signal of a cell or pixel may be normalized to a signal from a microbead detected within a time interval of the cell or pixel, such as 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 the time the cell or pixel was detected. Alternatively or additionally, if the microbeads used as standards include microbeads in different populations with different metals, the normalization of the mass label signal can be based on one or more microbeads whose metals are detected in the same mass channel as the mass label. Alternatively or additionally, if the microbeads used for the standard include microbeads in different populations having different amounts of metal, the normalization of the signal from the mass label containing the same metal may be based on one or more microbeads providing similar signal intensities (e.g., at or less than ten times difference, at or less than five times difference, at or less than twice difference, etc.) for the same mass channel. As described herein, the metal may be an enriched isotope. Thus, a microbead standard comprising a population of microbeads having different metals and/or amounts of metals may be used. Such normalization may be similar to the EQ4 provided by Fluidigm for normalization of mass spectrometry data (e.g., data that normalizes FCS files obtained by mass spectrometry) ^TM The use of beads. However, the microbeads of the present disclosure may together 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).

The microbeads may be used as a standard for quantifying the amount of one or more mass-labeled antibodies (or other mass-labeled biomolecules), such as when a known (or estimated) number of metal atoms from a mass label are associated with an antibody (or other biomolecule). For example, the number of metal atoms associated with a mass-labeled antibody (or other biomolecule) that may have mass tags of more than one instance may be determined by: starting from a known number of metal/mass tags (e.g., on a polymeric mass tag) and UV/visible spectrum or ICP-MS signal characteristics; and further analyzing the fraction of mass-labeled antibodies (or other biomolecules) by UV or visible spectroscopy or ICP-MS. This analysis can be performed on fractions obtained by flash protein liquid chromatography. The quantitative mass-labeled antibodies can thus be based on the average number of metal atoms of each mass-labeled antibody and the detected mass spectrum signal from the mass-labeled antibodies and microbeads. The microbeads may have a known number of metal atoms (e.g., as determined herein). The microbeads and mass-labeled antibodies (or other biomolecules) may comprise the same metal. Thus, quantifying a mass-labeled antibody can be calculated as the number of metals in the microbead multiplied by the ratio of the signal from the mass-labeled antibody (or other biomolecule) to the signal from the metal of the microbead and divided by the average number of metal atoms/antibody (or other biomolecule). Quantification may be performed on individual cells, assay beads or pixels.

Embodiments of the present disclosure include a computer-readable medium configured to perform one or more of calibration, normalization, or quantification as described herein.

In some embodiments, the detecting step may include inductively coupled plasma mass spectrometry (ICP-MS), such as for suspension mass spectrometry flow cytometry or imaging mass spectrometry. Wherein the mass spectrum may be by simultaneous mass spectrometry, such as time of flight mass spectrometry (TOF-MS) or magnetic sector mass spectrometry.

In some embodiments, the detection microbeads may be subjected to imaging mass spectrometry (e.g., imaging mass spectrometry). The microbeads (e.g., microbead standard) can be fused (e.g., melted) to the solid surface prior to the detection step. The imaging mass spectrometry may be, for example, by laser ablation ICP-MS or Secondary Ion Mass Spectrometry (SIMS).

The biological sample may comprise any sample having biological properties that need to be analyzed. For example, a sample may include a biomolecule, tissue, fluid, and cells of an animal, plant, fungus, or bacteria. They may also include molecules of viral origin. Typical samples include, but are not limited to, sputum, blood cells (e.g., PBMCs), tissue or fine needle biopsy samples, urine, peritoneal fluid and pleural fluid or cells therefrom. Biological samples also include tissue sections, such as frozen sections taken for histological purposes. Another typical source of biological samples is viruses and cell cultures of animals, plants, bacteria, fungi, where the status of gene expression can be manipulated to explore the relationship between genes. In some cases, other samples, such as artificial samples, may be interrogated. Certain aspects of the present disclosure are particularly useful when interrogating samples of human origin, and are particularly useful when interrogating samples of human peripheral blood.

The mass-labeled oligonucleotide may be hybridized directly or indirectly to the target oligonucleotide. For example, one or more intermediate oligonucleotides may provide a scaffold on which multiple mass-labeled oligonucleotides may hybridize, thereby amplifying the signal. Aspects of the application thus include signal amplification based on oligonucleotides for hybridization.

In some aspects, the sample biomolecule may be a target oligonucleotide, such as a DNA or RNA molecule (such as a coding RNA, a small interfering RNA, or a microrna) of a cell or bead. The target oligonucleotide may be single stranded. The target oligonucleotide may have a known specific sequence (or may have homology to a known specific sequence).

In some aspects, the reporter may include a non-oligonucleotide biomolecule (such as an antibody or derivative thereof) that may be conjugated to an oligonucleotide, such as a synthetic single stranded DNA oligonucleotide comprising a known sequence. In such cases, both the antibody and the oligonucleotide may be referred to as part of the reporter.

The signal from the target oligonucleotide or the reporter comprising the non-oligonucleotide biomolecule conjugated to the oligonucleotide may be amplified by a hybridization protocol. Hybridization may be branched or straight chain. In certain aspects, the polymerase can extend the first oligonucleotide along the template to provide additional sites for attaching element tags (such as additional hybridization sites for element labeling oligonucleotides). The mass-labeled oligonucleotide may comprise a single labeling atom, or may comprise a polymer comprising a plurality of labeling atoms, and may be referred to as a reporter. The mass-labeled oligonucleotide may include a labeling atom, such as a heavy metal atom, in the chemical structure of the oligonucleotide itself.

Signal amplification can be uniquely beneficial for bead-based assays, where the same reporter label (labeled metal element or isotope) can be amplified and used in different beads and their target analytes.

Where the assay biomolecule is an oligonucleotide, the element-labeled reporter oligonucleotide may be hybridized to another portion of the target RNA or DNA, thereby providing a signal when the target RNA or DNA is bound to the bead. Where the assay biomolecule is an affinity reagent (such as an antibody) that binds to the analyte at a first epitope, the elemental labeled reporter affinity reagent (e.g., a reporter antibody) may bind to another epitope on the analyte, thereby providing a signal when the target analyte binds to the bead. The analyte may be further bound by a reporter, such as an element-labeled reporter antibody or an oligonucleotide. The reporter may comprise a highly sensitive (e.g., intensity) elemental tag that provides a high abundance of isotopes (e.g., more than 50, 100, 200, 500, 1000 copies of a single isotope) to enable detection of a smaller number of target analytes bound to the assay beads. Such highly sensitive element tags may include nanoparticles (e.g., including metal nanocrystalline surfaces functionalized to bind biomolecules such as antibodies or oligonucleotides) or hyperbranched polymers. For example, a plurality of reporter biomolecules comprising the same nanoparticle elemental tag will provide a high signal and take advantage of the fact that individual reporter biomolecules comprising the same elemental tag can be distinguished by assaying barcodes (e.g., of beads presenting analytes for which they have specificity). In certain aspects, nanoparticle labels (e.g., gold nanoparticles) can be associated with reporter probes by biotin-avidin (e.g., biotin-streptavidin) interactions. For example, nanoparticles (e.g., gold nanoparticles) can be conjugated to streptavidin. The reporter may also contain a low sensitivity elemental tag that provides a low abundance isotope (e.g., less than 100, 50, 30, 20, 10, or 5 copies of the isotope) that is different from the high abundance isotope, thereby allowing for the detection/quantification of the amount of analyte that is so high that the high abundance isotope will saturate the detector. In certain aspects, the high-abundance isotope and the low-abundance isotope have a difference in mass (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. The reporter (e.g., an antibody comprising a target analyte bound to a different assay bead) for a different analyte may comprise the same isotope or combination of isotopes, as the analytes will be distinguished by the unique assay barcode of the bead.

In certain aspects, the reporter can include a reporter system that provides signal amplification through correlation of multiple instances of an elemental tag with a single instance of a target analyte (e.g., a sample biomolecule). Signal amplification may be by enzymatic deposition, hybridization (e.g., branched hybridization, strand hybridization, and/or hybridization of multiple reporter oligonucleotides to a single long intermediate oligonucleotide), extension (e.g., single extension, rolling circle extension), and/or a series of branched conjugation. In certain aspects, multiple (e.g., all) analytes detected with an assay bead can be detected with the same reporter system. In certain aspects, the signal amplification reporter system can have a highly sensitive elemental tag.

For example, an elemental tag comprising an enzyme substrate moiety may be deposited from solution onto a bead (or a molecule attached to a bead) by an enzyme attached to a reporter biomolecule. This reaction may be through covalent binding of the tyramine element tag by horseradish peroxidase bound to the reporter biomolecule.

Aspects include hybridization schemes that allow multiple element-labeled oligonucleotides to indirectly hybridize (via 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.

As described herein, mass flow cytometry may enable enough detection channels (mass channels) to detect barcodes of both the sample and assay in the beads, while allowing additional channels for reporter (e.g., for detection of assay targets). Thus, the bead assays described herein may be sample and/or assay barcoded for use in mass flow cytometry. For example, a variety of different conditions (e.g., drug candidates such as enzymes or agonists or antagonists of one or more enzymes) may be applied to the biological sample and their effect on the various targets may be detected with the enzyme assay beads. Individual conditions can be identified using sample barcodes shared between different assay beads exposed to the same conditions. The barcoded beads can be combined prior to analysis, such as prior to exposure to conditions. The sample barcoded beads may be combined prior to analysis.

In certain aspects, the enzyme may be a protease, a kinase, a phosphatase, or a DNA modifying protein such as a DNA methyltransferase. The target can be a substrate that is acted upon by an enzyme, and the reporter (e.g., a reporter biomolecule as described herein) can bind only to the target (e.g., a sample biomolecule) 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 may increase in abundance when acted upon by a kinase or decrease in abundance when acted upon by a phosphatase. When the enzyme is a protease, the reporter may be associated with the end of the peptide substrate and removed from association with the bead when the substrate is cleaved (such that a decrease in the reporter element tag indicates an increase in protease activity).

The sample bar code may be used to indicate which of a plurality of enzymes (or agonists/antagonists thereof) are tested in a particular assay. For example, candidate enzymes, agonists, antagonists may be added to a biological fluid such as a cell lysate, after which the sample is contacted with assay barcoded beads to detect the activity of the enzyme. Alternatively, the candidate may be administered to a cell (e.g., directly or by genetic engineering) or organism such as a patient or mammalian test subject, and a sample obtained from a counter-source may be contacted with the assay beads. Sample barcodes allow for parallel screening of many such candidates. In either case, sample barcodes may be added as described herein for the beads to identify candidates. For example, more than 10, more than 20, more than 50, more than 100, more than 500, or more than 1000 different samples may be barcoded. For example, 12 different isotopes in 6 unique combinations provide 924 different combinations (e.g., for barcoding up to 924 samples). Another 12 different isotopes can be used for barcoding up to 1000 assays. Thus, more than 10, more than 20, more than 50, more than 100, more than 500, or more than 1000 different assay beads (e.g., beads that detect the amount of different substrates acted upon by a candidate) may be barcoded. At least one channel will be left for detection of a substrate by a reporter, as described herein. This may allow for unprecedented screening with immediate readout by mass spectrometry.

Post-translational modification of proteins is performed by enzymes within living cells. Known post-translational modifications include protein phosphorylation and dephosphorylation, and 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.

Bead-based kinase assays for mass flow cytometry are discussed in U.S. patent publication US20070190588 (which is incorporated by reference) and summarized below. However, such bead-based assays have not been proposed for barcoding of both samples and assays, which offer screening advantages and are uniquely achieved by high renaturation mass spectrometry.

Kinase function is used to transfer phosphate groups (phosphorylations) from a high energy donor molecule (such as ATP) to a specific target molecule (substrate). Enzymes that remove phosphate groups from a target are referred to as phosphatases. The largest group of kinases are protein kinases, which act on specific proteins and modify their activity. Various other kinases that act on small molecules (lipids, carbohydrates, amino acids, nucleotides, etc.) are often named for their substrates and include: adenylate kinase, creatine kinase, pyruvate kinase, hexokinase, nucleoside diphosphate kinase, thymidine kinase.

Protein kinases catalyze the transfer of phosphate from Adenosine Triphosphate (ATP) to a targeted peptide or protein substrate at serine, threonine or tyrosine residues. Protein kinases are distinguished by their ability to phosphorylate substrates on discrete sequences. Commercially available kinases can be in active form (phosphorylated by the vendor) or in inactive form and require phosphorylation by another kinase.

Protein phosphatases hydrolyze phosphomonoesters at phosphoserine, phosphothreonine or phosphotyrosine residues into phosphate ions and protein or peptide molecules with free hydroxyl groups. This effect is completely opposite to that of protein kinases. Examples include: protein tyrosine phosphatases (which hydrolyze phosphotyrosine residues), alkaline phosphatases, serine/threonine phosphatases and inositol monophosphate.

Another aspect of the present disclosure is to provide a kit for detecting and measuring an element in a sample, wherein the measured element comprises an element tag attached to a phosphorylated substrate, an element of a metal ion coordination complex, and an element of a uniquely labeled support, the kit comprising: an element tag for directly labeling a phosphorylated substrate; a plurality of phosphorylated substrates; uniquely labeling the support; a metal ion coordination complex; optionally phosphatase, phosphatase buffer and ADP.

Another aspect of the present disclosure is to provide a method for kinase assay, comprising: incubating ATP, at least one kinase, a free metal ion coordination complex, and a plurality of non-phosphorylated substrates immobilized on an element-labeled support in a manner that allows a single type of non-phosphorylated substrate to be attached to a single type of element-labeled support under conditions that allow the kinase to phosphorylate the substrates; separating the plurality of phosphorylated substrates immobilized on the element-labeled support with the attached metal ion coordination complex from the free metal ion coordination complex and the plurality of immobilized non-phosphorylated substrates; and measuring by elemental analysis a plurality of phosphorylated substrates immobilized on an elemental-labeled support having an attached metal ion coordination complex.

Another aspect of the present disclosure is to provide a kit for detecting and measuring an element in a sample, wherein the measured element comprises an element tag attached to a non-phosphorylated substrate and an element of a metal ion coordination complex, the kit comprising: an element tag for directly labeling a non-phosphorylated substrate; a non-phosphorylated substrate solid support; a metal ion coordination complex; optionally kinase, kinase buffer and ATP.

Pharmacological modulation of enzymes has become a key element in the identification of possible therapeutic agents. Proteases are a subset of protein degrading enzymes that have recently been shown to play a critical role in signaling pathways, and their deregulation may lead to cancers, cardiovascular diseases and neurological disorders. Of the approximately 400 known human proteases, ten or more are being investigated as potential drug candidates. Small molecule inhibitors of proteases are now considered valuable therapeutic precursors for the treatment of degenerative diseases, for the treatment of cancer, as antibacterial, antiviral and antifungal agents. Bead-based protease assays for mass flow cytometry are discussed in U.S. patent publication US20170023583 (which is incorporated by reference) and summarized below. However, such bead-based assays have not been proposed for barcoding of both samples and assays, which offer screening advantages and are uniquely achieved by high renaturation mass spectrometry.

There is a need for a robust, sensitive and quantitative enzyme assay that allows simultaneous measurement of multiple enzyme reactions. Such an assay may allow for preservation of valuable biological samples and reagents, achieve high throughput and reduced assay time, and reduce the overall cost of enzymatic analysis.

One aspect of the invention is a method for detecting protease activity in a biological fluid. The method may include attaching a coding bead to a first amino acid of a peptide substrate to form an immobilized peptide substrate, the peptide substrate comprising the first amino acid and a last amino acid and being a substrate for a protease; attaching an element tag to the last amino acid of the peptide substrate to form a labeled peptide substrate; incubating the immobilized labeled peptide substrate with a biological fluid; and detecting the elemental signature and the encoded beads in the biological fluid by elemental analysis.

The encoded microbeads may be both assay and sample barcoded, as discussed herein.

The protease assay kit may comprise an assay encoding a first amino acid attached to a peptide substrate (immobilized peptide substrate), which may comprise the first amino acid and the last amino acid and may be a substrate for a protease. An elemental tag may be attached at or near the last amino acid of the peptide substrate to form a labeled peptide substrate. The encoded beads may be barcoded for both the assay and the sample, as discussed herein.

The mixture of assay beads can together target at least 5, 10, 20, 50, 100, 200, 500, 1000 or more analytes (sample biomolecules). In certain aspects, sample barcodes may distinguish between assay barcode beads and/or cells from at least 5, 10, 20, 50, or 100 or more different samples.

The sample barcoded reagent for a cell may include one or more elemental labeled antibodies (that bind to multiple cell types or a majority of cells in the sample), elemental tags that are functionalized to bind cells non-specifically (e.g., through covalent bond interactions), and/or metals in solution. The sample barcoded reagent for the cell may further include a reagent for entering the sample bar code into the cell (e.g., DMSO, a cell permeabilizing reagent such as a detergent or alcohol, etc.). Sample barcoded reagents for assaying barcoded beads may be present within the beads, on the surface of the beads, or may be applied to the beads. If used for application to the beads, the sample barcoded reagents may contain functional groups as described herein to bind to the surface of the beads (e.g., to bind to functional groups presented by the beads or to capping reagents present on the bead surface). The sample barcoded reagents for a given sample may contain a unique combination of isotopes specific for that sample. In certain aspects, cells from the same sample (e.g., a separate blood sample) and the assay barcoded beads can be labeled with the same assay barcode. The same assay barcode used to label cells and beads may contain the same combination of isotopes and/or the same means of same attachment (e.g., functional groups).

The sample barcoded reagents may be provided as a mixture with or with a set of antibodies, such as a lyophilized set of antibodies. The sample barcoded reagent may be provided as a mixture with or with the assay barcoded beads. The assay barcoded beads may be provided as a mixture with or with the antibody set. The assay barcoded beads and sample barcoded reagents may be provided as a mixture with or with a set of lyophilized antibodies (e.g., where the sample barcoded reagents bind to the assay barcoded beads and cells in the sample). In certain embodiments of the above, the sample barcoded reagents may be provided in, on, or with the sample and/or assay barcoded beads.

