Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
  • C08K3/00—Use of inorganic substances as compounding ingredients
  • C08K3/02—Elements
  • C08K3/08—Metals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
  • G01N33/587—Nanoparticles
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L25/00—Compositions of, homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Compositions of derivatives of such polymers
  • C08L25/18—Homopolymers or copolymers of aromatic monomers containing elements other than carbon and hydrogen
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/0027—Methods for using particle spectrometers
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/10—Ion sources; Ion guns
  • H01J49/105—Ion sources; Ion guns using high-frequency excitation, e.g. microwave excitation, Inductively Coupled Plasma [ICP]
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
  • C08F212/02—Monomers containing only one unsaturated aliphatic radical
  • C08F212/04—Monomers containing only one unsaturated aliphatic radical containing one ring
  • C08F212/06—Hydrocarbons
  • C08F212/08—Styrene
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
  • C08F212/34—Monomers containing two or more unsaturated aliphatic radicals
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L25/00—Compositions of, homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Compositions of derivatives of such polymers
  • C08L25/02—Homopolymers or copolymers of hydrocarbons
  • C08L25/04—Homopolymers or copolymers of styrene
  • C08L25/08—Copolymers of styrene
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N2015/1006—Investigating individual particles for cytology
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
  • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
  • G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
  • G01N23/2255—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident ion beams, e.g. proton beams
  • G01N23/2258—Measuring secondary ion emission, e.g. secondary ion mass spectrometry [SIMS]

Definitions

• the present disclosure relates to metal labelled polymer microbeads and in particular to lanthanide labelled polymer microbeads for mass cytometry bead based assays and multiplexing applications.
• Mass Cytometry is an emerging analytical technique employing an inductively coupled plasma time-of-flight mass spectrometer (ICP-TOF-MS) to analyze the signals of isotopic labels on cell and microbead samples.
• ICP-TOF-MS inductively coupled plasma time-of-flight mass spectrometer
• Bead-based assays are attractive as an analytical technique due to their high sample volume efficiency for multiplex assays with high throughput capacity.
• a wide variety of applications have been developed using bead-based assay technology, including capture sandwich immunoassays, competitive immunoassays, serology, gene expression profiling and genotyping.
• bead-based assays employ colloidal suspensions of particles as solid supports for different affinity reagents that can target different molecules as the analytes. By encoding the beads and creating a library of beads as classifiers for the analytes, a variety of captured analytes can be tracked by decoding and identifying individual beads throughout an experiment.
• Luminex has commercialized a library of 500 differently labelled polystyrene microspheres for bead-based assays by flow cytometry with 3 different colors at 10 different levels of intensities, with surface functionality for attaching bioaffinity reagents. 3-5
• Mass cytometry is an emerging multiparameter analytical technique that combines the features of flow cytometry and elemental mass spectrometry to determine the properties of single-cells or bead samples.
• cell and bead samples are labelled with heavy metal isotopes and individually introduced to the plasma torch of an inductively coupled plasma time-of-flight mass spectrometer (ICP-TOF-MS) to analyze the signals of metal isotope labels.
• ICP-TOF-MS inductively coupled plasma time-of-flight mass spectrometer
• MC is able to accurately detect different metal isotopes based on their atomic masses without channel overlap, and with the low abundance of heavy metal isotopes in biological samples, MC avoids complicated signal compensation processes associated with flow cytometry.
• K refers to the levels of metal concentration levels in the microbeads (including a concentration of zero); M is the number of different isotopes encoded in the beads.
• the term (-1) refers to beads with zero metal content for all the isotopes, which cannot be detected by MC.
• Lanthanide isotopes are an attractive choice as mass tags to encode PS microbeads because of their similar chemical and physical properties, low natural abundance as well as their high detection sensitivity in MC. 8 There are 15 lanthanide elements, whose isotopes covers at least 36 detection channels from 139 amu to 176 amu, theoretically available to be employed as bead labels.
• a library of beads with a variety of thousands can in principle be created for MC bead- based assays.
• metal containing microbeads employed in MC applications have to be in a size range of around 1 ⁇ 5 pm in diameter with very narrow size distributions. Smaller microbeads tend to contain fewer metal isotopes which may not be enough to be detected as single events in MC, whereas larger microbeads may not be consistently and fully consumed in the ICP torch.