In some cases, the barcoded reagents may be provided in a preconfigured form by: barcoded reagents are prepared having a plurality of unique combinations of assay barcodes and sample barcodes. In such cases, each unique barcoded reagent may be stored in a different container, such as a different well of a well plate. In one example, the well plate may be established such that all wells along a particular column (or row) share the same assay bar code, while wells along a particular row (or column) share the same sample bar code. In another example, an aperture plate may be established such that each filled aperture contains a barcoded reagent having a specific unique sample bar code and various combinations of assay bar codes. Thus, a first well may contain barcoded reagents that all have a first sample bar code but each have a different measurement bar code, and a second well may contain barcoded reagents that all have a second bar code but each have a different measurement bar code. In some cases, pre-configuring the barcoded reagents may require the production of thousands of unique sets of beads.

In some cases, the barcoded reagents (e.g., beads) may be provided in a semi-configured form by: a barcoded reagent having a unique assay barcode is prepared and functionalized to bind to the surface of the sample barcode. In such cases, the barcoded reagent biomolecules (e.g., antibodies) of each set can be coupled to biomolecules (e.g., antibodies) having a targeting function associated with the assay barcode of the barcoded reagent of the set.

When a semi-configured barcoded reagent is provided, the sample bar code can be combined with the barcoded reagent prior to combining the barcoded reagent with the sample. In one example, different barcoded reagents may be mixed together and then placed in a set of receptacles (e.g., wells in an well plate). A unique sample barcode may then be added to each container, the results of which may be mixed with the unique sample to perform an assay barcode identifiable assay on the sample and simultaneously label the sample with the sample barcode.

When a semi-configured barcoded reagent is provided, the sample bar code may be combined with the barcoded reagent after combining the barcoded reagent with the sample. In one example, the semi-configured barcoded reagents may be provided together or otherwise mixed together. Barcoded reagents may then be added to each of the sample sets. Separately, a unique sample barcode may be mixed with each of a set of samples before or after the addition of the barcoded reagent. The sample barcode may label the barcoded reagents and/or cells or particles of the sample.

In one example case, the barcoded reagent may comprise an assay barcoded bead functionalized with polydopamine for attachment of capture antibodies. Another molecule (e.g., avidin) may be added with the capture antibody. After the capture antibodies are added to the assay barcoded beads, the beads can be mixed and divided into aliquots of each sample. For sample barcodes, a unique combination of elemental tags that are functionalized (e.g., with biotin) to bind the molecule may be added.

In some cases, elemental analysis may be performed on an individual particle basis, referred to as particle elemental analysis. Particle elemental analysis includes determining elemental composition of individual particles (e.g., cell by cell), such as using a mass spectrometer-based flow cytometer. Certain aspects of the present disclosure utilize particle elemental analysis on a cell-by-cell basis, which may be referred to as cytometry elemental analysis. In some cases, elemental analysis may be performed on an as-received basis, referred to as bulk elemental analysis or solution elemental analysis. Bulk elemental analysis involves determining the elemental composition of the entire volume of the sample.

Elemental analysis may be used to interrogate a sample, such as a biological sample. If the sample is marked with a known elemental signature, detection of the elemental signature during elemental analysis may indicate a characteristic of the sample associated with the elemental signature.

As referred to herein, mass spectrometry is any method of detecting elemental tags (mass tags) in a biological sample, such as detecting multiple distinguishable mass tags simultaneously with single cell resolution. Mass flow cytometry may include mass-labeled beads independent of or in addition to cell analysis. Any of the kits and methods of the invention may include or be suitable for mass spectrometry. Mass flow cytometry includes suspension mass flow cytometry and imaging mass flow cytometry (IMC).

Suspension mass spectrometry includes analysis of elemental-labeled cells and/or beads by mass spectrometry (e.g., by atomic mass spectrometry) and is described in U.S. patent publications, including US20050218319, US20150183895, US20150122991, which are incorporated herein by reference in their entirety.

Imaging mass flow cytometry (IMC) includes any imaging mass spectrum (e.g., imaging atomic mass spectrum) of an elemental-labeled biological sample, such as a tissue slice or a cell smear. IMC may be capable of atomizing and ionizing mass labels of a cell sample by one or more of laser radiation, ion beam radiation, electron beam radiation, and/or Inductively Coupled Plasma (ICP). Mass flow cytometry can detect different mass tags from a single cell simultaneously, such as by time of flight (TOF) or magnetic sector Mass Spectrometry (MS). Examples of mass flow cytometry include suspension mass flow cytometry (where cells flow into ICP-MS) and imaging mass flow cytometry (where a cell sample (e.g., a tissue slice) is sampled, for example, by laser ablation (LA-ICP-MS) or by a primary ion beam (e.g., for SIMS). Laser-based IMCs are described in U.S. patent publications US20160131635, US20170148619, US20180306695, and US20180306695, all of which are incorporated herein by reference. In certain aspects, when the sample is a cell smear analyzed by IMC, the cells may be treated as described herein, such as by staining with a lyophilization set, sample barcoding, and/or assaying barcoding. Similarly, the assay beads described herein can be analyzed by IMC alone or in a mixture with cells.

The mass labels may be sampled, atomized and ionized prior to elemental analysis. For example, mass labels in biological samples may be sampled, atomized, and/or ionized by radiation (such as a laser beam, ion beam, or electron beam). Alternatively or additionally, the mass label may be atomized and ionized by a plasma, such as an Inductively Coupled Plasma (ICP). In suspended mass spectrometry flow cytometry, whole cells including a mass tag can be flowed into ICP-MS (such as ICP-TOF-MS). In imaging mass spectrometry, the form of radiation can remove (and optionally ionize and atomize) portions (e.g., pixels, regions of interest) of a solid biological sample (such as a tissue sample) that include a mass label. Examples of IMCs include LA-ICP-MS and SIMS-MS of mass-labeled samples. In certain aspects, the ion optics may deplete the ions of the mass label rather than the isotopes. For example, ion optics may remove lighter ions (e.g., C, N, O), organic molecule ions. In ICP applications, the ion optics may remove gases (such as Ar and/or Xe), such as by a high pass quadrupole filter. In certain aspects, IMCs may provide images of mass tags (e.g., targets associated with mass tags) at cellular or subcellular resolution.

The present disclosure also provides the following embodiments:

embodiment 1. A metal-encoded microbead comprising:

a copolymer, the copolymer comprising:

structural monomers, and

a metal chelating monomer comprising a metal and a chelating agent;

wherein the chelator coordinates to the metal at least at 3 sites; and is also provided with

Wherein the structural monomer does not comprise the chelating agent.

Embodiment 2. Microbeads according to embodiment 1, wherein the structural monomer is selected from the group consisting of substituted or unsubstituted styrene, alpha-methylstyrene, acrylic acid and esters and amides thereof, methacrylic acid and esters and amides thereof and derivatives thereof, optionally the structural monomer is styrene.

Embodiment 3. The microbeads of embodiments 1 or 2, wherein said metal-chelating monomer has the structure of formula I prior to polymerization

Wherein the ligand is the chelator, L is the linker, X is the polymerizable end group, M is the metal, and n is 1 or an integer greater than 1, wherein the metal chelating monomer is neutral in charge prior to polymerization.

Embodiment 4. Microbeads according to embodiment 3, wherein L is selected from the group consisting of 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, alkylaryl, alkylheteroaryl, a C3-C8 cycloalkylheteroaryl, C (O) O, or mixtures thereof, optionally, each of said alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl is independently unsubstituted or substituted with one or more substituents selected from the group consisting of: 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 5. Microbeads according to embodiment 3 or 4, wherein L is attached to the chelator by an amide or an ester.

Embodiment 6. The microbead of any of embodiments 3-5, wherein said polymerizable end group is selected from the group consisting of aryl vinyl, styrene, alpha-methyl styrene, acrylate, methacrylate, acrylamide, 2-methacrylamide, and mixtures thereof, optionally said polymerizable end group is aryl vinyl or styrene.

Embodiment 7. The microbead of any of embodiments 1-6, wherein said chelator is tetradentate, pentadentate, hexadentate, heptadentate, or octadentate, optionally said chelator is hexadentate or octadentate.

Embodiment 8. Microbeads according to any of embodiments 1 to 7, wherein the chelating agent comprises an amino polyacid moiety or a derivative thereof.

Embodiment 9. The microbead of embodiment 8, wherein said amino polyacid moiety is selected from the group consisting of amino polycarboxylic acids, amino polyphosphonic acids, or combinations thereof.

Embodiment 10. The microbead of embodiment 8 or 9, wherein said amino polyacid moiety is a substituted oligomer of one or more of ethyleneimine, acrylamide or mixtures thereof, said oligomer being substituted with two or more carboxylic and/or phosphonic acids, optionally said oligomer being a crown ether or an aza crown ether.

Embodiment 11. The microbead of embodiment 10, wherein said oligomer is further substituted with one or more substituents selected from the group consisting of: 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 of embodiments 1 to 11, wherein the chelating agent is selected from DFO, EDTA, DTPA, EGTA, EDDS, EDDHA, BAPTA, H neupa, H6 phospha, H4CHXoctapa, H4octapa, H2 CHXddpa, 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.

Embodiment 13. The microbead of embodiment 12, wherein said TACN-type chelating agent is selected from NOTA, NOPO, TRAP or a derivative thereof.

Embodiment 14. Microbeads according to embodiment 12, wherein said DOTA of the cyclen type chelating agent or derivatives thereof.

Embodiment 15. The microbead of embodiment 12, wherein said cyclam-type chelating agent is selected from TETA, cross-linked bridged TETA, diAmSar, or derivatives thereof.

Embodiment 16. The microbead of embodiment 12, wherein said (13) aneN 4-type chelating agent is selected from TRITA or derivatives thereof.

Embodiment 17. The microbead of embodiment 12, wherein said 1, 10-diaza-18-crown-6-type chelator is selected from MACROPA or a derivative thereof.

Embodiment 18. Microbeads according to 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 said derivative of DTPA comprises DTPA wherein two adjacent carbon atoms are linked together with the atoms therebetween to form a 5-or 6-membered ring, optionally a cycloalkyl ring, an aryl or heteroaryl ring.

Embodiment 20. The microbead of embodiment 18, wherein said metal-chelating monomer is, prior to polymerization

Wherein L and X are as defined in any one of embodiments 4 to 6.

Embodiment 21. The microbead of embodiment 20, wherein said metal-chelating monomer is selected from the group consisting of

/>

Or a mixture thereof.

Embodiment 22. The microbead of any of embodiments 1-9, wherein said chelator comprises porphyrin or phthalocyanine.

Embodiment 23. The microbead of embodiment 22, wherein said chelator is a substituted or unsubstituted porphyrin.

Embodiment 24. The microbead of embodiment 22 or 23, wherein said metal-chelating monomer prior to polymerization is selected from the group consisting of

/>

Or mixtures thereof, and wherein n is an integer from 1 to 4.

Embodiment 25. Microbeads according to embodiment 24, wherein L is aniline.

Embodiment 26. The microbead of embodiment 24 or 25, wherein n is at least 2.

Embodiment 27. The microbead of embodiment 3, wherein said metal-chelating monomer is selected from the group consisting of

/>

Embodiment 28. The microbead of any of embodiments 1-27 further comprising a steric stabilizer, optionally selected from PVP, polyvinyl alcohol, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, polyacrylic acid, a water-soluble homopolymer of an acrylate, a water-soluble homopolymer of a methacrylate, a water-soluble homopolymer of acrylamide, a homopolymer of methacrylamide, a water-soluble copolymer steric stabilizer, or mixtures thereof.

Embodiment 29. The microbeads of embodiment 28, wherein said water-soluble copolymer steric stabilizer is selected from the group consisting of copolymers of acrylic acid esters, methacrylic acid esters, acrylamide or methacrylamide with methyl acrylate and/or ethyl acrylate, or mixtures thereof.

Embodiment 30. Microbeads according to any of embodiments 1 to 29, wherein said copolymer is crosslinked.

Embodiment 31. The microbead of any of embodiments 1-30, wherein said metal is a plurality of metals.

Embodiment 32. The microbead of embodiment 31, wherein said plurality of metals comprises one or more enriched isotopes.

Embodiment 33. The microbead of embodiment 31 or 32, wherein said plurality of metals comprises at least 2 metals, at least 3 metals, or at least 4 metals.

Embodiment 34. The microbead of any of embodiments 31-33, wherein the amount of each metal of said plurality of metals is within about 20% or about 10% of the amount of another metal of said plurality of metals.

Embodiment 35. Microbeads according to any of embodiments 1 to 34, wherein the metal comprises indium, bismuth or a rare earth metal, optionally selected from the lanthanide series metals, yttrium or mixtures thereof.

Embodiment 36. The microbead of embodiment 35 wherein said metal comprises a rare earth metal selected from the group consisting of Y, la, ce, pr, nd, pm, sm, eu, gd, tb, dy, ho, er, tm, yb, lu, different isotopes thereof, and mixtures thereof.

Embodiment 37. The microbead of embodiment 36, wherein said rare earth metal is selected from 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, 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.

Embodiment 38. The microbead of any of embodiments 1-37, wherein said metal is distributed throughout the microbead.

Embodiment 39. The microbead of any of embodiments 1-38, wherein said microbead has a glass transition temperature of about 60 ℃ or above 60 ℃, optionally about 70 ℃ or above 70 ℃, about 80 ℃ or above 80 ℃, about 90 ℃ or above 90 ℃, about 100 ℃ or above 100 ℃, about 115 ℃ or above 115 ℃, about 125 ℃ or above 125 ℃, or about 135 ℃ or above 135 ℃.

Embodiment 40. The microbead of any of embodiments 1-39, wherein said microbead has 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.

Embodiment 41. The microbead of any of embodiments 1-40, wherein said microbead is colloidally stable in water.

Embodiment 42. The microbead of any of embodiments 1-41, wherein the surface of said microbead comprises functionalization for attachment to a biomolecule.

Embodiment 43. The microbead of embodiment 42 wherein said attachment is covalent attachment.

Embodiment 44. The microbead of embodiments 42-43 wherein said biomolecule is selected from the group consisting of a protein, an oligonucleotide, a small molecule, a lipid, a carbohydrate, or a mixture thereof.

Embodiment 45. The microbead of embodiments 42-43, wherein said biomolecule is an affinity reagent, optionally wherein said affinity reagent is an antibody.

Embodiment 46. The microbead of embodiment 45, wherein said antibody is specific for a cytokine, optionally a chemokine, an interferon, a lymphokine, a monokine, an interleukin such as IL-1-36, tumor necrosis factor, and colony stimulating factor.

Embodiment 47. The microbead of embodiment 44, wherein said antigen is a viral antigen.

Embodiment 48. The microbead of any of embodiments 42-47, wherein said functionalization comprises a coating of silica on the surface of said microbead, optionally said functionalization further comprises functionalizing said coating of silica.

Embodiment 49. The microbead of embodiment 42 wherein said attachment to said biomolecule is a non-covalent attachment.

Embodiment 50. The microbead of any of embodiments 42-49, wherein the surface of said microbead is functionalized with avidin, streptavidin, neutravidin, or a mixture thereof.

Embodiment 51. The microbead of any of embodiments 42-50, wherein the surface of said microbead is conjugated to said biomolecule.

Embodiment 52. The microbead of any of embodiments 42-50, wherein said metal provides a barcode identifying said biomolecule.

Embodiment 53. A population of microbeads as defined in any of embodiments 1 to 52.

Embodiment 54. The population of microbeads of embodiment 53, wherein said population has a size distribution with a Coefficient of Variation (CV) of about 10% or less than 10%.

Embodiment 55. The population of microbeads of embodiment 54, wherein said coefficient of variation is less than 5%.

Embodiment 56. The population of microbeads according to any of embodiments 53-55, wherein each microbead comprises a plurality of metals, the average amount of each metal of said plurality of metals in the entire population of microbeads being within about 10% or 10% of the average amount of another metal of said plurality of metals.

Embodiment 57. The population of microbeads of embodiment 56, wherein said plurality of metals comprises one or more enriched isotopes.

Embodiment 58. The population of microbeads according to any of embodiments 53-57, wherein the amount of each metal of one microbead in the population of microbeads is within about 20% or 20% of the amount of the same metal of another microbead in the population of microbeads, alternatively within about 10% or 10%, alternatively within about 5% or 5%.

Embodiment 59. The population of microbeads according to any of embodiments 53-57, wherein the amount of each metal in said population of microbeads has a distribution with a coefficient of variation of about 20% or less than 20%.

Embodiment 60. The population of microbeads of embodiment 59, wherein the amount of each metal in said population of microbeads has a distribution coefficient of variation of about 10% or less.

Embodiment 61. The population of microbeads according to any of embodiments 58-60, wherein the microbeads in said population of microbeads comprise substantially the same amount of the same metal.

Embodiment 62. The population of microbeads according to embodiment 61, wherein said same metal is a plurality of metals and said microbeads comprise substantially the same amount of each of said plurality of metals.

Embodiment 63 a kit comprising a plurality of different populations of microbeads as defined in any of embodiments 53 to 62.

Embodiment 64 the kit of embodiment 63, wherein the population of each microbead is distinguishable from the population of another microbead based on the metal or metals of the microbeads.

Embodiment 65 the kit of embodiment 63 or 64, wherein the microbeads in the population of at least one microbead comprise a metal or metals that is different from the metal or metals of the microbeads in the population of another microbead.

Embodiment 66. The kit of embodiment 63 or 64, wherein the microbeads in the population of at least one microbead and the microbeads in the population of another microbead comprise different ratios of multiple metals.

Embodiment 67 the kit of any one of embodiments 64 to 66, wherein the microbeads in each population of microbeads are conjugated to different biomolecules.