• Microbeads can be used for various qualitative and quantitative applications for instance for analyte detection. Some cytokines are believed to play a critical role in the pathology of COVID-19. However, there are limitations to current research tools to fully investigate the mechanism of cytokine generation and action. 34
• Cytokines are soluble signaling protein molecules produced by cells at picomolar to nanomolar concentrations to regulate immune responses and modulate cellular activities. They are an exceptionally large and diverse group of pro- and anti-inflammatory factors in the body. 35 Deep characterizations of cytokines in blood samples can provide critical details of immune responses to disease or infections, orto a vaccine, therapy and intervention. Enzyme- linked immunosorbent assays (ELISAs) are the most extensively used monoplex Ab-based immunoassays to analyze cytokines in biological samples. However, ELISA assays require substantial amounts of sample and are time consuming when large numbers of cytokines are analyzed at once. 36 37
• Bead-based immunoassays are a technology platform that offers information-rich multiparametric analysis in a high-throughput setting.
• Bead-based assays employing fluorescent dyes for signal detection are the current gold standard for multiplexed cytokine analysis, and kits for cytokine analysis are commercially available.
• polymer microbeads are labeled with 2 or 3 different fluorescent dyes at different concentrations to well-defined intensities.
• Successful antigen detection is recognized by a reporter antibody (Ab) labeled with dye with a distinct emission.
• microbeads that can be synthesized by multi-stage dispersion polymerization and comprise a structural monomer and a metalchelating monomer that comprises a chelator that is chelated to a metal prior to being subjected to polymerization.
• the microbead comprises, and is formed by polymers including copolymers.
• microbeads obtained have a narrow size distribution and defined amounts of different metals.
• the quantity of metal incorporated into the microbead is substantially consistent from bead to bead; allowing for substantially uniform populations of microbeads to be obtained.
• the metal can be incorporated throughout the microbead as opposed to only being present on the surface.
• the microbeads of the present disclosure can be functionalized on the surface of the microbeads and also conjugated to biomolecules such as antibodies or other affinity reagents.
• a metal encoded microbead comprises at least one metal element, and may comprising a plurality of metal elements.
• a metal element may comprise a natural abundance of isotopes of the element or may comprise one or more enriched isotopes of the element.
• lanthanide-encoded microbeads were made by employing polymerizable metal-complexes as the ligands in a 2-stage dispersion polymerization.
• diethylenetriamine pentaacetic acid was functionalized with a polymerizable monomer by reacting DTPA dianhydride with 4-vinylbenzyl amine (VBA). Then, various lanthanide metal-ion complexes of this DTPA derivative were prepared.
• the metal-encoded microbeads were synthesized by introducing these polymerizable metal-DTPA complexes into dispersion polymerization reactions of monomers such as styrene.
• the microbeads obtained have very small bead-to-bead variations in their size and metal content. Similar metal incorporation efficiencies were found in bead syntheses with different metal ions complexed to this polymerizable chelator.
• microbeads prepared using this approach were linearly dependent on the metal complex feed in the bead syntheses regardless the type of metal. Batches of microbeads encoded with four lanthanides at three different concentrations were prepared. These microbeads generated MC signal intensities at three different levels with very good baseline resolution.
• the surface of a batch of microbeads was surface functionalized and conjugated to an antibody and used to detect a target analyte (e.g. sample biomolecule).
• a target analyte e.g. sample biomolecule
• a multiplexed bead-based assay was developed for the detection of analytes including cytokines.
• the classifier beads were labeled with heavy metal isotopes at different levels of metal incorporation.
• Each classifier bead carried a different Ab on its surface.
• the reporter can be a metal or metal oxide nanoparticle (NP) with an appropriate Ab of other biorecognition element bound to its surface.
• Samples were injected stochastically into the plasma torch of an inductively coupled plasma time of flight mass spectrometer. The instrument is capable of single mass resolution over the range m/z 85 to m/z 209. Thus, as shown, a very high level of multiplexing was possible.
• cytokines were used as the exemplary targets.
• PBMCs peripheral blood mononuclear cells
• the present disclosure includes a metal-encoded microbead comprising: a copolymer comprising: a structural monomer, and a metal-chelating monomer comprising a metal and a chelator; wherein the chelator coordinates the metal at least at 3 sites; and wherein the structural monomer does not comprise the chelator.
• the present disclosure includes a population of microbeads of the present disclosure.
• the present disclosure includes a kit comprising a plurality of distinct populations of microbeads of the present disclosure.
• the present disclosure includes a method of preparing 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 chelator attached to at least one polymerizable end group to obtain a second mixture, wherein the chelator coordinates the metal at least at 3 sites and wherein the metalchelating monomer is polymerizable with the structural monomer; and polymerizing the second mixture to form a copolymer of the microbead; wherein the structural monomer does not comprise the chelator.