Embodiment 68. A method of making a metal-encoded microbead comprising:

polymerizing the second mixture to form a copolymer of the microbeads;

wherein the structural monomer does not comprise the chelating agent.

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 a completion of about 5% to about 20% based on the structural monomer.

Embodiment 71 the method of any one of embodiments 68 to 70, wherein the polymerization of the second mixture occurs to about 75% to about 100% completion, about 80% to about 99% completion, about 85% to about 95% completion, 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 said metal is as defined in any one of embodiments 29 to 35.

Embodiment 76 the method of any one of embodiments 68-75 further comprising functionalizing said microbeads.

Embodiment 77 the method of embodiment 76, wherein said functionalizing of said microbeads comprises

polymerizing the third mixture.

Embodiment 78. The method of embodiment 77, wherein the reactive functional group is selected from the group consisting of alcohols, aldehydes, carboxylic acids, epoxides, vinyl, alkynes, maleimides, or mixtures thereof.

Embodiment 79 the method of embodiment 76, wherein said functionalizing of said microbeads comprises coating said microbeads with silica.

Embodiment 80. The method of embodiment 76 wherein said functionalizing of said microbeads further comprises functionalizing a coating of said silica.

Embodiment 81 the method of any one of embodiments 68 to 80, further conjugating the microbeads to a biomolecule.

Embodiment 82. The method of 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 diameters 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.

Embodiment 84 the method of any one of embodiments 68 to 83, wherein said microbeads are as defined in any one of embodiments 1 to 52.

Embodiment 85. A microbead prepared by the method of any of embodiments 68 to 83.

Embodiment 86. The microbead of any of embodiments 1-52, wherein the internal structure of said microbead comprises said copolymer.

Embodiment 87. The microbead of any of embodiments 1-52 and 86, wherein said metal-chelating monomer chelates a single metal atom, but not multiple metal atoms.

Embodiment 88. The microbead of any of embodiments 1-52, 86, and 87, wherein said microbead comprises a polymer seed that does not comprise said metal-chelating monomer.

Embodiment 89. The microbead of embodiment 88, wherein said polymer seed comprises a structural monomer; optionally, wherein the structural monomer of the polymer seed is structurally the same as the structural monomer of the copolymer.

Embodiment 90. The method of embodiment 68, wherein the internal structure of the microbead comprises the copolymer.

Embodiment 91 a method of making a metal-encoded microbead, said method comprising:

contacting the aqueous dispersion with a monomer comprising a structural monomer and a metal chelating monomer, wherein the metal chelating monomer comprises a metal and a chelating agent attached to at least one polymerizable end, wherein the chelating agent coordinates to the metal at least at 3 sites, and wherein the structural monomer does not comprise the chelating agent;

Initiating polymerization of the monomer in the aqueous dispersion of the swollen seed particles.

Embodiment 92. The method of embodiment 91, wherein the structural monomer is selected from the group consisting of: acrylic, methacrylic and vinyl monomers selected from the group consisting of styrene, divinylbenzene (DVB), ethylvinylbenzene, vinylpyridine, amino-styrene, methyl-styrene, dimethylstyrene, ethylstyrene, ethyl-methyl-styrene, 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 to 94, wherein providing an aqueous dispersion of swellable seed particles comprises preparing monodisperse swellable seed particles by emulsion polymerization.

Embodiment 96 the method of any one of embodiments 91 to 95 wherein the aqueous dispersion of swellable seed particles further comprises an organic compound having a molecular weight of less than 5000 daltons and less than 10 at 25 ℃ and optionally an organic solvent in which the organic compound is soluble ^-2 g/L water solubility.

Embodiment 97 the method of any of embodiments 91 to 96, wherein the swellable seed particles are monodisperse swellable seed oligomer particles.

Embodiment 98 the method of any 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 label, wherein the reporter specifically binds to a sample biomolecule specifically bound by at least one of the different biomolecules.

Embodiment 102. The kit of embodiment 101, wherein the sample biomolecule is an oligonucleotide, and wherein at least one of the different biomolecules 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 a plurality of mass-labeled oligonucleotides to the sample biomolecule.

Embodiment 104. The kit of any one of embodiments 101 to 103, wherein at least one of the different biomolecules is an affinity reagent such as an antibody.

Embodiment 105 the kit of embodiment 104, wherein the sample biomolecule is a viral particle, wherein at least one of the different biomolecules is a first antibody that specifically binds the viral particle, and wherein the reporter comprises a second antibody that specifically binds the viral particle.

Embodiment 106. The kit of embodiment 104, wherein the sample biomolecule is a cytokine, wherein at least one of the different biomolecules is a first antibody that specifically binds the cytokine, and wherein 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, wherein at least one of the different biomolecules is a first antibody that specifically binds the cancer biomarker, and wherein the reporter comprises a second antibody that specifically binds the cancer biomarker.

The kit of any one of embodiments 101-107, wherein at least one of the different biomolecules comprises a viral antigen, wherein the sample biomolecule is an antibody that specifically binds the viral antigen, and wherein the reporter comprises a second antibody that binds the sample biomolecule.

Embodiment 109 the kit of any one of embodiments 101 to 108, further comprising a plurality of different reporters, wherein each of the different reporters binds a sample biomolecule that is specifically bound by a different biomolecule.

Embodiment 110 the kit of embodiment 109, wherein each of the plurality of different reporters comprises the same mass tag.

Embodiment 111 the kit of any one of embodiments 101 to 110, wherein the mass label comprises a metal nanoparticle.

Embodiment 112 the kit of any one of embodiments 101 to 110, wherein the mass label comprises a metal chelating polymer.

Embodiment 113 the kit of embodiment 67, wherein at least one of the biomolecules is an enzyme substrate of a sample biomolecule.

Embodiment 114 the kit of embodiment 113, further comprising a reporter that specifically binds to the enzyme substrate when the enzyme substrate has been modified by the sample biomolecule.

Embodiment 115 the kit of embodiment 113, further comprising a reporter that specifically binds to the enzyme substrate when the enzyme substrate has not been modified by the 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 the sample biomolecule.

Embodiment 117 the kit of any one of embodiments 101 through 116, wherein microbeads from a plurality of different populations are in a first microbead mixture.

The kit of embodiment 118, further comprising a second mixture of microbeads comprising the same biological molecule as the first mixture of microbeads, wherein the microbeads of the first mixture of microbeads and the microbeads of the second mixture of microbeads comprise different sample barcodes.

Embodiment 119 the kit of embodiment 118 wherein the sample barcode is internal to the microbead.

Embodiment 120. The kit of embodiment 119, wherein said sample barcode is a metal subgroup sequestered by said metal-chelating monomer of said microbeads of said first mixture and said second mixture.

Embodiment 121. The kit of embodiment 118, wherein the sample barcode is 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 partitions, the plurality of sample barcodes functionalized to bind to surfaces of microbeads from a plurality of different populations.

Embodiment 123 the kit of any one of embodiments 101 to 122, wherein the kit further comprises a set of mass-labeled antibodies mixed with each other, wherein at least some of the antibodies of the set are specific for a cell surface marker.

Embodiment 124 the kit of embodiment 123, wherein the plurality of antibodies are mixed with microbeads of the kit.

Embodiment 125 the kit of embodiments 123 or 124, wherein at least some of the mass-labeled antibodies comprise the same metal as the metal chelated by the metal chelating monomer of the microbeads of the kit.

Embodiment 126 the kit of any one of embodiments 101-125, further comprising a steric stabilizer mixed with the microbeads of the kit.

Embodiment 127 the kit of embodiment 126, wherein the steric stabilizer is polyvinylpyrrolidone.

Embodiment 128 the kit of any one of embodiments 101 to 127, wherein the microbeads of the kit are in a buffer solution having a pH of 5, 9, or between 5 and 9.

Embodiment 129 the kit of any one of embodiments 101 to 125, wherein the microbeads are lyophilized.

Embodiment 130 the kit of 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 film.

Embodiment 133 the kit of any one of embodiments 101-132, further comprising one or more of a buffer, an anticoagulant, an immobilization reagent, and a permeabilization reagent.

Embodiment 134. A method comprising detecting a population of microbeads according to any of embodiments 53-62 by mass spectrometry.

Embodiment 135 the method of embodiment 134, further comprising calibrating a mass spectrometer for detecting the microbeads based on mass spectra obtained from the microbeads.

The method of embodiment 134, further comprising normalizing mass spectral signals obtained from a plurality of mass tags based on mass spectra obtained from the microbeads.

Embodiment 137 a method of mass spectrometry comprising:

mixing a population of different microbeads as in any of embodiments 53-62 with a sample, wherein the microbeads in each population of microbeads bind to different biomolecules, and wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or metals of the microbeads;

binding different sample biomolecules of said sample to said different biomolecules of a population of different microbeads;

binding a reporter directly or indirectly to each of the different sample biomolecules, wherein the reporter bound to each of the different sample biomolecules comprises a mass label; and detecting the metal and the mass label of each microbead by mass spectrometry.

The method of embodiment 138, further comprising attaching the different biomolecules to the microbeads in the different population of microbeads prior to the step of mixing the different population of microbeads with the sample.

Embodiment 139. The method of embodiment 137 or 138, wherein the different sample biomolecules comprise oligonucleotides.

The method of any one of embodiments 137-139, wherein said different sample biomolecules comprise antibodies.

Embodiment 141 the method of any one of embodiments 137 to 140, wherein said different sample biomolecules comprise cytokines.

Embodiment 142 the method of any one of embodiments 137-141, wherein said different sample biomolecules comprise cancer biomarkers.

Embodiment 143. A method of mass spectrometry using the kit of any one of embodiments 101-133, comprising:

mixing a population of different microbeads with the sample, wherein the microbeads in each population of microbeads bind to different biomolecules, and wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or metals of the microbeads;

Embodiment 144 a method of mass spectrometry of a cell sample using the microbeads of any of embodiments 63-74, comprising:

providing a plurality of mass-labeled antibodies, wherein each of the mass-labeled antibodies is conjugated to a polymeric mass label that chelates a plurality of atoms of a metal or enriched isotope thereof;

contacting the sample with the plurality of mass-labeled antibodies;

detecting the mass-labeled antibodies bound to the sample and the microbeads by mass spectrometry.

Embodiment 145 the method of embodiment 144, further comprising calibrating the mass spectrometer used in the detecting step, wherein the calibrating is based on a mass spectrometry signal obtained from the microbeads.

The method of embodiment 146, further comprising normalizing a mass spectrometry signal obtained from the mass tag based on a mass spectrometry signal obtained from the microbead.

Embodiment 147 the method of embodiment 144, further comprising quantifying the mass-labeled antibodies based on the average number of metal atoms of each of the mass-labeled antibodies and the detected mass spectrum signals from the mass-labeled antibodies and the microbeads.

Embodiment 148 the method of embodiment 146 or 147, wherein the microbeads and the mass-labeled antibody comprise the same metal.

Embodiment 149 the method of any one of embodiments 134-148, wherein the detecting step comprises imaging mass spectrometry.

Embodiment 150 the method of embodiment 149, further comprising quantifying or normalizing the mass labeled antibodies at each pixel or bound to each cell based on a mass spectrometry signal detected from the microbeads.

Embodiment 151 the method of embodiment 149 or 151, wherein the microbeads are melted to a solid surface prior to the detection step.

Embodiment 152 the method of any one of embodiments 134-151, wherein said detecting step comprises suspension mass spectrometry.

Embodiment 153 the method of any one of embodiments 134-152, wherein said detecting step comprises inductively coupled plasma mass spectrometry (ICP-MS).

Embodiment 154 the method of any one of embodiments 149-151, wherein the detecting step comprises laser ablation ICP-MS or Secondary Ion Mass Spectrometry (SIMS).

Embodiment 155 the method of any one of embodiments 134-154, wherein said mass spectrum is time of flight mass spectrum (TOF-MS).

The foregoing disclosure generally describes the present disclosure. A more complete understanding can be obtained by reference to the following specific examples. These embodiments are described for illustrative purposes only and are not intended to limit the scope of the present disclosure. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms are employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Examples

The following non-limiting examples are illustrative of the present disclosure:

example 1

Polystyrene (PS) microbeads with one or more lanthanoid elements chelated by DTPA

Metal-encoded PS microbeads were synthesized by introducing the polymerizable metal-DTPA complex as a second stage aliquot into the dispersion polymerization of styrene in ethanol. The synthesis of the components and microbeads and the materials used are described.

Material

Diethylene triamine pentaacetic anhydride (DTPA dianhydride, 98%), 2' -azobis (2-methylpropanenitrile) (AIBN, 98%), polyvinylpyrrolidone (PVP, mw-55 kDa), triton-X305 (TX 305, 70% aqueous solution), benzylamine (BA, 99%), allylamine (ALA, 99%), N- (3-aminopropyl) methacrylamide hydrochloride (APMAm, 98%), triethylamine (TEA, 99%), sodium acetate (anhydrous, > 99%), ammonium acetate (> 98%), sodium carbonate (> 99%), hydrogen peroxide solution (H) ₂ O ₂ ，30％H ₂ O solution) and metal salts having a purity of 99.99% or more (trace metal basis) (including yttrium (III) chloride hexahydrate (YCl) ₃ ·6H ₂ O), cerium (III) chloride heptahydrate (CeCl) ₃ ·7H ₂ O,), europium(III) chloride hexahydrate (EuCl) ₃ ·6H ₂ O), holmium (III) chloride hexahydrate (HoCl) ₃ ·6H ₂ O), lutetium (III) chloride hexahydrate (LuCl) ₃ ·6H ₂ O) and deuterium oxide (D) ₂ O, 99.9%) was purchased from Sigma-Aldrich. 4-vinylbenzylamine (VBA,. Gtoreq.92%) is supplied by TCI America. Absolute ethanol (EtOH) was produced by commercial alcohols. Nitric acid (trace metal grade, 68% -69%), sulfuric acid (trace metal grade), sodium hydroxide and phosphate buffered saline (1 x PBS solution, fisher BioReagents) were purchased from Fisher Scientific. All of the above chemicals were used without further purification. Styrene (St, sigma-Aldrich,. Gtoreq.99%) was purified by passing through a packed column of alumina (Sigma-Aldrich, activated, neutral). A standard solution of single elements for inductively coupled plasma mass spectrometry (ICP-MS) calibration was purchased from PerkinElmer (Pure Plus). Seven-element encoded microbeads (average per bead containing) ⁸⁹ Y(69×10 ⁶ )、 ¹¹⁵ In(43×10 ⁶ )、 ¹⁴⁰ Ce(17×10 ⁶ )、 ¹⁵¹ Eu(10×10 ⁶ )、 ¹⁵³ Eu(11×10 ⁶ )、 ¹⁶⁵ Ho(5.8×10 ⁶ )、 ¹⁷⁵ Lu(7.5×10 ⁶ ) And ²⁰⁹ Bi(5.8×10 ⁶ ) Described in Liu et al 2020 ¹⁵ . For mass flow cytometry (MC) EQ ^TM A four element (EQ 4) calibration bead is provided by the Fluidigm Canada friendly. Deionized water was produced by a Millipore purification system with a minimum resistivity of 18mΩ.cm. Compressed nitrogen (99.998%, praxair) was used as a protective atmosphere for the polymerization reaction.

Synthesis of functional DTPA-bis (amide) derivatives

To incorporate the DTPA metal complex into Polystyrene (PS) microbeads, DTPA is first functionalized by: by reacting DTPA dianhydride with 4-vinylbenzylamine, benzylamine, allylamine or aminopropyl methacrylamide (R-NH) ₂ = VBA, BA, ALA or APMAm) was reacted in a 1:2 stoichiometric ratio. The synthetic method was developed with minor modifications from the schemes reported below: zhang et al ¹⁶ Reported DTPA-bis (vinylbenzylamide) (DTPA-VBAm ₂ ) Aime et al ¹⁷ Reported DTPA-bis (benzylamide) (DTPA-BAm ₂ ) And Shuhendler et al ¹⁸ Reported DTPA-bis (allylamide) (DTPA-ALAm ₂ )。

In a typical experiment to prepare 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 eq.) in ethanol was added to the reaction to form the trisodium salt of DTPA-VBAm2, after which the reaction mixture was diluted with 45mL of acetone to precipitate the DTPA salt. The precipitated DTPA salt was then collected by sedimentation and redissolved in ethanol. Three cycles of dissolution-precipitation-sedimentation were performed to purify the product. The product was dried at room temperature under reduced pressure overnight to remove residual solvent and at room temperature by drying at D ₂ Proton nuclear magnetic resonance using a Varian 500MHz instrument (Agilent) in O solution ¹ H-NMR).

Synthesis of metal complexes of DTPA-bis (amide) derivatives

Metal complexes of DTPA-bis (amide) derivatives for bead synthesis (M (DTPA-R) were prepared by ₂ )): loading of metal ions to DTPA-R in aqueous solution ₂ And (3) upper part. In a typical metal chelation experiment, 0.30mmol DTPA-R was used ₂ Trisodium salt and equimolar amount of metal chloride (0.30 mmol) were separately dissolved in water (3 mL). The metal salt solution was then added to the DTPA derivative solution while monitoring the pH and adjusting it to 5.0-6.0 with 0.01M HCl and 0.01M NaOH solution. At Ce ³⁺ Ion chelation to DTPA-VBAm ₂ The clear solution of the mixture slowly became opaque as the metal chloride solution was added. In 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 3h. Isolation of M (DTPA-R) by acetone precipitation and sedimentation ₂ ) A complex. Note that the unchelated metal ions are soluble in the acetone-water mixture, whereas the metal complexes of DTPA derivatives have low solubility in acetone. The precipitate was then dissolved in ethanol and precipitated with acetone for further purification. The final product of the metal complex was collected by sedimentation and depressurized at room temperature Drying overnight. By using ¹ H-NMR to characterize the metal complex.

Two stage dispersion polymerization

Metal-encoded Polystyrene (PS) microbeads are prepared using two-stage dispersion polymerization (2-stage DisP) 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 mild solution of the required amount of DTPA metal complex in ethanol was introduced at 2h as a second stage aliquot. The reaction was terminated 24h after initiation, with styrene conversion above 90%. Table 1 describes a typical formulation of this 2-stage DisP for the preparation of microbeads.

TABLE 1 typical formulation for two stage dispersion polymerization of styrene for bead synthesis

a. The reaction was initiated by immersing the flask in a 70 ℃ oil bath. The reaction solution was purged with nitrogen for 30min before initiation.

b. A second stage aliquot was introduced to the reaction 2h after initiation.

c. A desired amount of DTPA derivative-metal complex (M (DTPA-R) ₂ ) Is introduced into the aliquot. Details of the metal addition are described in table 2.

Example 2

Using the method and materials of example 1 and different amounts of M (DTPA-R) as feed in the second stage ₂ ) Twelve batches of bead synthesis were prepared. These complexes were modified with different functional groups and loaded with different types of metal ions as described in table 2.

TABLE 2 DTPA derivative-metal complex (M (DTPA-R) ₂ ) A) feeding.

Vbam represents vinylbenzylamide;

bam represents benzylamide;

ALAm represents allylamine;

AmPMAm represents amidopropyl methacrylamide

e. Smaller amounts of Ho and Lu complexes were added to these syntheses to avoid saturation of the MC detector by the resulting microbeads.

f. The amount of metal complex addition was designed to achieve microbeads that produced similar intensity levels across all five isotopes.

After termination of the reaction, the microbead dispersion was washed twice with absolute ethanol and four times with water by a sedimentation-redispersion cycle to remove free stabilizer, unreacted monomer and any smaller diameter particles. These dispersion of washed microbeads were used for MC characterization and aliquots were freeze-dried to measure the solids content.

Example 3

Surface modification and secondary antibody attachment

The microbeads are coated with a silica shell and conjugated to a secondary antibody.

Materials for silica coating and bioconjugation

Tetraethylorthosilicate (TEOS, 99%), (3-aminopropyl) triethoxysilane (APTES, 99%), succinic anhydride (99%), anhydrous dimethyl sulfoxide (DMSO, 99.9%) were purchased from Sigma-Alderich. Ammonia solution 25% (NH) ₄ OH), MES buffer (0.5 m pH 5.5), phosphate buffered saline (1 x pbs, pH 7.4), N-hydroxysuccinimide (NHS), N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC), neutravidin (NAv), biotin-xx-goat anti-mouse (h+l IgG), and Bovine Serum Albumin (BSA) were ordered from ThermoFisher Scientific. ¹⁷⁵ Lu-labeled mouse anti-tnfα (human) (clone MAb 11), cell staining buffer and cell harvesting solution were provided by Fluidigm Canada friendly.

Preparation of metal-encoded microbeads functionalized with goat anti-mouse (antibody) through silica coating.

First byProcess metal-encoded microbeads (Eu-1) are coated with Silica (SiO) ₂ ). Typically, an aliquot of the bead dispersion containing 50mg of beads (solids) is washed in a centrifuge tube by a sedimentation-redispersion cycle with 5mL of ethanol-ammonia solution (ethanol: ammonia=99:1 volumes). TEOS (75. Mu.L) was added to the bead dispersion to initiate the silica condensation reaction. The reaction was stirred on a sample rotator (40 rpm, ambient temperature) for 20 hours and quenched by four sedimentation-redispersion cycles with 98% volume ethanol (5 mL). Silica coated Eu-1 beads (Eu-1/SiO) ₂ ) An aliquot of the dispersion (1 mL, about 10mg of solid) was treated in an ultrasonic bath for 5min and was prepared by adding APTES (5. Mu.L) to Eu-1/SiO ₂ Bead dispersion for further use with amino (NH) ₂ ) Functionalization. NH is added to ₂ The coating reaction was stirred in an oven at 40 ℃ for 20 hours and quenched by four cycles of sedimentation-redispersion wash with absolute ethanol (1 mL). To NH ₂ Modified microbeads (Eu-1/NH) ₂ ) The functional group on the catalyst is converted into Carboxyl (COOH), and freshly prepared Eu-1/NH ₂ An aliquot of the microbead dispersion (800 μl, about 8mg of solid) was washed with ethanol-TEA solution (1 mL, containing 0.2% by volume TEA) and subjected to four sedimentation-redispersion cycles, followed by addition of freshly prepared succinic anhydride solution in DMSO (100 μl, 10% by weight succinic anhydride). The COOH modification reaction was stirred at 40 ℃ overnight on a sample rotator (40 rpm) and then terminated by four cycles of sedimentation-redispersion washes with water (800 μl).

Next, NAv was conjugated to COOH-modified Eu-1 beads (Eu-1/COOH) by EDC/NHS coupling. In the conjugation reaction, an aliquot of Eu-1/COOH dispersion (100. Mu.L, about 1% solids) was washed four times with MES buffer (100. Mu.L, 0.1M, pH 5.5) to exchange solvent. EDC/NHS activation was initiated by adding EDC/NHS solution (100. Mu.L) containing EDC (8 mg) and NHS (12 mg) in MES buffer to the washed Eu-1/COOH dispersion. After 15min incubation, the reaction solution was quickly switched to 1 XPBS buffer (100. Mu.L; pH 7.4) by 2 cycles of centrifugation and then NAv solution (50. Mu.L, 2 mg/mL) was added. NAv conjugation reactions were incubated for 4 hours, after which unattached NAv was removed by 4 cycles of centrifugation washing with PBS buffer (100 μl).

Goat anti-mouse (GAM) secondary antibodies were attached to Eu-1 microbeads by: NAv modified Eu-1 beads (about 2 million beads) were incubated in BSA-PBS solution (100. Mu.L, 0.5 wt% BSA in PBS) containing biotin-xx-GAM (20. Mu.g) for 2 hours. Excess GAM was then removed by four cycles of centrifugation wash with BSA-PBS solution (100 μl).

Example 4

The size and size distribution and metal distribution of microbeads prepared using the methods and materials of examples 1 and 2 are characterized.

Characterization of microbead size and size distribution

Scanning Electron Microscope (SEM) images of the bead dispersion were collected using a Hitachi S-5200 microscope to characterize bead diameters and diameter distribution. Typically, 2 μl of the diluted bead dispersion is 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, see equation 2) are calculated based on at least 300 measurements.

Acid digestion of microbeads

The microbead dispersion was treated with sulfuric acid and H prior to ICP-MS measurement ₂ O ₂ And (5) digestion. In a typical digestion experiment, sulfuric acid (500 μl) and the microbead dispersion (100 μl,0.5% -2% solids) were mixed in a 20mL glass vial. The microbead dispersion in sulfuric acid was then heated on a hot plate to 250 ℃ and with 30% h ₂ O ₂ The addition of the solution (50. Mu.L) was maintained under magnetic stirring for 40min. For ICP-MS analysis, 2% HNO was then used ₃ The digestion solution is diluted.

Instrument for measuring and controlling the intensity of light

Inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS (iCAP-Q, thermo Scientific) was used to quantify the metal ion concentration in the samples. The samples were analyzed in kinetic energy discrimination (kend) measurement mode. Standard solution of element is treated with 2% HNO ₃ Serial dilutions were made to concentration series of 40, 20, 10, 1 and 0.1ppb as calibration solutions. The metal content in each solution sample was determined based on the calibration fitted curve. The detection limit of the element of interest is estimated to be below 10ppt.

Mass flow cytometry (MC). Through mass spectrometry flow cytometry systemCyTOF, fluidigm) characterizes the metal content of the microbeads on a bead-by-bead basis. Sample of seven-element encoded microbeads (7E 1) (which is reported in Liu et al 2020 ¹⁵ In) are used as internal standards and are mixed with a diluted sample of the microbead dispersion. The mixture consisting of the 7E1 reference beads and microbead sample was then introduced into the MC system at a rate of 30. Mu.L/min, and the beads were introduced into the ICP individually but randomly. After signal acquisition, unimodal events were identified and gated on the dot plot generated by MC. The mean, median, robust Standard Deviation (RSD) and Robust Coefficient of Variation (RCV) of the unimodal signal are reported as raw data that are not normalized. Robust statistics provide alternative pathways for classical statistical estimates such as mean, SD, and CV.

Results

The styrene conversion of the microbeads prepared using the methods of examples 1-3 was above 90%.

Four different types of DTPA derivatives were prepared as shown in scheme 1 below. This incorporation efficiency was tested using Ce (III) -DTPA complex as described below.

Functional DTPA-R ₂ Synthesis of derivatives

To covalently incorporate the metal complex of DTPA into PS microbeads, DTPA is modified with functional groups that can react with styrene during DisP. In this design, two of the five carboxylic acid esters on DTPA are substituted with functional groups, so the remaining three carboxylic acid esters in the functionalized DTPA derivative can form charge neutral complexes with the lanthanide (III) ions, which promotes binding stability and ethanol solubility of its metal complex in DisP.

The metal complex was synthesized by the method outlined in scheme 1. DTPA dianhydride reacts effectively with primary amine to produce DTPA bisamide ^21,22 . The bis (amide) derivative of DTPA is an octadentate chelator that binds strongly to lanthanide ions. It is assumed that the binding between metal ions and DTPA-bisamide is highly stable with respect to metal dissociation and exchange during bead synthesis in which different types of metal complexes of DTPA-bisamide are mixed in the reaction.

Scheme 1 was used for the synthesis of two-stage dispersion polymerized DTPA derivative-metal complexes. The metal chelator DTPA is functionalized by reacting DTPA anhydride with an amine containing a functional molecule in DMSO. Different types of metal ions are loaded onto the DTPA derivative at pH 5-6.

Two DTPA-bisamides that can be reacted with styrene via chain transfer reactions are prepared via: from DPTA-bis-benzylamide (DTPA-BAm) ₂ ) Benzyl carbon or DTPA-bis-allylamine (DTPA-ALAm) ₂ ) Allyl carbon of (2) ^25,26 Hydrogen abstraction was performed. The results were compared with DPTA-bis-4-vinylbenzylamide (DTPA-VBAm) ₂ ) Wherein it is expected that the 4-vinylphenyl group will have similar reactivity to styrene ^27–29 ) Those obtained in the above. Passing the resulting product through ¹ H-NMR characterization to confirm its structure.

FIG. 1 shows Na ₃ (DTPA-VBAm ₂ ) A kind of electronic device ¹ H-NMR spectra showing different chemical shifts of protons in vinyl groups at 5.3 (b, 2H), 5.8 (a, 2H) and 6.8 (c, 2H). Chemical shifts at 4.4ppm (f, 4H) indicate the formation of amide linkages. Integration of protons from vinyl, benzyl and DTPA moieties confirms the double substitution of vinylbenzyl groups per DTPA molecule. Shown in FIGS. 8A and 8B ¹ H-NMR spectra are described in the literature ^18,19,30 DTPA-BAm reported in ₂ And DTPA-ALAm ₂ And confirm successful synthesis of both DTPA-bis (amide) derivatives.

Test pass Ce (DTPA-R) ₂ ) Incorporation of metal complexes into PS microbeads

Ce(DTPA-R ₂ ) Synthesis of metal complexes. To examine the doping activity of different DTPA derivatives, ce (DTPA-VBAm) was prepared ₂ )、Ce(DTPA-BAm ₂ ) And Ce (DTPA-ALAm) ₂ ) Ce complex of (a). By mixing CeCl in a molar ratio of 1:1 ₃ Is added to water at pH 5.0-6.0 ₂ To synthesize a metal complex. Precipitation of Ce (DTPA-R) by addition of acetone ₂ ) Complexes, and passing them through ¹ H-NMR characterization to confirm metal chelation. These Ce complexes ¹ The H-NMR spectra are shown in FIGS. 9A to 9C. Because of Ce ³⁺ Is a paramagnetic NMR shift reagent, so the chemical shift of protons from these Ce complexes shifts and widens to some extent, indicating Ce ³⁺ Is a chelate interaction of (a) and (b). Several researchers reported several M (DTPA-BAm ₂ ) The crystal structure of the complex (m=in, Y, and Lu), and the metal coordination In the complex was confirmed using X-ray diffraction ^17,30 . Due to the similarity of material properties, a material is similar to that described herein (DTPA-R ₂ ) The metal coordination of the chelator may be similar to that reported for M (DTPA-BAm ₂ ) A complex.

Using Ce (DTPA-R) ₂ ) 2-DisP of metal complexes. Three kinds of Ce (DTPA-R) were examined ₂ ) Efficiency of incorporation of the complex into PS microbeads. As described in Table 2, an equimolar amount of Ce (DTPA-VBAm ₂ )、Ce(DTPA-BAm ₂ ) And Ce (DTPA-ALAm) ₂ ) Dissolved in ethanol and aliquoted as the second stageThe samples were introduced into the Ce-1, ce-2 and Ce-3 bead synthesis, respectively. From each of these syntheses, colloidally stable microbeads are obtained that retain their colloidally stability in water. The average diameter (d) of Ce-1 microbeads was 2.9 μm with a narrow size distribution (cv=1.2%) as shown in fig. 2. As summarized in Table 3, the Ce-2 and Ce-3 microbeads were also uniform in size (CV<1.5%) but with a slightly smaller average diameter (d=2.1 and 2.5 μm, respectively), possibly due to the chain transfer effect of benzyl and allyl groups, thus inhibiting the chain growth of PS ³¹ 。

The metal ion incorporation in the reaction was determined by ICP-MS. In use H ₂ SO ₄ +H ₂ O ₂ The total metal ion content in the reaction was determined after digestion of the sample. The microbeads in the reaction stock were separated by centrifugal sedimentation. The metal content in the supernatant was also analyzed by ICP-MS to quantify the metal ions remaining in the solution. Such as 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. This metal incorporation efficiency reflects the incorporation of the Ce complex into PS beads, since Ce is fed to the reaction as a Ce-DTPA complex, and Ce ions in all three Ce-complexes are expected to remain sequestered with DTPA derivatives during bead synthesis, due to their relatively high binding stability ²³ 。

The Ce incorporation efficiencies for the three microbead syntheses are presented in table 3. In Ce-1 synthesis, ce (DTPA-VBAm) was added to 74% of the reaction ₂ ) The complex was incorporated into PS beads. However, more than 97% of Ce (DTPA-BAm after the reaction ₂ ) And Ce (DTPA-ALAm) ₂ ) Remaining in solution. In other words, only 2.7% of Ce (DTPA-BAm) added to the Ce-2 and Ce-3 synthesis was incorporated respectively ₂₎ And 2.8% Ce (DTPA-ALAm) ₂ ) A complex.

As a supplementary measurement of Ce content, a composition containing a known amount was used ¹⁴⁰ Ce calibration microbeads as quantitative standards Ce-1, ce-2 and Ce-3 microbeads were examined by MC on a bead-by-bead basis. The Ce content in the sample beads was evaluated by: assuming MC signal intensities from different microbeads and in the same measurementProportional to the metal content, comparing the signal intensity between the sample and calibration beads ¹⁵ . Table 3 summarizes the Ce content of the three batches of microbeads as determined by MC.

In MC, ce-1 microbeads give rise to sharp and strong ¹⁴⁰ Ce signal (see fig. 11), where the value intensity is 6000 counts/bead and RCV is 6.2% (see fig. 11A). The Ce content in the Ce-1 microbeads was estimated to be 5.44X10 ⁷ Ce ions/beads (see table 3). In contrast, ce-2 and Ce-3 microbeads show very weak ¹⁴⁰ Ce signal intensity (fig. 11B and 11C, respectively). Ce content of Ce-2 and Ce-3 microbead samples was essentially the same at 1.6X10 ⁶ And 1.6X10 ⁶ Ce ions/beads.

To confirm the incorporation mechanism of the polymerizable Ce complex in the bead synthesis, another Ce complex Ce (DTPA-Ambam) was prepared using methacrylamide as a functional group copolymerizable with styrene ₂ ). Such as in Ce (DTPA-VBAm) ₂ ) In the preparation of (2), ce (DTPA-AmPMAm is prepared by ₂ ) Complexes: firstly reacting DTPA dianhydride with APMAm in DMSO, and then chelating Ce in water at pH 5-6 ³⁺ Ions. Fig. 8 (c) and 9 (d) ¹ H-NMR spectra confirm Na ₃ (DTPA-AmPMAm ₂ ) And Ce (DTPA-AmPMAM) ₂ ) Is a structure of (a). In the Ce chelate experiments, ce (DTPA-AmPMAm ₂ ) The seemingly specific Ce (DTPA-VBAm) ₂ ) Is more hydrophilic because of Ce (DTPA-AmPMAM ₂ ) The chelating solution without ethanol added is completely soluble. Under similar conditions as for the synthesis of Ce-1, ce-2 and Ce-3, equal amounts of Ce (DTPA-AmPMAm were used ₂ ) The complex was used as a ligand to prepare Ce-4 microbeads. Thus, ce-4 microbeads also have an average diameter of 2.9 μm, have a narrow size distribution (cv=1.3%) and produce a bright color ¹⁴⁰ Ce signal, median intensity 4000 counts/bead and RCV 5.7% (see fig. 11D).

Ce(DTPA-VBAm ₂ ) And Ce (DTPA-AmPMAM) ₂ ) Both complexes are efficiently incorporated into PS beads during DisP due to the reactive copolymerization between vinylbenzylamide/methacrylamide and styrene. Ce (DTPA-BAm) ₂ ) And Ce (DTPA-ALAm) ₂ ) The much less efficient incorporation of the complex is due to the low reactivity between benzylamide/allylamide as chain transfer agent and styrene under bead synthesis conditions.