• the present disclosure includes a microbead prepared by a method of the present disclosure.
• the present disclosure includes a method of preparing metal-encoded microbeads, the method comprising: providing an aqueous dispersion of swellable seed particles and an anionic surfactant; contacting the aqueous dispersion with monomers comprising a structural monomer and a metal-chelating monomer, wherein the metal-chelating monomer comprises a metal and a chelator attached to at least one polymerizable end, and wherein the chelator coordinates the metal at least at 3 sites; allowing the monomers to diffuse into the seed particles to form an aqueous dispersion of swollen seed particles; and initiating polymerization of the monomers in the aqueous dispersion of swollen seed particles; wherein the structural monomer does not comprise the chelator.
• Fig. 1 is a schematic showing an 1 H-NMR spectrum of Na 3 (DTPA-VBAm 2 ) molecule dissolved in D 2 0.
• the structure of Na 3 (DTPA-VBAm 2 ) given has protons labelled corresponding to the represented chemical shifts.
• Fig. 4A shows a graph depicting the MC signal intensities
• the error bars in a) and b) represent the RSD of MC signal intensities and the SD of metal content evaluated from MC signal intensities.
• Fig. 5 is a graph depicting the linear dependence of the metal content concentration in microbeads on the feed concentration of metal complexes in the second stage aliquots.
• the solid symbols represent data from the preliminary bead syntheses (Y-1 , Ce-1 , Eu-1 , Ho-1 , Lu- 1 , and 5E1).
• the solid line is the linear regression of these solid data points.
• the open symbols represent the data from bead syntheses samples 4E1 , 4E2, and 4E3, where we used the linear relationship observed in preliminary bead syntheses as a guide for the bead synthesis design.
• the x-axis in each figure 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 describe the signal from 4E1, 4E2, and 4E3 microbeads, respectively.
• Fig. 7A is a schematic showing an antigen detection agent using M(DTPA-VBAm 2 )- encoded microspheres (Eu-1).
• Eu-encoded microbeads surface functionalized with a goat-anti-mouse-lgG are incubated with 175 Lu-labeled mouse IgG as the reporter. Washed microbeads are then examined by MC for both 153 Eu and 175 Lu signals as a proof of reporter detection.
• Fig. 7B depicts a histogram of MC measurements generated using the antigen detection agent of Fig. 7A showing the 175 Lu signal intensity for Goat Anti-Mouse (GAM)-modified Eu-1 microbeads (Eu-1/GAM).
• GAM Goat Anti-Mouse
• NAv-modified Eu-1 microbeads (Eu-1/NAv) without GAM gave the weak signal shown in magenta as a negative control.
• Fig. 8A is a 1 H-NMR spectrum of Na 3 (DTPA-BAm 2 ) and Fig. 8B is a 1 H-NMR spectrum of Na3(DTPA-ALAm 2 ) measured in D 2 0. Fig. 8C is a 1 H-NMR spectrum of Na 3 (DTPA- AmPMAm 2 ). The structure of these molecules given has protons labelled corresponding to the represented chemical shift.
• Fig. 9A is a ⁇ -NMR (500 MHz) spectrum of Ce(DTPA-VBAm 2 )
• Fig. 9B is a 1 H-NMR (500 MHz) spectrum of Ce(DTPA-BAm 2 )
• Fig. 9C is a 1 H-NMR (500 MHz) spectrum of Ce(DTPA- ALAm 2 )
• Fig. 9D is a 1 H-NMR (500 MHz) spectrum of Ce(DTPA-AmPMAm 2 ) measured in D 2 0.
• the resonance peaks in the figure were broadened and shifted because Ce(lll) is a paramagnetic NMR shift reagent.
• Fig. 10A is a 1 H-NMR (500 MHz) spectrum of Y(DTPA-VBAm 2 )
• Fig. 10B is a 1 H-NMR (500 MHz) spectrum of Eu(DTPA-VBAm 2 )
• Fig. 10C is a 1 H-NMR (500 MHz) spectrum of Ho(DTPA- VBAm 2 )
• Fig. 10D is a 1 H-NMR (500 MHz) spectrum of Lu(DTPA-VBAm 2 ) measured in D 2 0.
• the resonance peaks in b) and c) were broadened and shifted because Eu(lll) and Ho(lll) are paramagnetic NMR shift reagents.