TABLE 3 preparation of the metal chelators by Ce complexes of styrene with different DTPA derivatives (Ce (DTPA-VBAm) ₂ )、Ce(DTPA-BAm ₂ )、Ce(DTPA-ALAm ₂ ) And Ce (DTPA-AmPMAM) ₂ ) Four batches of Ce-encoded microbeads prepared from 2-stage DisP.

a. The average diameter (d) and Coefficient of Variation (CV) of the microbeads were characterized by SEM images of the microbead sample after sedimentation-redispersion wash;

b. metal content/microbeads were measured by MC using calibration beads as standards; the mean and standard deviation of the metal content of the microbeads were evaluated based on the median and robust standard deviation of their MC signal intensities;

C. the efficiency of incorporation of metal ions in the synthesis of the beads was determined by: the total metal ion content in the reaction was compared to the metal content remaining in the supernatant after the beads settled at the end of the reaction.

Evaluation of DTPA-VBAm for incorporation of other lanthanide metal ions into PS microbeads ₂ Chelating agents.

Using M (DTPA-VBAm) ₂ ) Preparation of metal-encoded microbeads of metal complexes

Synthesis of DTPA-VBAm ₂ With four other metal ions (Y ³⁺ 、Eu ³⁺ 、Ho ³⁺ And Lu ³⁺ ) Is a complex of (a) and (b). These syntheses were performed as described above for Ce (DTPA-VBAm ₂ ) The described method is similar. Y (DTPA-VBAm) ₂ )、Eu(DTPA-VBAm ₂ )、Ho(DTPA-VBAm ₂ ) And Lu (DTPA-VBAm) ₂ ) A kind of electronic device ¹ The H-NMR spectra are presented in FIGS. 10A to 10D, respectively. Eu (Eu) ³⁺ And Ho ³⁺ Is a paramagnetic NMR shift reagent. Eu (DTPA-VBAm) ₂ ) And Ho (DTPA-VBAm) ₂ ) Chemistry in NMR spectra of (C)The shift presents a shift profile similar to the spectrum of Ce (DTPA-VBAm 2). In contrast, Y ³⁺ And Lu ³⁺ Is diamagnetic, Y (DTPA-VBAm) in FIGS. 10S and 10D ₂ ) And Lu (DTPA-VBAm) ₂ ) No displacement effect is shown. The chemical shift of protons in vinylbenzyl functions in these spectra (5-8 ppm) is similar to those of chelators, while the chemical shift of protons of DTPA in the aliphatic region (1-4 ppm) is broadened, possibly due to the different DTPA-VBAm associated with metal chelation ₂ Interconversion of isomers ^30,32 . In the metal chelating experiments, attention should be paid to Ce (DTPA-VBAm) ₂ ) In contrast, ho (DTPA-VBAm) ₂ ) And Lu (DTPA-VBAm) ₂ ) Is less soluble in ethanol but more soluble in water. Without wishing to be bound by theory, this observation suggests that Ho (DTPA-VBAm ₂ ) And Lu (DTPA-VBAm) ₂ ) The polarity of the complex is higher than that of Ce (DTPA-VBAm) ₂ ) Possibly due to the stronger binding of Ho and Lu to DTPA chelators ²³ . It will be appreciated that preferably all of the metal complex is completely dissolved in the second stage aliquot during bead synthesis.

To examine the incorporation of these metal complexes into 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) in each synthesis were prepared using each of these different M (DTPA-VBAm 2) complexes, as described in Table 2. Microbeads synthesized from Y-1, eu-1, ho-1 and Lu-1 were uniform and similar in size (see Table 4). The MC signal intensity of the coding elements from these microbeads is powerful and narrowly distributed, as presented in fig. 12. The incorporation of a macroelement into each microbead was confirmed by the metal content assessed by comparing the MC signal intensities from the sample microbeads and calibration beads in the same measurement, as summarized in table 4. The incorporation efficiencies of metals in these bead syntheses assessed as described above were all in the range of 65% to 72%, with a slight increase in this range for lanthanide metal ions with larger ionic radii. (see FIG. 3A).

A batch of five-element encoded microbeads (5E 1) was synthesized by: 5mg of each M (DTPA-VBAm) ₂ ) Complex (m=y, ce, eu,Ho and Lu) was dissolved in 15g ethanol for use as a second stage aliquot in the DisP reaction. To investigate the incorporation interference between different elements when they were mixed in one reaction, the incorporation efficiency of individual metals in the synthesis of the beads was characterized by ICP-MS.

The average diameter of the 5E1 microbeads was 2.7 μm with a narrow distribution (cv=1.2%). The incorporation efficiency of these five metals shown in fig. 3A indicates that 63% -70% of the metal complex introduced into the 5E1 reaction was incorporated into the beads. The different metal ions added to the 5E1 bead reaction were incorporated into the microbeads with similar efficiency. Microbeads of seven-element code as calibration standard and MC signal intensity of isotope encoded in 5E1 bead ¹⁵ Measured together. Fig. 4A summarizes the median MC signal intensity of 5E1 beads, with error bars representing RSD of signal intensity. ¹⁴⁰ Ce、 ^151,153 Eu、 ¹⁶⁵ Ho sum ¹⁷⁵ The signal intensity of Lu is within the optimal MC sensitivity range>300 counts/bead) with narrow signal distribution (RCV<10%). From 5E1 beads ⁸⁹ The signal intensity of Y is less than 100 counts/bead due to the low transmission coefficient of 89amu channels in MC ¹⁴ . The metal content/5E 1 beads were calculated using MC signal intensity from the calibration beads. The results are presented in fig. 4B. The content of Ce, eu, ho and Lu in the 5E1 beads is in a similar range, 3.4 to 4.0X10 ⁶ Individual ions/beads. Due to 5mg Y (DTPA-VBAm) ₂ ) Middle ratio of 5mg other Ln (DTPA-VBAm) ₂ ) More Y atoms are present in the sample and therefore the Y content is slightly higher (5.7X10 ⁶ Individual ions/beads).

Control of metal ion incorporation in microbead synthesis

FIG. 3A summarizes the use of M (DTPA-VBAm ₂ ) Metal complexes as ligands Metal incorporation efficiency results in the bead Synthesis batches (Y-1, ce-1, eu-1, ho-1, lu-1 and 5E 1) described in Table 2. The x-axis in fig. 3 represents the ion radius of the incorporated metal ions. Despite the variation in metal feed in these bead syntheses, the incorporation efficiencies of the different metals in these batches overlap in the very close range 62-74%. It appears that the reactivity of the 4-vinylbenzylamide group is independent of the metal ion bound to the chelating agent. Thus, when M (DTPA-VBAm 2) is metal complexWhen the compound is used as a ligand, the metal incorporation efficiency in the bead synthesis is consistent.

M (DTPA-VBAm) ₂ ) Relationship between the feed of the complex and the metal content of the resulting PS microbeads. In fig. 5, the metal content present in the microbeads is plotted in solid symbols with respect to the amount of metal complex fed to the synthesis. Considering the slight batch-to-batch variation in bead size, the metal content in each bead was divided by the volume of the bead as the metal concentration in the bead shown as the y-axis in fig. 5. The solid data points in fig. 5 show the linear dependence of the metal content in the microbeads on the feed of the metal complex in the bead synthesis. Linear regression model fitting of these data served as a convenient guide to the change in the M (DTPA-VBAm) introduced in each reaction ₂ ) The level of metal content in the metal-encoded microbeads is designed. This is a significant improvement in experimental design and performance over the use of metal salts + acrylic acid as a means of preparing metal-containing PS microbeads. By this new method, a library of metal-encoded microbeads with various levels of metal content can be prepared, which can be individually identified as classifier beads for bead-based MC bioassays.

Preparation of a set of metal-encoded classifier microbeads with varying levels of metal content

In order to prepare a library of classifier beads with maximum variability, multiple metal isotopes must be encoded into microbeads with finely controlled metal content so that microbeads with various metal content can be resolved individually in the MC. A set of reaction conditions is provided that is capable of synthesizing classifier beads containing three different levels of metal ions with baseline separation as described in example 2. Intensity levels that differ by three times are selected. However, other intensity levels may be selected. For example, intensity levels differing by a factor of 2 or 2,5 may be selected.

Y(DTPA-VBAm ₂ ) (70. Mu. Mol) requires a longer time to dissolve in ethanol (15 g), possibly due to its high polarity. Therefore, ions other than Y ions are 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 different from the metal Chelating monomers are used together. For example, Y may be used in different concentrations. Three samples of four element PS microbeads containing Ce, eu, ho and Lu were synthesized at concentration levels differing by three times. These samples are represented as 4E1, 4E2 and 4E3 as shown in table 2. The purpose is to obtain microbeads produced by ¹⁴⁰ Ce、 ¹⁵¹ Eu、 ¹⁵³ Eu、 ¹⁶⁵ Ho sum ¹⁷⁵ MC signal intensity levels of Lu were 0.2, 0.6 and 1.8 times that of the 7-element encoded microbeads, respectively ¹⁵ 。

Microbeads obtained from the synthesis of 4E1, 4E2 and 4E3 are colloidally stable in the reaction stock and free of aggregates. The average diameter of these microbeads after washing by sedimentation-redispersion was in the range of 2.8-3.0 μm, with a narrow size distribution (CV < 1.5%), as shown in table 4. As presented in fig. 3B, the incorporation efficiencies of the different metal ions in the three bead syntheses were consistent and close to the levels observed in the synthesis described in fig. 3A.

The MC signal intensities of five encoded lanthanide isotopes from these three batches of microbeads are presented in fig. 6, with groups sorted by element. From 4E3 beads ¹⁴⁰ Ce、 ¹⁵¹ Eu、 ¹⁵³ Eu、 ¹⁶⁵ Ho sum ¹⁷⁵ The average MC signal intensity of Lu was highest among the three batches of beads and at similar levels of 2560, 2053, 2590, 2540 and 2530 counts/bead, respectively. The signal intensity distribution of the 4E3 beads was narrow, with an RCV value of less than 9%, indicating a narrow distribution of the lanthanide elements encoded in the beads. For all the encoded isotopes, the 4E1 and 4E2 beads also produced a sharp and narrowly distributed MC signal with intensities at about 200 and 700 counts/bead, respectively. The histogram in fig. 6 shows the clear baseline resolution between the three batches of microbeads. The signal intensity of the three batches of beads increased approximately three times from 4E1 to 4E3 beads.

The metal content of the three microbeads was evaluated by comparing the MC signal intensity with a 7 element calibration bead. Values are plotted in fig. 5 as open symbols against metal feed in the bead synthesis. These hollow symbol data points follow the linear trend line observed from the previous bead synthesis presented in fig. 5.

The synthesis of these three microbeads served as a display for the use of M (DTPA-VBAm) in bead synthesis ₂ ) As a convenient example of a polymerizable metal complex to prepare a library of five types of lanthanide isotope-encoded beads at three different concentration levels. The maximum variability of this pool of beads shown in fig. 6 is 1023 according to equation 1.

The doping efficiency between different metals has been shown to be more consistent than the previous method for synthesizing 7-element beads in Liu 2020. In Liu 2020, the incorporation efficiency varies greatly between different metals, making control labeling of microbeads difficult. For example, in Liu 2020, lu ³⁺ With the highest incorporation of all seven elements, about 50%, and Ce ³⁺ With the lowest efficiency. Ce added to the reaction mixture ³⁺ Only about 15% was incorporated into the beads. The doping efficiency of the lanthanoid element basically shows a tendency that smaller ions have higher doping efficiency.

As shown herein, construct M (DTPA-VBAm ₂ ) Is effectively copolymerized with styrene in a two-stage dispersion polymerization in ethanol in the presence of polyvinylpyrrolidone. This reaction resulted in PS microbeads with a diameter of about 2 μm and a very narrow size distribution (cv≡1% -2%). The metal complex incorporation efficiency is about 60% to 70%, which range increases slightly for lanthanide metal ions with larger ionic radii. This efficiency appears to be independent of the amount of metal complex introduced into the reaction for the concentration range examined. This reaction feature allows for the adjustment of specific metal content for samples of PS microbeads. This is a useful strategy for preparing calibration beads for MC and for preparing classifier beads for bead-based MC assays as described in example 6. Eu-labeled beads with goat anti-mouse antibody bound to the surface are effective in detecting Lu-labeled mouse IgG.

Example 5

Metal leakage from microbeads

Microbeads prepared according to examples 1 and 2 were stable with respect to metal ion leaching to aqueous mediaEvaluation was performed under several experimental conditions. Will be achieved by using M (DTPA-VBAm ₂ ) Sample 4E3 beads prepared from stage 2 DisP were used for this test. Washed microbead samples (0.5% solids) were suspended in 30mL of the following three different buffers also containing 1% PVP: sodium acetate (50 mM, pH 3.0), ammonium acetate (10 mM, pH 7.0), sodium carbonate (200 mM, pH 10.5). The samples were stored at 4 ℃. At various time intervals, each sample was first vortexed and then 2mL aliquots were taken. These aliquots were centrifuged and the supernatant collected to measure the metal content by ICP-MS. The percentage of metal that has leached from the beads was determined by ICP-MS based on a comparison of the metal content in the supernatant to the metal content in the bead dispersion. In parallel, a batch of microbeads prepared by the 2-stage DisP AA method was subjected to the same experimental conditions.

A batch of M (DTPA-VBAm 2) encoded beads (4E 3) was tested for stability against metal leaching during storage and under typical application conditions. As depicted in fig. 13, these DTPA-beads were stable (< 1.5%) relative to the loss of metal ions when stored in buffers ranging from pH 3 to 10.

Leaching stability of incorporated metals

The metal-encoded microbeads used in MC must be stable with respect to metal ion leaching during storage and under typical application conditions. Samples of these microbeads at 0.5 wt.% were dispersed in three aqueous buffers (sodium acetate, pH 3.0; ammonium acetate, pH 7.0; sodium carbonate, pH 10.5) and stored at 4 ℃. In parallel, another set of microbead samples were tested in the same buffer, but these beads were prepared by 2-stage DisP incorporating metal ions using Acrylic Acid (AA) as ligand, as reported in Abdelrahman et al 2009 ⁹ . Leakage of each element from the 4E3 beads into these solutions was monitored over time by ICP-MS.

Also monitored from M (DTPA-VBAm) stored in non-buffered water in the presence of 1 wt% PVP ₂ ) Metal leaching of the encoded microbeads.

FIG. 13 presents the results of a metal leaching experiment, wherein solid symbols refer to DTPA-beads (4E 3) and open symbols refer to AA-beads as a comparison . As depicted in fig. 13B, very little detectable leakage of any incorporated element was observed from both microbeads in pH 7 buffer for more than 100 days<0.06%). For 4E3 DTPA-beads stored in PVP solution at 4℃very little release of metallic elements was detected within 100 days (up to 0.3%) while AA-beads lost slightly more metal ions (for Ce) when stored under the same conditions ³⁺ About 1.3%). Aged microbeads in acidic (pH 3) and basic (pH 10.5) buffers showed higher levels of ion loss. Here, the leakage of elemental ions reached a plateau in 20 days, with no further loss in the next 80 days. Eu (Eu) ³⁺ 、Ho ³⁺ And Lu ³⁺ The loss from DTPA-beads was less in pH 3 buffer<0.2%) for Ce ³⁺ Higher (about 1.5%), presumably due to Ce ³⁺ The binding stability with DTPA chelators is weak. Overall, DTPA-beads showed less metal ion leakage and greater stability under four test conditions.

TABLE 4 preparation of the compositions by using M (DTPA-VBAm ₂ ) Metal content in microbeads prepared from styrene 2-stage DisP of Metal Complex

b. Metal content/microbeads were measured by MC using calibration beads as standards.

Example 6

Classifier beads

Surface modification and antibody attachment

Use by MC ¹⁷⁵ Lu-labeled Ab reporter assay secondary goat anti-mouse (GAM) as described in example 3Antibody (Ab) functionalized M (DTPA-VBAm ₂ ) Specific binding between GAM modified Eu-1 microbeads of the encoded beads.

Testing specific binding between classifier beads and reporter by mass spectrometry

For the specific binding experiments, GAM modified microbeads (Eu-1/GAM) as a classifier were first dispersed in BSA blocking solution (50. Mu.L, 3% BSA in PBS). After 30min incubation, will contain as reporter ¹⁷⁵ A cell staining solution (50. Mu.L) of Lu-labeled mouse IgG (6.25. Mu.g) was introduced into the Eu-1/GAM dispersion. The dispersion of classifier and reporter was incubated for 2 hours, followed by two cycles of centrifugal washing with cell harvesting solution (100 μl) to remove unbound reporter. Binding of the reporter on the classifier beads was checked by MC. To test for non-specific binding, NAv modified Eu-1 beads (without GAM attachment) were used as negative controls for the same binding experimental conditions and also checked by MC.

Fig. 14 illustrates a strategy for surface functionalization of a bead sample and subsequent attachment of abs to the bead surface. The microbeads were first coated with a thin silica shell as described in example 3, followed by the process described by Abdelrahman ³³ It is reported that amino groups are introduced in a two-step silica sol-gel reaction. TEOS and APTES are used in the silica coating process. Obtaining NH with silica crust and surface amino groups about 10nm thick ₂ Modified Eu-1 microbeads. The amino groups on the surface of the microbeads were then converted to carboxyl groups (COOH) by reaction with succinic anhydride in DMSO. The carboxylate groups act as functional groups for attachment of the bioaffinity agent and help reduce non-specific binding of the reporter to the microbead surface. Neutravidin (NAv) was covalently conjugated to COOH groups on the bead surface via EDC/NHS coupling. Finally, biotinylated GAM was attached to NAv modified microbead surfaces with strong biotin-avidin affinity.