• Fig. 11 A to D are histograms of MC 140 Ce signal intensity counts in Ce-1 , Ce-2, Ce-3, and Ce-4 microbeads respectively: the x-axis is the 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 microbead samples for Y-1 ;
• Fig. 12B is a histogram of isotope signal intensity counts in microbead samples for Eu-1.
• Fig. 12C is a histogram of isotope signal intensity counts in microbead samples for Ho-1 , and
• Fig. 12D is a histogram of isotope signal intensity counts in microbead samples for Lu-1.
• the x- axis is the isotope signal intensity; the y-axis is the number of beads normalized to 100.
• Fig. 13A is a graph depicting the release profiles of metal ions Ce 3+ (square), Eu 3+ (circle), Ho 3+ (up-pointing triangle) and Lu 3+ (down-pointing triangle), from 4E3 microbeads (solid symbols and solid lines) at 0.5% solids content into a pH 3.0 buffer solution: 50 mM sodium acetate determined by ICP-MS.
• Fig. 13B is a graph depicting the release profiles of metal ions Ce 3+ (square), Eu 3+ (circle), Ho 3+ (up-pointing triangle) and Lu 3+ (down-pointing triangle), from 4E3 microbeads (solid symbols and solid lines) at 0.5% solids content into a pH 7.0 buffer solution: 10 mM ammonium acetate determined by ICP-MS.
• Fig. 13B is a graph depicting the release profiles of metal ions Ce 3+ (square), Eu 3+ (circle), Ho 3+ (up-pointing triangle) and Lu 3+ (down-pointing triangle), from 4E3 microbeads (solid symbols and solid lines) at 0.5% solids content into a pH 7.0 buffer solution: 10 mM ammonium acetate determined by ICP-MS.
• 13C is a graph depicting the release profiles of metal ions Ce 3+ (square), Eu 3+ (circle), Ho 3+ (up-pointing triangle) and Lu 3+ (down-pointing triangle), from 4E3 microbeads (solid symbols and solid lines) at 0.5% solids content into a pH 10.5 buffer solution: 200 mM sodium carbonate/bicarbonate determined by ICP-MS.
• Fig. 13D is a graph depicting the release profiles of metal ions Ce 3+ (square), Eu 3+ (circle), Ho 3+ (up-pointing triangle) and Lu 3+ (down-pointing triangle), from 4E3 microbeads (solid symbols and solid lines) at 0.5% solids content into a 1% PVP solution determined by ICP- MS.
• the open symbols and dash lines represent the release profiles of metal ions from a batch of microbeads prepared by AA approach under the same conditions as 4E3 DTPA-beads.
• Fig. 14 is a schematic of a multi-step strategy to functionalize microbead surface with Goat Anti-Mouse (GAM) by silica coating.
• GAM Goat Anti-Mouse
• Fig. 15 is a schematic of a microbead assay of the present disclosure.
• FIG. 16 is a schematic illustrating an exemplary multiplexed bead-based sandwich immunoassay by MC carried out in a 96-well filter plate.
• Fig. 17 are dot-plot diagrams of 11 types of classifier microbeads (C1 to C11), where panel A shows the 140 Ce- 142 Ce isotopic dot-plot diagram of a mixture of 11 types of classifier microbeads (C-1 to C-11). The oval circle isolates the singlet events of 11 types of microbeads.
• Panels b-k are dot-plot-diagrams showing the gating strategies to individually identify C-1 to C-11 microbeads by MC.
• Fig. 18 are histograms of the reporter signal intensities on IL-4 classifier beads (C-5) in a series of four-plex assays of standard solutions at various IL-4 concentrations (a), (b), and (c) AuNP was employed as the reporter in four-plex assays of standard solutions containing IL-4 at concentrations of 0, 1.2, and 20 pg/mL, respectively (d), (e), and (f) NanoGold was employed as the reporter in four-plex assays of standard solutions containing IL-4 at 0, 1.2, and 20 pg/mL, respectively.
• Fig. 19 are standard curves of two sets of four-plex assays for (a) IL-4, (b) IL-6, (c) IFNy, and (d) TNFa.
• the x-axis in each plot represents the analyte concentration and the and y-axis represents the median MC signal intensity of NPs attached to the corresponding classifier beads.
• Two different types of streptavidin-conjugated reporter (AuNP and NanoGold) were investigated in these four-plex assays. The results are presented for the AuNP as circles ( ⁇ ) and for the NanoGold as squares ( ⁇ ). Negative events with 197 Au signal intensities of ⁇ 1 count per bead were excluded from the statistical analysis for median intensities.
• the dose-response curves were drawn by fitting the experimental results with a four-parameter logistic regression model.
• Fig. 20 shows standard curves of four sets of nine-plex assays for (a) II_-1b, (b) IL-4, (c) IL-6, (d) IL-10, (e) IL- 18, (f) IFNy (g) TNFa, (h) CD163 and (i) CXCL-9 at different concentrations of biotinylated anti-CD163 and anti-CXCL-9 in the detection Ab cocktails.
• the x-axis in each plot represents the analyte concentration.
• the y-axis in each plot represents the median MC signal intensity of AuNP attached to the corresponding type of classifier beads.
• the concentrations of biotinylated anti-CD 163 and anti-CXCL9 in the detection Ab cocktails were reduced from 2.5 to 2.0, 1 .0 and 0.5 pg/mL, while the concentrations of other detection Abs were kept constant at 2.5 pg/mL in the cocktails.
• the results of these assays are plotted for Ab concentrations of 2.5 pg/mL with filled circles ( ⁇ ), of 2.0 pg/mL with filled squares ( ⁇ ), of 1 .0 pg/mL with filled triangles (A), and of 0.5 pg/mL with filled diamonds ( ⁇ ).
• Negative events with 197 Au signal intensities of ⁇ 1 count per bead were excluded from the statistical analysis for median intensities.
• the dose-response curves were drawn by fitting the experimental results with a four-parameter logistic regression model.
• Fig. 21 are histograms showing median 197 Au signal intensities of AuNP reporter attached to classifier beads in nine-plex assays for the analysis of (a) IL-1 b, (b) IL-4, (c) IL-6, (d) IL-10, (e) IL-18, (f) IFNy, (g) TNFa, (h) CD163, and (i) CXCL-9 in the stimulated and unstimulated PBMC samples at different sample dilution ratios. Solid columns in the figure represent the assay results of the stimulated samples, while striped columns represent the assay results of the unstimulated samples.
• Fig.22(a) is a SEM image of C-1 microbeads prepared by two-stage DisP.
• Fig. 23 are standard curves of three sets of four-plex assays for (a) IL-4, (b) IL-6, (c) IFNy and (d) TNFa at different reporter (NP) concentrations.
• the x-axis in each plot represents the analyte concentration.
• the y-axis in each plot represents the median MC signal intensity of AuNP attached to the corresponding classifier beads.
• Three concentrations of AuNP with 200x, 400x, and 800* dilutions from the stock solution were investigated in these four-plex assays. Their results are presented for dilutions of 200* with circles ( ⁇ ), of 400* with squares ( ⁇ ), and of 800* with triangles (A).
• Fig. 24 is a summary chart of median MC signal intensities of AuNP attached to classifier beads in a series of nine-plex assays of blank samples in the absence of analyte molecules (0 pg/mL).
• Fig. 25 are standard curves of the nine-plex assays for I L- 1 b , IL-4, IL-6, IL-10, IL- 18, IFNy, TNFa, CD163 and CXCL-9 using the same assay conditions for the analysis of stimulated and unstimulated PBMC samples.
• the dose-response curves were drawn by fitting the experimental results with a four-parameter logistic regression model.
• Fig. 26 are graphs showing cytokine concentrations in the stimulated and unstimulated PBMC samples calculated based on the dose-response standard curves in Fig. 25. Some measured MC intensity values presented in Fig. 21 are lower than the minimum values of the 4P-LR modeled standard curves presented in Fig. 25. No concentration is calculated from these values.
• the second component as used herein is chemically different from the other components or first component.
• a metal chelated to a second component can be different from a metal chelated to a first component, when the second component and the first component can have the same chelator.
• a “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.
• suitable means that the selection of the particular compound or condition would depend on the specific synthetic manipulation to be performed, the identity of the molecule(s) to be transformed and/or the specific use for the compound, but the selection would be well within the skill of a person trained in the art.
• the compounds described herein may have at least one asymmetric center. Where compounds possess more than one asymmetric center, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present disclosure. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (for example, less than 20%, suitably less than 10%, more suitably less than 5%) of compounds of the present disclosure having an alternate stereochemistry. It is intended that any optical isomers, as separated, pure or partially purified optical isomers or racemic mixtures thereof are included within the scope of the present disclosure.
• the compounds of the present disclosure may also exist in different tautomeric forms and it is intended that any tautomeric forms which the compounds form, as well as mixtures thereof, are included within the scope of the present disclosure.
• the present description refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.
• alkyl as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups.