To verify the functionality of GAM modified Eu-1 microbeads as classifier, selection was made ¹⁷⁵ Lu-labeled mouse IgG was used as a reporter and incubated with Eu-1/GAM beads. As a second Ab, GAM has mouse IgG against the reporter As indicated in fig. 7 (a). To test antigen recognition, a suspension of GAM modified microbeads (Eu-1/GAM) was first dispersed in a solution containing bovine serum albumin (50. Mu.L, 3% BSA in PBS). After 30min incubation, will contain as reporter ¹⁷⁵ A cell staining solution (50. Mu.L) of Lu-labeled mouse IgG (6.25. Mu.g) was introduced and incubated at 23℃for 2h, followed by two cycles of centrifugation wash with cell harvesting solution (100. Mu.L) to remove unbound reporter. Samples were checked by MC. As shown in FIG. 7B, the tip on the Eu-1/GAM microbeads ¹⁷⁵ The Lu signal peak, with a median intensity of 460 counts/bead (rcv=15%), indicates strong specific binding between GAM modified microbeads and the reporter. In contrast, the signal obtained for the samples of Eu-1/NAv microbeads without GAM attached gave a signal that was statistically minimized to zero.

Example 7

With CE/DTPA (DTPA-AmPMAm) using methacrylamide derivative Ce ₂ ) Encoded microbeads

To prepare the 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 into APMAm-DMSO solution and the reaction was stirred at room temperature overnight. NaOH (1 m,3 eq.) in ethanol was added to the reaction to form trisodium salt of DTPA-Ambam 2, followed by filtration with a syringe filter (0.2 μm). The filtrate was diluted with 45mL of acetone to precipitate the product. The precipitated DTPA salt was then collected by sedimentation and dissolved in ethanol. Three cycles of dissolution-precipitation-sedimentation were performed to purify the product. The product was dried at room temperature under reduced pressure overnight to remove residual solvent. By using ¹ H-NMR confirmed the structure of the product.

To prepare DTPA-AmaPMAM ₂ Ce complex of (D) with DTPA-AmaPMAm ₂ (0.3 mmol) and CeCl ₃ 7H ₂ O (0.3 mmol) was dissolved separately in 5mL and 1mL DI H, respectively ₂ O. The Ce solution was then added to DTPA-AmPMAM ₂ The pH was monitored simultaneously and adjusted to 6.0 with 0.01M NaOH solution. The mixture was stirred at room temperature for 3h. Separation of Ce (DTPA-AmPMAm by precipitation with acetone and sedimentation ₂ ) A complex. The precipitate was then dissolved in ethanol and reprecipitated with acetone for further purification. Also by ¹ H-NMR characterizes the final product.

Preparation of Ce (DTPA-AmPMAm) by typical two-stage dispersion polymerization ₂ ) Complex-encoded Polystyrene (PS) microbeads. PVP-55 (1.00 g), AIBN (0.25 g), triton X305 (0.35 g) was added to the flask and dissolved completely in a mixture of styrene (6.25 g) and ethanol (18.75 g). The solution was sealed in a flask and stirred with an overhead mixer. After purging with nitrogen at room temperature for 30min, the polymerization was initiated by immersing the flask in an oil bath at 70 ℃. 2h after initiation of the reaction, ce (DTPA-AmPMAm ₂ ) A gentle solution of the complex (54 mg, about 70. Mu. Mol) in ethanol (15.0 g) was injected into the reaction. The reaction was terminated 24h after initiation. A stable, coagulum-free bead dispersion was obtained and stored at 4 ℃.

The particles obtained in this example were agglomerate free, with a narrow size distribution (cv=1.3%) and an average diameter of 2.9 μm. Ce content in microbeads measured by mass flow cytometry was about 2.7x10 ⁷ Ions/beads. The 49% Ce added in the reaction remained in solution after termination of the reaction as assessed by ICP-MS. Thus, ce (DTPA-AmPMAM) ₂ ) The incorporation efficiency of the complex in the microbeads was estimated to be 51%.

A similar method can be used for other Ln ions.

M=ln ions, such as Y, ce, eu, ho and Lu

Scheme 2 was used for the synthesis of 2-stage dispersion polymerized DTPA derivative-metal complexes. The metal chelator DTPA is functionalized by reacting DTPA anhydride with an amine containing a functional group molecule in DMSO. Different types of metal ions are loaded onto the DTPA derivative at pH 5-6.

Example 8

Bioconjugation of microbead-nucleic acids

It will be appreciated that nucleic acids or oligonucleotides may be conjugated to microbeads using methods available in the art. For example, the 3' -hydroxyl group can be conjugated to a carboxylic acid (such as succinic acid) functional group on the functionalized microbead. For example, phosphoramidite derivatives of nucleotides can be used for conjugation. Oligonucleotides can be functionalized for conjugation (e.g., to microbeads or biomolecules as described herein), and are readily commercially available, such as from Integrated DNA Technologies (IDT). For example, oligonucleotides functionalized with biotin (e.g., desthiobiotin), amine, alkyne modifications, thiol modifications, acrydite, or NHS esters (such as azide) can be conjugated as described further herein.

Example 9 bead-based multiplexing assay by MC

Experiment

Material

The synthesis and characterization of the metal-encoded microbeads employed in this example are presented in example 10. The buffer used in this example was 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 (AuNP, 10nm,10 OD) were purchased from Abcam. Frozen human Peripheral Blood Mononuclear Cells (PBMC) were purchased from Immunospot (CTL, LP-188HHU 20130715).Streptavidin (Nanoprobe), EQ ^TM Four element calibration beads (EQ 4) and Cell-ID ^TM The Pd barcoded kit is provided by Fluidigm Canada friends.

Table 5 material formation: cytokine standards, capture abs and detection of biotinylated abs used in this study

a. The suppliers: r & D systems.

b. The suppliers: bioLegend.

Preparation of a set of metal-encoded classifier beads

To develop a bead-based assay in MC, a library of microbeads labeled with various metal ions, which can be individually identified by MC, was prepared. Described herein is the preparation of a polymer by introducing a polymer having the structure M (DTPA-VBAm) in a 2-stage dispersion polymerization (DisP) reaction ₂ ) A method of encoding microbeads with controlled levels of metal ions. Experimental details of the preparation and processing of metal-encoded classifier beads are presented in example 10. A set of 11 binary metal-encoded 3 μm Polystyrene (PS) microbeads was prepared using the method of the present disclosure as described in example 10.

To prepare classifier beads that can capture target analytes in an immunoassay, each type of metal-encoded bead is modified with one type of Ab specific for cytokine analytes.

Stimulation of PBMC for cytokine secretion

Frozen commercial human PBMC samples (CTL Immunospot) were thawed and added to pre-warmed RPMI serum-free medium (10 mL) containing 0.5mL CTL anti-aggregation wash supplement (20×). PBMCs in the samples were spun down by centrifugation (8 min,300 rpm). After centrifugation the supernatant was pipetted. PBMCs were then resuspended with mild serum-containing complete RPMI (10 mL). An aliquot of this PBMC suspension in RPMI (4.8 mL, 4.8X10) ⁶ Individual cells) were transferred to PS tubes and stimulated with PBS solution (100 μl) containing phorbol myristate acetate (PMA, 25 nmol) and ionomycin (100 nmol). Samples with stimulus were incubated at 37℃and 5% CO ₂ Incubate for 5h. The stimulated PBMC suspension was then centrifuged to settle the cells. The supernatant was collected as stimulated sample and stored at-80 ℃ prior to bead-based assay. In addition to the addition of the stimulus (PBS only), another aliquot of PBMC suspension in RPMI medium was treated in parallel under the same conditions. Will not be stimulatedSample collection was the control in this experiment.

Bead-based sandwich immunoassays for mass flow cytometry

To optimize assay conditions, several bead-based multiplexed assays were performed using a series of solutions with mixtures of cytokines and chemokines of known concentrations as standard samples. These mixtures were analyzed by MC to draw a standard curve. Fig. 16 shows the assay procedure developed in this study.

A series of quadruple assays were performed to analyze IL-4, IL-6, TNF alpha and IFN gamma in 10 standard samples. In these experiments, four types of capture Ab coated metal-encoded classifier microbeads were first mixed in approximately equal amounts in BSA solution (0.5% BSA in PBS). The classifier bead dispersion was then transferred to 10 wells in a 96-well filter plate (filter cut-off: 0.45 μm). One standard sample can be analyzed for each well containing about 2 million beads in a dispersion (50 μl). A series of 10 solutions (50 μl each) consisting of four analytes at 0, 0.31, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000, 20000pg/mL were added to the wells, respectively. The mixture of standard solution and classifier beads in each well was first stirred with a pipette and then incubated at room temperature on a microplate shaker (1200 rpm for 30s, then 900rpm for 2 h). After incubation, the solution in the mixture was removed by means of reduced pressure filtration through a built-in filter at the bottom of the well. Classifier beads with analytes captured on their surface were purified by washing with wash buffer (200 μl, 0.025% in PBS 20 For example, a liquid sample) to remove possible uncaptured analytes. Once the wash buffer was removed from the wells by filtration, a detection Ab mixture (100 μl, 0.5% BSA in PBS) containing four types of biotinylated abs (2.5 μg/mL for each Ab) was transferred to each well. Ab was detected by pipette agitation to re-separate classifier beads. The mixture of classifier beads and detection abs was incubated on a microplate shaker (900 rpm) for 1h at room temperature. At incubationThereafter, the mixture was filtered and washed by 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 (AuNP) as a reporter. The reporter dispersion was prepared by diluting (e.g., 200 times) the AuNP dispersion from the vendor with 0.5% BSA buffer. The mixture of classifier beads and AuNP reporter was incubated on a shaker (900 rpm) for 1h and washed by two filter-redispersion cycles with wash solution and two cycles with PBS buffer (100 μl). To measure multiple assays in a single MC run, the assay samples (100. Mu.L) in each well were barcoded with a unique palladium barcoded solution (40. Mu.L in Cell-ID ^TM 3 x dilutions of the stock solution in the Pd barcoded kit). The barcoded staining reaction was incubated for 30min with stirring and quenched by two filtration-redispersion cycles with 0.5% BSA solution (200 μl) and two cycles with water (100 μl). A total of 10 barcoded assay samples were combined in one test tube and checked by MC using EQ4 beads as calibration standard.

In a typical multiplex assay of samples with unknown analyte concentrations, different types of metal-encoded classifier beads that can capture target analytes are first mixed in equal amounts in BSA solution and then transferred to a 96-well filter plate. In each well, one unknown sample can be analyzed by adding an aliquot (50 μl) of sample solution to the well, the classifier bead dispersion (50 μl). The assay was then incubated, washed, stained with detection Ab and AuNP reporter in a similar procedure as described in the above assay for the standard sample.

Instrument for measuring and controlling the intensity of light

Mass flow cytometry (MC). The microbead sample was subjected to mass flow cytometry (Helios ^TM System, fluidigm) bead by bead characterization. In a typical MC measurement, a barcoded immunoassay sample and EQ4 beads as internal standards are pooled into a test tube and Is introduced into the MC system at a rate of 30. Mu.L/min. After signal acquisition, MC signals were normalized using the signal from EQ4 beads and de-barcoded to separate the results from the different assays. To analyze MC results of one assay sample, the single peaks of all classifier beads included in the assay are first identified and in ¹⁴⁰ Ce- ¹⁴² Gating on Ce dot plot. Each type of classifier bead is then gated based on its metal-encoded signature. Reporters, e.g. AuNP ¹⁹⁷ The median signal strength of Au is reported as the result.

Results

Bead-based multiplexed immunoassays in MC were developed. Metal-encoded microbeads are used as solid supports for immunoassays and gold (Au) NPs are used as reporters. The capture Ab of each immunoassay was coupled to one of a set of microbeads, each of which consisted of microbeads with uniform, different content of heavy metal isotopes. Once coupled with the capture Ab, microbeads from different groups as classifiers can be pooled together for multiplex assays and later separated after data acquisition. Collecting data on MC and passing through FlowJo ^TM And (5) software analysis. (see FIG. 16 for experimental design) immunoassays result in variability of Au NP reporter ¹⁹⁷ Au signal intensity, which is proportional to the amount of analyte bound to the surface of each microbead. Because MC quantifies the amount of different heavy metal isotopes in each microbead, a pool of microbeads can be separated into individual bead sets with the median value of NP reporter for each bead set ¹⁹⁷ Au signal intensity. Because of this feature, many assays can be performed simultaneously, allowing multiple quantitative analytes to be multiplexed in a single measurement. In addition, the concentration of the analyte in the sample may be determined by extrapolation from the internal standard. Synthesis of metal-encoded classifier beads by dispersion polymerization

It is envisaged that synthesis is carried out by altering the five types of lanthanide metal ions (La ³⁺ 、Pr ³⁺ 、Tb ³⁺ 、Ho ³⁺ And Tm ³⁺ ) Is binary coded 32 (2) ⁵ =32) types of classifier microbeads with intensity levels of 0 or about 1000 (±20%) counts/bead.In addition, ce can be ³⁺ Ions are incorporated into each bead at similar levels. Cerium isotopes ¹⁴⁰ Ce and ¹⁴² ce may act as a bead identifier to gate classifier signals in MC results. As a demonstration, a set of 11 binary metal-encoded 3 μm Polystyrene (PS) microbeads for bead-based assays were synthesized by a series of two-stage dispersion polymerizations (disps) following the bead synthesis protocol described herein in example 4.

Bead synthesis and characterization are described in example 10. The scanning electron microscope image of the bead sample C-1 (see Table 6) is presented in panel (a) of FIG. 22. These microbeads were shown to be uniform and have an average diameter of 3.0 μm and a CV of 1%. The median intensity level of the MC signal encoding the isotope is in the range of 1000-1200 counts/bead. The table provides a summary of the beads prepared, their average diameter (and CV value), and the pattern of markers and signal intensity in MC detection. These 11 types of microbeads are uniform (CV 1%) and share similar dimensions, with average diameters in the range of 2.8 to 3.0 μm. For antibody attachment, the beads were coated with antibodies, and abs attached to the corresponding classifier beads are listed in the table.

Table 6 summary of particle size, median intensity of MC signal and target analyte for classifier microbeads prepared by 2-stage DisP of styrene and M (DTPA-VBAm 2) metal complex.

a. The average diameter (d) and Coefficient of Variation (CV) of the bead samples were evaluated by measuring the diameter of at least 300 beads in their SEM images;

b. the median signal intensity of the microbeads was measured by MC using EQ4 beads as calibration standard. The Robust Coefficient of Variation (RCV) of signal intensity for each of these microbeads is in the range of 7% -9% (not shown in the table);

c. For capturing target analytes in an assay sample, a capture Ab is covalently coupled to the surface of the microbead

* No target analyte was assigned to the microbeads in this study. The microbeads may be surface modified with a bioaffinity reagent to capture the analyte.

In addition, metal ions incorporated into these microbeads produce MC signals at similar intensity levels of 800 to 1200 counts per bead. Because all microbeads (C1 to C11) carry Ce which generates Ce signal at similar level in MC ³⁺ Thus the mixture of all beads was analyzed in a single MC measurement and was measured from ¹⁴⁰ Ce- ¹⁴² Ce dot patterns separate the single peaks of 11 types of microbeads in one gating. See FIG. 17 (a)]The dot plot in fig. 17 (b-k) demonstrates a gating strategy to identify each type of classifier microbead individually based on the metal-encoded signature. Because the microbeads in the classifier beads of this group are uniform and have MC signal intensities at similar levels, gating templates are created in FlowJo software based on this microbead gating strategy to simplify the data analysis process for multiple assays.

Initial optimization of bead-based sandwich immunoassay conditions

To test and optimize the reagents and conditions used in multiplexed sandwich immunoassays, these immunoassays are used to measure cytokine levels in a range of solutions with known concentrations of cytokines as standard solutions. The effect of different assay reagents and conditions on the assay results was evaluated by examining the standard curve from these measurements. In the first stage of assay development, a series of quadruple assays using standard solutions test candidate reporters, which are commercially available streptavidin conjugated nanoscale Au reporters, and are used to optimize the concentration of the reporter in the assay. The assay was then extended to a nine-fold assay to investigate the effect of detecting Ab concentration on assay performance.

In an exemplary experiment, a series of solutions containing known concentrations of target cytokines were prepared by diluting the standard solution with 0.5% BSA buffer (0.5% BSA in PBS). Each of these standard solutions (50 μl) was mixed with a mixture of Ab-modified classifier beads (50 μl) in the filter plate. During incubation, the Ab-modified classifier beads in the assay are allowed to capture their target analytes in the sample. After the 1-hour incubation period, the incubation period,the classifier beads were washed by filtration to remove unbound molecules in the sample. The mixture 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 mixture can recognize the analyte molecules captured on the surface of the classifier beads. These classifier beads in the assay were then washed on a filter to eliminate unbound detection Ab. Next, streptavidin conjugated Au mass label dispersion (100 μl) was applied as a reporter to the assay. These mass tags are capable of detecting abs by biotinylation attached to classifier beads by streptavidin-biotin interactions ⁴⁷ . After washing away unattached reporter particles, the metal content of the sample in each microbead event was determined by MC examination. An exemplary design of this bead-based sandwich immunoassay is shown in fig. 16.

After signal acquisition, the measurement results were analyzed in FlowJo software by its dot plot. Figure 17 shows the gating strategy employed in the study. The unimodal events with classifier beads were first isolated in group (a) of fig. 17. Each classifier bead in the gated unimodal event was then identified individually by a series of gating steps in the dot plot shown in panel (b-k) of fig. 17. Then check for each classifier event ¹⁹⁷ Au signal and reported as assay signal.

And (3) selecting a reporter. To find the appropriate metal-labeled mass label to report analyte binding on classifier beads, two types of commercially available streptavidin conjugated reporters were examined, namely small diameter gold clusters @d.apprxeq.1.4 nm) and slightly larger diameter gold nanoparticles [ AuNP, d.apprxeq.10 nm). These were used as reporters in a series of quadruple assays to analyze standard solutions consisting of four analytes at 12 concentrations.