• the number of carbon atoms that are possible in the referenced alkyl group are indicated by the prefix “Cn1-n2”.
• C1 -1 Oalkyl means an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
• alkylene whether it is used alone or as part of another group, means straight or branched chain, saturated alkylene group, that is, a saturated carbon chain that contains substituents on two of its ends.
• the number of carbon atoms that are possible in the referenced alkylene group are indicated by the prefix “Cn1-n2”.
• C2- 6alkylene means an alkylene group having 2, 3, 4, 5 or 6 carbon atoms.
• available refers to atoms that would be known to a person skilled in the art to be capable of replacement by a substituent.
• amine or “amino,” as used herein, whether it is used alone or as part of another group, refers to groups of the general formula NR'R", wherein R' and R" are each independently selected from hydrogen or C1-6alkyl.
• cycloalkyl as used herein, whether it is used alone or as part of another group, means a saturated carbocyclic group containing one or more rings.
• the number of carbon atoms that are possible in the referenced cycloalkyl group are indicated by the numerical prefix “Cn1-n2”.
• C3-10cycloalkyl means a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
• aryl refers to carbocyclic groups containing at least one aromatic ring.
• the aryl group contains from 6, 9 or 10 carbon atoms, such as phenyl, indanyl or naphthyl.
• heterocycloalkyl refers to cyclic groups containing at least one non-aromatic ring in which one or more of the atoms are a heteroatom selected from O, S and N. Heterocycloalkyl groups are either saturated or unsaturated (i.e. contain one or more double bonds). When a heterocycloalkyl group contains the prefix Cn1-n2 this prefix indicates the number of carbon atoms in the corresponding carbocyclic group, in which one or more, suitably 1 to 5, of the ring atoms is replaced with a heteroatom as defined above.
• heteroaryl refers to cyclic groups containing at least one heteroaromatic ring in which one or more of the atoms are a heteroatom selected from O, S and N.
• a heteroaryl group contains the prefix Cn1-n2 this prefix indicates the number of carbon atoms in the corresponding carbocyclic group, in which one or more, suitably 1 to 5, of the ring atoms is replaced with a heteroatom as defined above.
• All cyclic groups including aryl and cyclo a 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, spirofused or linked by a bond.
• a first ring being “fused” with a second ring means the first ring and the second ring share two adjacent atoms there between.
• a first ring being “bridged” with a second ring means the first ring and the second ring share two non-adjacent atoms there between.
• a first ring being “spirofused” with a second ring means the first ring and the second ring share one atom there between.
• halo refers to a halogen atom and includes fluoro, chloro, bromo and iodo.
• atmosphere refers to atmosphere
• MS mass spectrometry
• protecting group refers to a chemical moiety which protects or masks a reactive portion of a molecule to prevent side reactions in those reactive portions of the molecule, while manipulating or reacting a different portion of the molecule. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not degrade or decompose the remaining portions of the molecule.
• PG protecting group
• the selection of a suitable protecting group can be made by a person skilled in the art. Many conventional protecting groups are known in the art, for example as described in “Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W.
• EDTA refers to ethylenediaminetetraacetic acid.
• DTPA diethylenetriaminepentaacetic acid
• EGTA as used herein refers to egtazic acid.
• EDDS refers to ethylenediamine-N, N'-disuccinic acid.
• EDDHA ethylenediamine-N, N'-bis(2- hydroxyphenylacetic acid).
• BAPTA refers to 1 ,2-bis(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid.
• TACN as used herein refers to 1 ,4,7-triazacyclononane.
• TACD as used herein refers to 1 ,5,9-triazacyclododecane.
• cyclen refers to 1 ,4,7,10-tetraazacyclododecane.
• cyclam refers to 1 ,4,8,11-tetraazacyclotetradecane.
• (13)aneN4 refers to 1 ,4,7, 10-tetrazacyclotridecane.
• DFO desferrioxamine
• TACD-type chelator refers to a chelator that comprises a specified base structure (i.e. TACD, TACN, cyclen, etc.) where the specified base structure can be further substituted at available hydrogen atoms.
• swellable polymer seed refers to a polymer particle that is capable to increasing in volume.
• a swellable polymer seed can increase in volume when contacted with a swelling agent.
• the swelling agent can be for example an anionic surfactant and/or an organic compound.
• substantially oxygen-free conditions refers to reaction conditions wherein the oxygen content is low or non-existent.
• substantially oxygen-free conditions can refer to a reaction condition where the reaction is carried out under an inert atmosphere, for example a noble gas (e.g. helium, argon) or nitrogen atmosphere.