The histogram shown in FIG. 18 demonstrates the signal strength of the reporter on the IL-4-classifier bead in the quadruple assay. AuNP was used as a reporter in the assays shown in FIGS. 18 (a-c). When IL-4 is not present in the blank solutionSome positives ¹⁹⁷ The Au signal is recorded in fig. 18 (a). The median value of all events in this figure is reported as background noise control to reflect the non-specific binding of reporter NP to classifier beads. IL-4-classifier bead ¹⁹⁷ The intensity peak of the Au signal shifted to the high field in groups (b) and (c) of FIG. 18 as the IL-4 concentration in the assay increased from 1.2 to 20 pg/mL. At 1.2pg/mL, there were nearly 0 counts/bead ¹⁹⁷ Some events of Au signal intensity are recorded in fig. 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 below the MC detection limit. To eliminate uncertainty and simplify the data analysis process, positive events (reporter signal intensity) in positive samples were reported in this study>1 count/bead) and plotted.

The histograms shown in sets (d), (e) and (f) of FIG. 18 will beUsed as a reporter to analyze standard solutions containing IL-4 at concentrations of 0, 1.2 and 20pg/mL, respectively. Compared to the results of assays using AuNP as a reporter, IL-4-classifier beads were observed ¹⁹⁷ Au>The intensity of the reporter signal is much weaker. More events with signal intensity of-0 exist in the histogram shown in group (e) compared with group (b) of fig. 18.

When the analyte concentration is low, the typical log-log standard curve for bead-based assays begins with a low and flat region. The curve then rises with increasing analyte concentration, followed by a plateau at higher concentrations ^41,43,48 . In this embodiment, the detection range of the assay is estimated based on the slope region of the standard curve. Within this slope region, the analyte concentration is typically measurable.

Figure 19 summarizes the median MC signal intensity for different types of NPs attached to classifier beads at different analyte concentrations. In the low concentration region (about 1pg in height)Per mL) the signal intensity of both types of reporter is low (-10 counts/bead) and insensitive to increases in analyte concentration. At higher analyte concentrations ranging from 1 to 1000pg/mL, the signal intensity of these reporters increases significantly with increasing analyte concentration. The AuNP signal intensity approaches the plateau at analyte concentrations above 1000pg/mL, and is usedThe curve of the assay as a reporter shows a similar trend but much lower intensity than using AuNP as a reporter. Because much higher signal intensities were obtained at the lower analyte concentrations in these assays, auNP was chosen as the reporter candidate for the subsequent bead-based assays in this study to optimize the lower detection limit.

Optimization of reporter concentration. The effect of NP concentration on the measured signal intensity level was then studied. In this study, stock AuNP dispersions were diluted 200, 400 and 800-fold with 0.5% BSA buffer. These dilutions were then used as reporter solutions in three sets of quadruple assays to analyze standard solutions. Fig. 23 shows standard curves for quadruple assays performed at different AuNP concentrations. The signal intensity of AuNP at all three dilutions showed little difference at analyte concentrations above 20 pg/mL. The detection limit of the assay stained with 200 Xdiluted AuNP dispersion was as low as 0.3pg/mL for IL-4, IL-6 and TNF. Alpha. And slightly higher at 1.2pg/mL for IFN. Gamma. 200 XAuNP dilutions were chosen for bead-based assays in this study.

Optimization of detection Ab concentration (in a nine-fold assay). After using a series of quadruple assays as proof of concept experiments, the multiplexing capacity of the assays was extended to nine analytes by adding the assays of IL-1 beta, IL-10, IL-18, CD163 and CXCL-9 to the group. Experimental conditions developed in the quadruple assay were suitable for analysis of a series of standard solutions containing nine analytes. In the nine-fold assay of the first set, the concentration of each type of biotinylated Ab in the detection Ab mixture was 2.5ug/mL. The results are plotted as filled circles (+) in fig. 20. The median intensity levels of AuNP signal on all types of classifier beads were observed to be higher (100 counts/bead) compared to the quadruple assay at low analyte concentrations (. Ltoreq.1.22 pg/mL). Without wishing to be bound by theory, these relatively high levels of signal intensity at low analyte concentrations may be attributed to background noise in the assay, which may be the result of non-specific binding.

To minimize non-specific binding in the nine-fold assay, three sets of nine-fold assays were performed in which the concentration of biotinylated anti-CD 163 and anti-CXCL-9 was reduced to 2.0, 1.0, and 0.5 μg/mL, while the concentration of other detection abs was maintained at 2.5 μg/mL in the detection Ab mixture. The concentrations of anti-CD 163 and anti-CXCL-9 detected were reduced in these assays, in part because at high Ab concentrations CXCL-9 and CD163 may cross-react with Interleukin (IL) and interfere with detection Ab potentially resulting in more non-specific binding. These nine-fold measured standard curves are plotted in figure 20. For assays containing standard solutions of low concentrations (. Ltoreq.1.22 pg/mL) of analyte, the median signal intensity in assays with low concentrations (0.5 and 1.0. Mu.g/mL) of biotinylated anti-CD 163 and anti-CXCL-9 Ab were significantly lower than those obtained with higher concentrations (2.0 and 2.5. Mu.g/mL) of detection Ab. In the determination of standard solutions with high analyte concentrations (. Gtoreq.1250 pg/mL), the signal intensity level and the sensitivity of the determination are similar under all experimental conditions. In general, when the concentration of biotinylated anti-CD 163 and anti-CXCL-9 Ab in the detection Ab mixture is reduced to 0.5 and 1.0 μg/mL, the detection sensitivity and detection range of the analyte at low analyte concentrations is improved. As shown in fig. 24, the background noise in these nine-fold assays for the blank solution was also significantly reduced as the concentration of biotinylated anti-CD 163 and anti-CXCL-9 Ab was reduced from 2.0 to 0.5 μg/mL. Based on these results, it appears that non-specific binding of biotinylated anti-CD 163 and anti-CXCL-9 to classifier beads can be minimized by reducing its detection Ab concentration in the assay.

Bead-based sandwich immunoassay for cytokines in biological samples

To examine the performance of nine assays on biological samples, commercial PBMC samples (from healthy donors) were used. PMA/ion was used to suspend one PBMC sample in cell culture Medium (RPMI)The mycin was stimulated and the supernatant of stimulated PBMC suspension was analyzed for extracellular release of cytokines using the above optimized assay conditions. PMA has a structure similar to diacylglycerol and can diffuse into the cytoplasm through the cell membrane. In the cytoplasm, PMA activates protein kinase C. When used in combination with ionomycin, a calcium ionophore that triggers calcium release, appropriate levels of cytokines are released from the cells. The 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 2 x 4 x, 16 x, 64 x, and 256 x diluted in the assay to change the analyte concentration in the measurement so that some assays produced MC intensity values within the range of the standard curve. Median value of AuNP attached to classifier beads in these assays ¹⁹⁷ Au signal intensities are presented in fig. 21. In a typical assay, a sample solution (50 μl) classifier dispersion (50 μl) is diluted without dilution or at a ratio of 1:2, 1:8, 1:32, or 1:128. The concentration of anti-CD 163 and anti-CXCL 9 detection Ab in the assay was 0.5 μg/mL and 1.0 μg/mL, while the concentration of other detection Abs was 2.5 μg/mL. Significantly higher detection from IL-4, IFNγ and TNFα -classifier beads in assays of stimulated samples at all dilutions compared to unstimulated samples ¹⁹⁷ Au signal. This result is consistent with the fact that the stimulation process can lead to an elevated release of IL-4, IFNγ and TNF α by PBMC, and is consistent with Ai et al ¹⁸ The findings reported for samples stimulated by similar processes are substantially consistent.

A series of standard solutions containing nine target cytokines at known concentrations were measured using the same assay conditions. A series of standard curves were then developed by fitting the results of the standard solution measurements to a four-parameter logistic regression (4P-LR) model. (see FIG. 25) the concentration of these cytokines in the unstimulated and stimulated samples was assessed using these standard curves. However, some of the measurements in fig. 21 were below the minimum of the standard curve presented in fig. 25. Therefore, the concentration cannot be inferred from these values. Figure 26 summarizes the preliminary findings of cytokine concentrations by fitting the assay results presented in figure 21 to the standard curve presented in figure 25. Because the dilution factor is considered in the calculation of cytokine concentrations presented in fig. 26, theoretically similar concentration values for the same sample measured should be observed from different dilutions. For measurement of IL-4 concentration in stimulated samples, samples diluted at ratios of 1:1, 1:2, 1:8, 1:32 and 1:128 reported IL-4 concentrations of 600, 430, 460, 640 and 1110pg/mL in stock solutions of stimulated samples, whereas for unstimulated samples, no measurement was able to generate a signal intensity sufficient to evaluate IL-4 concentration, possibly due to low IL-4 concentrations in unstimulated samples. For the measurement of ifnγ concentration in stimulated samples, the assays at different dilutions all reported very high signal intensities, which were higher than the signal intensity of the most concentrated standard solution.

Conclusion(s)

A set of 11 types of lanthanide-encoded microbeads were synthesized by 2-stage DisP. The metal content of the six metals in these microbeads was carefully controlled by varying the feed of the metal complex in the second stage of DisP to produce microbeads that produced a signal in MC with a median intensity of about 1000 counts per bead. These microbeads are of a size (CV _{Diameter of} <2%) and metal content (RCV<15%) and make them good candidates for classifier beads for use in bead-based assays.

In this bead-based assay by MC, cytokine levels were analyzed. To develop a bead-based multiplexed sandwich immunoassay for cytokines and chemokines in MC, the surface of metal-encoded microbeads is modified with different types of abs and used as classifier beads to capture target analytes. To develop a reporter system and optimize 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 the experiment, the MC signal intensity of the reporter NP was responsive to the concentration difference of the standard solution. These assays show high sensitivity at low analyte concentrations. However, some of the nonspecific binding of detection abs resulted in increased background noise problems. Background noise may compromise the detection limits of these assays. In addition, the supernatants of two PBMC samples were analyzed for cytokines and chemokines by the nine-fold assay developed in this study. The assay results showed that the supernatant of the PMA/ionomycin stimulated samples contained elevated levels of IL-4, IFNγ and TNFα compared to the unstimulated samples. This finding is substantially consistent with the results reported in the literature for cytokine detection measured by other assays. The results presented in this study show that bead-based sandwich immunoassays in MC can be used to detect different analytes simultaneously.

Example 10 additional experimental details and discussion of example 9

Material

Styrene (St, sigma-Aldrich, > 99%), polyvinylpyrrolidone (PVP, mw-55 kDa), 2' -azobis (2-methylpropanenitrile) (AIBN, 98%), triton-X305 (TX 305, 70% solution in water), diethylenetriamine pentaacetic anhydride (DTPA dianhydride, 98%), phorbol 12-myristate 13-acetate (PMA), ionomycin, hydrogen peroxide solution (H) ₂ O ₂ At H ₂ 30% of O), sulfuric acid (trace metal grade) and metal salts with a purity of 99.99% or more (trace metal basis) (including lanthanum (III) chloride heptahydrate (LaCl) ₃ ·7H ₂ O), cerium (III) chloride heptahydrate (CeCl) ₃ ·7H ₂ O), praseodymium (III) acetate hydrate [ Pr (OAc) ₃ ·xH ₂ O]Terbium (III) chloride hexahydrate (TbCl) ₃ ·6H ₂ O), holmium (III) chloride hexahydrate (HoCl) ₃ ·6H ₂ O) and thulium (III) chloride hexahydrate (TmCl) ₃ ·6H ₂ O)) was purchased from Millipore-Sigma. 4-vinylbenzylamine (VBA,. Gtoreq.92%) was purchased from TCI America. Absolute ethanol (EtOH) was purchased from Commercial Alcohols (misissauga, ontario). A standard solution of single elements for inductively coupled plasma mass spectrometry (ICP-MS) calibration was purchased from PerkinElmer (Pure Plus).

Preparation of metal-encoded classifier beads

DTPA-bis-vinylbenzylamide (DTPA-VBAm) ₂ ) Is used as a chelating agent for incorporating different types of metal ions into Polystyrene (PS) microbeads. Polymerizable DTPA-VBAm ₂ Metal materialThe chelant synthesis and metal loading procedure is described in example 4. To encode La, ce, pr, tb and Tm into microbeads, the beads are prepared by combining DTPA-VBAm ₂ Respectively with LaCl ₃ 、CeCl ₃ 、Pr(OAc) ₃ 、TbCl ₃ And TmCl ₃ Mixing to mix La (DTPA-VBAm ₂ )、Ce(DTPA-VBAm ₂ )、Pr(DTPA-VBAm ₂ )、Tb(DTPA-VBAm ₂ ) And Tm (DTPA-VBAm) ₂ ) Prepared in an aqueous solution. Passing the metal complexes through ¹ H NMR characterization.

Microbeads as classifier beads for bead-based assays were synthesized by a series of two-stage dispersion polymerizations (disps) as described in table 7. In a typical bead synthesis, the first stage of polymerization of styrene (6.25 g) in absolute ethanol (18.75 g) was initiated by AIBN (0.25 g) at 70 ℃ in the presence of PVP (1 g) and TX305 (0.35 g) as stabilizers. With N controlled by a gas quality controller (OMEGA) ₂ The reaction was protected by purging (3 mL/min). At 2 hours after initiation of the reaction, the reaction mixture will contain DTPA-VBAm ₂ Warm ethanol aliquots (15 g) of different types of metal complexes were added to the DisP reaction to incorporate metal ions into the microbeads. Optimizing M (DTPA-VBAm) in second stage aliquots ₂ ) To produce microbeads encoded with metal ions that produce a designed level of signal intensity in the MC. (see table 8) the reaction was terminated 24 after initiation and cooled to room temperature. The microbeads in the reaction dispersion were purified by two sedimentation-redispersion cycles with absolute ethanol and four cycles with water. The dispersion of purified microbeads was used for Scanning Electron Microscope (SEM) imaging, MC measurement and further surface modification.

The diameter and diameter distribution were characterized from SEM images of microbeads using a Hitachi S-5200 microscope. Typically, 2 μl of the diluted bead dispersion is 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) are calculated based on at least 300 measurements.

In the first two experiments, the feeding of metal complexes to the synthesis mixture was explored to obtain microbeads that can produce MC signals with intensities in the target range. These microbeads are denoted as test-1 and test-2. Based on the feed formulation developed in the experimental experiments, we then prepared 11 samples as classifier beads. These classifier beads are denoted as C-1, C-2 … C-11, respectively, and are listed in the table.

Acid digestion of microbeads

After termination of DisP, an aliquot of the C-1 reaction dispersion was treated with H at 250 ℃ ₂ SO ₄ /H ₂ O ₂ The total metal content in the reaction was digested and analyzed by inductively coupled plasma mass spectrometry (ICP-MS). For digestion of microbeads in the reaction dispersion, microbead dispersion (100 μl,0.5% -2% solids content) and 500 μl sulfuric acid were mixed in a 20mL glass vial, heated to 250 ℃ on a hot plate and held under magnetic stirring for 40min, after which 30% H was added ₂ O ₂ Solution (50. Mu.L). For ICP-MS analysis, 2% HNO was then used ₃ The digestion solution is diluted. 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 microbeads and the filtrate was collected for ICP-MS analysis. The metal incorporation efficiency in 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.

Instrument for measuring and controlling the intensity of light

Inductively coupled plasma mass spectrometry (ICP-MS). To quantify the metal content in the samples, an ICP-MS (iCAP-Q, thermo Scientific) system was used. Using 2% HNO ₃ The elemental standard solutions were diluted sequentially to concentrations of 40, 20, 10, 1 and 0.1ppb as calibration solutions. The metal content in each solution sample was determined based on a calibration fit curve, with a detection limit below 10ppt.

Additional results and discussion

Synthesis of metal-encoded classifier beads by dispersion polymerization

In this study, the polymerization of the metal complex M (DTPA-VBAm ₂ ) Metal-encoded microbeads were prepared with 2-stage DisP incorporating metal ions into PS microbeads. By reacting DTPA dianhydrideReaction with 4-vinylbenzylamine DTPA-VBAm in anhydrous DMSO ₂ Synthesis of chelating agents. La is subjected to ³⁺ 、Ce ³⁺ 、Pr ³⁺ 、Tb ³⁺ 、Ho ³⁺ And Tm ³⁺ Carried in an aqueous solution at pH 5-6 on DTPA-VBAm ₂ On the chelating agent. By passing through ¹ H-NMR characterizes these synthesized products. Details of the chelant synthesis and metal loading procedure are described in example 4.

To develop a formulation for classifier bead synthesis, the procedure was to optimize M (DTPA-VBAm) in the second stage aliquoting test ₂ ) To prepare the feed to MC at MC production ¹³⁹ La、 ¹⁴⁰ Ce、 ¹⁴¹ Pr、 ¹⁵⁹ Tb、 ¹⁶⁵ Ho sum ¹⁶⁹ The Tm signaling microbeads have an intensity level of 800-1000 counts per bead for each of these isotope channels. Based on the linear relationship between metal feed and metal content discussed herein in example 4, some experimental syntheses were performed to design feeds of six metal complexes. Based on the feed formulation developed in the experimental experiments, C-1 samples were prepared as a set of classifier beads encoded with six types of metal ions.

Furthermore, the metal incorporation efficiency of the metal ions into the C-1 beads was evaluated by ICP-MS. The results shown in group (b) of fig. 22 indicate that all six types of metal ions are effectively incorporated with an efficiency in the range of 63% -74%. The incorporation efficiency of each metal was evaluated by comparing the free metal content after synthesis with the total metal content in the reaction. A slight increase in efficiency was observed for lanthanide metal ions with larger ion radii, consistent with the findings described above in example 4. Generally, C-1 beads are suitable as candidate classifier beads.

The size of microbeads prepared by DisP varies from batch to batch, possibly due to the sensitivity of particle nucleation to reaction conditions. In order to optimize and prepare microbeads with small batch-to-batch variations in particle size, several control factors were explored in experimental conditions and it was found that reproducibility of particle size could be achieved by maintaining N between batches ₂ Purging and reaction heating procedures are consistent and improved. As summarized in Table 611 types of microbeads (C-1 to C-11) were prepared, which were uniform (CV<2%) and share similar dimensions, with average diameters in the range of 2.8 to 3.0 μm.

Following formulation optimisation, additional types of classifier beads were prepared using feeds that developed metal complexes for C-1 synthesis. In the first group of samples C-2 to C-6, two types of metal ions Ce (DTPA-VBAm) ₂ ) The second type of metal complex is added to the bead synthesis. The amount of each metal complex in the synthesis of these bimetallic beads was the same as in the synthesis of C-1 beads. The same principle is then also applied to the synthesis of some trimetallic coded microbeads (C-7 to C11).

Table 7A for styrene and DTPA-VBAm ₂ Typical formulation for two-stage dispersion polymerization of metal complexes of derivatives

b. The second stage aliquot was introduced into the reaction mixture 2h after initiation.

c. The required amount of DTPA-VBAm to be dissolved in ethanol ₂ Metal complexes ([ M (DTPA-VBAm) ₂ )]Is introduced into the aliquot. Details of the metal additions are described in table 8.

TABLE 8M (DTPA-VBAm) in the second stage of DisP for the synthesis of microbeads as classifier beads ₂ ) Is a summary of the feeds to (a)

a. First, DTPA-VBAm is used ₂ Is dissolved in ethanol (15 g) and then introduced as a second stage aliquot to DisP.

* "C" in the sample notation represents "classifier".

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 disclosure is not limited to the disclosed embodiments. On the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents, and patent applications are herein incorporated 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 by reference in its entirety. In particular, the sequences associated with each search 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.

Thus, the scope of 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.

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Claims

1. A metal-encoded microbead comprising:

a copolymer, the copolymer comprising:

structural monomers, and

a metal chelating monomer comprising a metal and a chelating agent;

Wherein the structural monomer does not comprise the chelating agent.

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

3. The microbead according to claim 1 or 2, wherein the metal chelating monomer has the structure of formula I before polymerization

Wherein the ligand is the chelator, L is the linker, X is the polymerizable end group, M is the metal, and n is 1 or an integer greater than 1, wherein the metal chelating monomer is neutral in charge prior to polymerization,

optionally, wherein L is selected from a bond, C3-C8 alkylamine, C3-C8 alkylene, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, 5-or 6-membered aryl or heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, C (O) O, or mixtures thereof, optionally, wherein each of the alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl is independently unsubstituted or substituted with one or more substituents selected from the group consisting of: 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, and/or

Optionally, wherein the polymerizable end group is selected from the group consisting of aryl vinyl, styrene, alpha-methyl styrene, acrylate, methacrylate, acrylamide, 2-methacrylamide, or mixtures thereof, optionally, the polymerizable end group is aryl vinyl or styrene.

4. The microbead of claim 3, wherein one or more of the following:

wherein L is attached to the chelating agent by an amide or ester, and

wherein the chelator is tetradentate, pentadentate, hexadentate, heptadentate or octadentate, optionally wherein the chelator is hexadentate or octadentate,

optionally, wherein the chelating agent comprises an amino polyacid moiety or derivative thereof, optionally, wherein the amino polyacid moiety is selected from amino polycarboxylic acid, amino polyphosphonic acid, or a combination thereof, optionally, wherein the amino polyacid moiety is a substituted oligomer of one or more of ethyleneimine, acrylamide, or a mixture thereof, the oligomer being substituted with two or more carboxylic and/or phosphonic acids, optionally, the oligomer being a crown ether or an azacrown ether.

5. The microbead according to claim 4 wherein said oligomer is further substituted by one or more substituents selected from the group consisting of: 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.

6. The microbead according to claim 4, wherein the chelating agent is selected from DFO, EDTA, DTPA, EGTA, EDDS, EDDHA, BAPTA, H neupa, H6 phosphoa, H4CHXoctapa, H4octapa, H2 CHXddpa, 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.

7. The microbead according to claim 6, wherein the chelating agent of the TACN type is selected from NOTA, NOPO, TRAP or a derivative thereof, wherein the chelating agent of the cyclen type is selected from DOTA or a derivative thereof, wherein the chelating agent of the cyclam type is selected from TETA, cross-bridged TETA, diAmSar or a derivative thereof, the chelating agent of the (13) aneN4 type is selected from TRITA or a derivative thereof, the chelating agent of the 1, 10-diaza-18-crown-6 type is selected from MACROPA or a derivative thereof, and/or wherein the chelating agent is selected from DTPA, cy-DTPA, ph-DTPA or a derivative thereof.

8. The microbead according to claim 3, wherein said metal chelating monomer is selected from the group consisting of

Optionally, wherein L is as defined in claim 3,

optionally, wherein the metal chelating monomer is selected from the group consisting of:

or a mixture thereof.

9. The microbead according to claim 4, wherein the chelating agent comprises porphyrin or phthalocyanine, optionally substituted or unsubstituted porphyrin, and

optionally, wherein the metal chelating monomer prior to polymerization is selected from

Or a mixture thereof, and wherein n is an integer from 1 to 4, optionally wherein n is at least 2,

optionally, wherein L is aniline.

10. The microbead according to claim 1, wherein the metal is a plurality of metals,

Optionally, wherein one or more of the following:

wherein the plurality of metals comprises one or more enriched isotopes, optionally the plurality of metals comprises one or more enriched isotopes,

wherein the plurality of metals comprises at least 2 metals, at least 3 metals, or at least 4 metals,

wherein the amount of each metal in the plurality of metals is within about 20% or about 10% of the amount of the other metal in the plurality of metals, and

wherein the metal is distributed throughout the microbeads and

optionally wherein the metal comprises indium, bismuth or a rare earth metal, optionally selected from the group consisting of lanthanide metals, yttrium or mixtures 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, different isotopes thereof or mixtures thereof, and

optionally, the rare earth metal is selected from 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, 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.

11. The microbead of any of claims 1-3, wherein one or more of the following:

wherein the microbeads have a glass transition temperature of about 60 ℃ or greater than 60 ℃, optionally about 70 ℃ or greater than 70 ℃, about 80 ℃ or greater than 80 ℃, about 90 ℃ or greater than 90 ℃, about 100 ℃ or greater than 100 ℃, about 115 ℃ or greater than 115 ℃, about 125 ℃ or greater than 125 ℃ or about 135 ℃ or greater than 135 ℃, and

wherein the microbeads have diameters 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

wherein the microbeads are colloidally stable in water.

12. The microbead according to claim 1-3, wherein the surface of the microbead comprises functionalization for attachment to a biomolecule,

optionally, wherein one or more of the following:

wherein the attachment is a covalent attachment or a non-covalent attachment,

wherein the surface of the microbead is functionalized with avidin, streptavidin, neutravidin or a mixture thereof, and

wherein the surface of the microbead is conjugated to the biomolecule,

optionally, wherein the biomolecule is selected from the group consisting of a protein, an oligonucleotide, a small molecule, a lipid, a carbohydrate, or a mixture thereof,

Optionally, the biomolecule is an affinity reagent, optionally, wherein the affinity reagent is an antibody, optionally, the 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, the antigen is a viral antigen, and

optionally, wherein the functionalization comprises a coating of silica on the surface of the microbead, optionally, the functionalization further comprises functionalizing the coating of silica.

13. The microbead according to claim 12, wherein the metal provides a bar code identifying the biomolecule.

14. A population of microbeads as claimed in any of claims 1-3,

optionally, wherein one or more of the following:

wherein the population has a size distribution with a Coefficient of Variation (CV) of about 10% or less than 10%, optionally, less than 5%,

wherein each microbead comprises a plurality of metals, the average amount of each metal of said plurality of metals throughout the population of microbeads being within about 10% or 10% of the average amount of another metal of said plurality of metals,

Optionally, wherein the plurality of metals comprises one or more enriched isotopes,

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% or about 10% or less than 10%,

wherein the amount of each metal of one microbead in the population of microbeads is within about 20% or within about 10% or within about 5% or within 5% of the amount of the same metal of another microbead in the population of microbeads, and

wherein the microbeads in the population of microbeads comprise the same metal in substantially the same amount, optionally wherein said same metal is a plurality of metals and said microbeads comprise each metal of the plurality of metals in substantially the same amount.

15. A kit comprising a plurality of different populations of microbeads as claimed in claim 14,

optionally, wherein one or more of the following:

wherein the population of each microbead is distinguishable from the population of another microbead based on the metal or metals of the microbeads, and

wherein the microbeads in the population of at least one microbead comprise a metal or metals that are different from the metal or metals of the microbeads in the population of another microbead, or wherein the microbeads in the population of at least one microbead and the microbeads in the population of another microbead comprise a different ratio of metals.

16. The kit of claim 15, wherein the microbeads in each population of microbeads are conjugated to different biomolecules.

17. A method of making a metal-encoded microbead, comprising:

polymerizing the second mixture to form a copolymer of the microbeads;

wherein the structural monomer does not comprise the chelating agent.

18. The method of claim 17, wherein one or more of:

wherein the metal is a plurality of metals,

wherein the structural monomer is polymerized in the nucleation stage to a completion of about 5% to about 20% based on the structural monomer, and

wherein the polymerization of the second mixture occurs to about 75% to about 100% completion, about 80% to about 99% completion, about 85% to about 95% completion, about 85% to about 93% completion based on the structural monomers.

19. The method of claim 17, further comprising functionalizing the microbeads,

optionally, wherein the functionalization of the microbead 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 group is selected from alcohols, aldehydes, carboxylic acids, epoxides, vinyl, alkynes, maleimides, or mixtures thereof, and

polymerizing the third mixture; and is also provided with

Optionally, wherein one or more of the following:

wherein said functionalizing of said microbeads comprises coating said microbeads with silica,

wherein said functionalization of said microbead further comprises a coating of functionalized silica, and

wherein the method further comprises conjugating the microbeads to a biomolecule.

20. A microbead prepared by the method of claim 17.

21. A method of making a metal-encoded microbead, said method comprising:

22. The method of claim 21, wherein one or more of:

wherein the structural monomer is selected from the group consisting of: acrylic monomers, methacrylic monomers and vinyl monomers selected from the group consisting of styrene, divinylbenzene (DVB), ethylvinylbenzene, vinylpyridine, amino-styrene, methyl-styrene, dimethylstyrene, ethylstyrene, ethyl-methyl-styrene, p-chlorostyrene or 2, 4-dichlorostyrene,

wherein the aqueous dispersion of swollen seed particles further comprises a steric stabilizer, optionally polyvinylpyrrolidone,

wherein providing an aqueous dispersion of swellable seed particles comprises preparing monodisperse swellable seed particles by emulsion polymerization,

wherein the aqueous dispersion of swellable seed particles further comprises an organic compound having a molecular weight of less than 5000 daltons and a water solubility of less than 10g-2g/L at 25 ℃ and optionally an organic solvent in which the organic compound is soluble,

Wherein the swellable seed particles are monodisperse swellable seed oligomer particles and

wherein the anionic surfactant is sodium dodecyl sulfate.

23. A method as claimed in claim 21 wherein the structural monomer is as defined in claim 2 and/or the metal chelating monomer is as defined in any one of claims 1, 3 and 8.

24. The kit of claim 16, further comprising a reporter comprising a mass label, wherein the reporter specifically binds to a sample biomolecule that is specifically bound by at least one of the different biomolecules,

optionally, wherein one or more of the following:

wherein the sample biomolecule is an oligonucleotide, and wherein at least one of the different biomolecules is an oligonucleotide that specifically hybridizes to the sample biomolecule,

wherein the reporter comprises a plurality of oligonucleotides that hybridize to indirectly bind a plurality of mass-labeled oligonucleotides to the sample biomolecule, and

wherein at least one of the different biological molecules is an affinity reagent, such as an antibody.

25. The kit according to claim 24,

Wherein one or more of the following:

wherein (i) the sample biomolecule is a viral particle, wherein at least one of the different biomolecules is a first antibody that specifically binds to the viral particle, and wherein the reporter comprises a second antibody that specifically binds to the viral particle, (ii) the sample biomolecule is a cytokine, wherein at least one of the different biomolecules is a first antibody that specifically binds to the cytokine, and wherein the reporter comprises a second antibody that specifically binds to the cytokine, or (iii) the sample biomolecule is a cancer biomarker, wherein at least one of the different biomolecules is a first antibody that specifically binds to the cancer biomarker, and wherein the reporter comprises a second antibody that specifically binds to the cancer biomarker,

wherein at least one of the different biomolecules comprises a viral antigen, wherein the sample biomolecule is an antibody that specifically binds to the viral antigen, and wherein the reporter comprises a second antibody that binds to the sample biomolecule, and

wherein the kit further comprises a plurality of different reporters, wherein each of the different reporters binds to a sample biomolecule that is specifically bound by a different biomolecule.

26. The kit of claim 25, wherein one or more of the following:

wherein each of the plurality of different reporters comprises the same mass label,

wherein the mass label comprises metal nanoparticles, and

wherein the mass label comprises a metal chelating polymer.

27. The kit according to claim 26,

wherein the kit further comprises a reporter that specifically binds to an enzyme substrate when the enzyme substrate has been modified by the sample biomolecule, or

Wherein the kit further comprises a reporter that specifically binds to the enzyme substrate when the enzyme substrate has not been modified by the sample biomolecule, optionally the enzyme substrate comprises a mass label that is removed when the enzyme substrate is modified by the sample biomolecule.

28. The kit of claim 24, wherein microbeads from a plurality of different populations are in a first microbead mixture,

optionally, wherein the kit further comprises a second mixture of microbeads comprising the same biological molecule as the first mixture of microbeads, wherein the microbeads of the first mixture of microbeads and the microbeads of the second mixture of microbeads comprise different sample barcodes.

29. The kit of claim 28, wherein the sample barcode is on the interior of the microbeads, optionally the sample barcode is a metal subgroup chelated by metal chelating monomers of microbeads of the first and second microbead mixtures, or

Wherein the sample bar code is on the surface of the microbeads of the first and second microbead mixtures.

30. The kit of claim 28, wherein one or more of the following:

wherein the kit further comprises a plurality of sample barcodes in separate partitions, the plurality of sample barcodes being functionalized to bind to surfaces of microbeads from a plurality of different populations, and

wherein the kit further comprises a set of mass-labeled antibodies mixed with each other, wherein at least some of the antibodies in the set are specific for a cell surface marker, optionally wherein the plurality of antibodies are mixed with microbeads of the kit, and

optionally, wherein at least some of the mass-labeled antibodies comprise the same metal as the metal chelated by the metal chelating monomer of the microbeads of the kit.

31. The kit of claim 24, wherein one or more of the following:

wherein the kit further comprises a steric stabilizer mixed with the microbeads of the kit, optionally the steric stabilizer is polyvinylpyrrolidone,

wherein the microbeads of the kit are in a buffer solution having a pH of 5, 9, or between 5 and 9, or the microbeads are lyophilized, and

wherein the kit further comprises one or more of a buffer, an anticoagulant, an immobilization reagent, and a permeabilization reagent.

32. The kit of claim 24, wherein the microbeads are fused to a solid support,

optionally, wherein the solid support is a microscope slide or an adhesive film.

33. A method comprising detecting the population of microbeads according to claim 14 by mass spectrometry.

34. The method of claim 33, further comprising calibrating a mass spectrometer for detecting the microbeads based on mass spectra obtained from the microbeads.

35. The method of claim 33, further comprising normalizing mass spectral signals obtained from a plurality of mass tags based on mass spectra obtained from the microbeads.

36. A method of mass spectrometry comprising:

Mixing different populations of microbeads according to claim 14 with the sample, wherein the microbeads in each population of microbeads bind to different biomolecules, and wherein each population of microbeads is distinguishable from the other population of microbeads based on the metal or metals of the microbeads;

binding different sample biomolecules of the sample to different biomolecules of a population of different microbeads;

binding a reporter directly or indirectly to each of the different sample biomolecules, wherein the reporter bound to each of the different sample biomolecules comprises a mass label; and

the metal and the mass label of each microbead are detected by mass spectrometry.

37. The method of claim 36, further comprising attaching different biomolecules to microbeads in the population of different microbeads prior to the step of mixing the population of different microbeads with the sample.

38. The method of claim 36 or 37, wherein the different sample biomolecules comprise oligonucleotides, antibodies, cytokines, and/or cancer biomarkers.

39. A method of mass spectrometry using the kit of claim 24, comprising:

Mixing a population of different microbeads with the sample, wherein the microbeads in each population of microbeads bind to different biomolecules, and wherein each population of microbeads is distinguishable from the other population of microbeads based on the metal or metals of the microbeads;

40. A method of mass spectrometry of a cell sample using the microbead of claim 12, comprising:

contacting the sample with the plurality of mass-labeled antibodies;

41. The method of claim 40, wherein the method comprises,

wherein the method further comprises:

calibrating a mass spectrometer used in the detecting step, wherein the calibrating is based on mass spectrometry signals obtained from the microbeads,

normalizing the mass spectral signal obtained from the mass tag based on the mass spectral signal obtained from the microbead, optionally wherein the microbead and the mass-labeled antibody comprise the same metal, or

Quantifying the mass-labeled antibodies based on the average number of metal atoms of each mass-labeled antibody and the detected mass spectrum signals from the mass-labeled antibodies and the microbeads, optionally wherein the microbeads and the mass-labeled antibodies comprise the same metal.

42. The method of claim 33, wherein the detecting step comprises imaging mass spectrometry and/or laser ablation ICP-MS or Secondary Ion Mass Spectrometry (SIMS),

optionally, wherein the method further comprises quantifying or normalizing the mass-labeled antibodies at each pixel or bound to each cell based on the mass-spectral signal detected from the microbeads, and/or

Optionally, wherein the microbeads are fused to a solid surface prior to the detection step.