• substantially oxygen-free conditions can refer to an oxygen content between about 0 ppm to about 5 ppm, about 0 ppm to about 3 ppm, about 0 ppm to about 2 ppm, or about 0 ppm to about 1 ppm, or about 0.01 ppm to about 2 ppm.
• antibody as used herein is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies and binding fragments thereof.
• the antibody may be from recombinant sources and/or produced in transgenic animals.
• Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
• Antibody fragments as used herein mean binding fragments
• oligonucleotide refers to a nucleic acid comprising, a sequence of nucleotide or nucleoside monomers consisting of naturally and non-naturally occurring bases, sugars, and intersugar (backbone) linkages, and includes single-stranded and double- stranded molecules, RNA and DNA. Oligonucleotides may be long (e.g. greater than 1000 monomers and up to 10K monomers), medium sized (e.g. between and inclusive of 200 and 1000 nucleotides) or short for example less than 200 monomers, 100 monomers, 50 monomers, including non-naturally occurring monomers.
• oligonucleotide includes, for example, single stranded DNA (ssDNA), genomic DNA (gDNA), complementary DNA (cDNA, reverse transcribed from an RNA), messenger RNA (mRNA), “antisense oligonucleotides” and “miRNA” as well as oligonucleotide analogues such as “morpholino oligonucleotides”, “phosphorothioate oligonucleotides”, or any oligonucleotide or analog thereof known to one of skill in the art.
• the present disclosure includes a metal-encoded microbead comprising: a copolymer comprising: a structural monomer, and a metal-chelating monomer comprising a metal and a chelator; wherein the chelator coordinates the metal at least at 3 sites, and wherein the structural monomer does not comprise the chelator.
• the microbead of the present disclosure can comprise substantially uniformly distributed metal.
• the metal chelated to the metal-chelating monomer is not limited to being surface bound but rather can be found distributed throughout and/or in an interior portion of the microbead of the present disclosure.
• the chelator coordinates the 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.
• DTPA is an octadentate ligand (capable of coordination at 8 sites).
• the structural monomer does not comprise any chelator (e.g., does not comprising any chelator that coordinates a metal at least at 2 sites).
• the structural monomer is metal-free.
• the structural monomer may not comprise a metal through chelation, through a covalent bond (such as a Tellurium in a carbon backbone of the structural monomer), or optionally through any other means.
• the structural monomer may not comprise a transition metal or a class of transition metals.
• the structural monomer may not comprise a rare earth metal (such as a lanthanide) and/or soft metal as described herein.
• metal-chelating monomer may comprise a heavy metal (e.g., of 80 amu or greater), while the structural monomer does not comprise a heavy metal (e.g., of 80 amu or greater).
• the structural monomer is selected from substituted or unsubstituted styrene, 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 styrene and/or combinations thereof.
• the metal-chelating monomer has a structure of Formula I prior to polymerization
• Ligand is the chelator
• L is a linker
• X is a polymerizable end group
• M is the metal
• n is 1 or an integer greater than 1 , wherein the metal-chelating monomer is neutral in charge prior to polymerization.
• metal is chelated to the chelator of the metal-chelating monomer through ionic, non-covalent interactions.
• metal is incorporated into the microbead of the present disclosure through non-covalent interactions.
• L is selected from a bond, C3-C8 alkyl amine, C3-C8 alkylene, C3- C8 cycloalkyl, C3-C8 heterocycloalkyl, 5-membered or 6-membered aryl or heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, C(O), C(0)0, or mixtures thereof.
• Each of the alkylene, aryl, alkylaryl, alkylheteroaryl, cycloalkyl, cycloalkylaryl, and cycloalkylheteroaryl can be independently unsubstituted or substituted with one or more substituents which can be selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3- C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
• the L can for example, be attached to the chelator through an amide or an ester group/linkage.
• the polymerizable end group is selected from arylvinyl, styrene, alpha-methylstyrene, acrylate ester, methacrylate ester, acrylamide, 2-methylacrylamide, and mixtures thereof, optionally the polymerizable end group is arylvinyl or styrene. In some embodiments, the polymerizable end group is arylvinyl or vinyl ester.
• the chelator can for example be tridentate.
• the chelator is tetradentate, pentadentate, hexadentate, heptadentate, oroctadentate, optionally the chelator is hexadentate or octadentate.
• the chelator comprises an aminopolyacid moiety, or a derivative thereof.
• the derivative of the aminopolyacid moiety includes amides of the aminopolyacid moiety.
• aminopolyacid moiety can be selected from aminopolycarboxylic acid, aminopolyphosphonic acid, or combinations thereof.
• L is a bond
• the chelator is an aminopolyacid
• the polymerizable end group is arylvinyl.
• the metal-chelating monomer can be vinylbenzene iminodiacetic acid coordinated to the metal or divinylbenzene iminodiacetic acid coordinated to the metal.
• the aminopolyacid moiety is a substituted oligomer of one or more of ethylene imine, propylene amine, or mixtures thereof, the oligomer being substituted with two or more carboxylic acids and/or phosphonic acids.
• the oligomer can be a crown ether or an aza-crown ether.
• the aminopolyacid moiety is a substituted oligomer of one or more of ethylene oxide, ethylene imine, propylene oxide, propylene amine, ethanolamine, propanolamine, aminophenol cyclohexane diamine, or mixtures thereof.
• the oligomer is further substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, halo, alcohol, amine, amide, ester, aryl, heteroaryl, akylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
• substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, halo, alcohol, amine, amide, ester, aryl, heteroaryl, akylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl
• the chelator is selected from DFO, EDTA, DTPA, EGTA, EDDS, EDDHA, BAPTA, H4neunpa, H6phospa, H4CHXoctapa, H4octapa, H2CHXdedpa, H5decapa, Cy-DTPA, Ph-DTPA, a TACN-type chelator, a TACD-type chelator, a cyclen-type chelator, a cyclam-type chelator, a (13)aneN4-type chelator, a 1,7-diaza-12-crown-4-type chelator, a 1,10-diaza-18-crown-6-type chelator, or derivatives thereof.
• the TACN-type chelator can be NOTA, NOPO, TRAP, or derivatives of any thereof.
• the cyclen-type chelator is DOTA or derivatives thereof.
• the cyclam-type chelator is selected from TETA, cross bridged- TETA, DiAmSar, or derivatives thereof.
• the (13)aneN4-type chelator is selected from TRITA or derivatives thereof.
• the 1,10-diaza-18-crown-6-type chelator is selected from MACROPA, or derivatives thereof.
• the chelator is selected from DTPA, Cy-DTPA, Ph-DTPA, or derivatives thereof.
• the derivative of DTPA can comprise DTPA having two adjacent carbon atoms joined together with atoms therebetween to form a 5- membered or 6-membered ring, optionally a cycloalkyl ring, an aryl or a heteroaryl ring.
• the monomers described herein Prior to polymerization, the monomers described herein are unreacted, i.e. the monomers comprise polymerizable end groups that can participate in polymerization.
• the metal-chelating monomer prior to polymerization, is wherein L and X are as described herein.
• the metal-chelating monomer prior to polymerization, is selected from r mixtures thereof.
• the mixture can comprise one or more said metal chelating monomers with different metals.
• the chelator comprises porphyrin or phthalocyanine.
• the chelator can be substituted or unsubstituted porphyrin.
• the porphyrin and phthalocyanine are each independently substituted with C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, alkylaryl, alkylheteroaryl, C3-C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, carboxylic acid, or mixtures thereof.
• the metal can be a soft metal.
• the soft metal can be cadmium, cobalt, copper, iron, zinc, nickel, tin, osmium, palladium, platinum, gold, thallium, mercury, or lead, including isotopes thereof, as well as mixtures thereof.
• the soft metal has an atomic mass of 80 amu or above.
• the metal-chelating monomer prior to polymerization is selected from
• n is an integer from 1 to 4.
• L can be aniline.
• n is 2 or at least 2.
• the metal-chelating monomer is selected from
• the microbead of the present disclosure can also further comprise a steric stabilizer.
• the steric stabilizer can be PVP, polyvinyl alcohol, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, polyacrylic acid, water soluble homopolymers of acrylic acid esters, water soluble homopolymers of methacrylic acid esters, water soluble homopolymers of acrylamide, homopolymers of methacrylamide, water soluble copolymer steric stabilizers, or mixtures thereof.
• the water-soluble copolymer steric stabilizer is selected from copolymers of acrylic acid ester, methacrylic acid ester, acrylamides or methacrylamide with methyl acrylate and/or ethyl acrylate, or mixtures thereof.
• the copolymer can also be crosslinked, as is achieved for example when the metalchelating monomer and/or the structural monomer have two or more polymerizable groups.
• a microbead of the present disclosure can comprise a plurality of metals.
• each metal can be incorporated by the same type of a different type of metalchelating monomer.
• the metal can be a plurality of metals.
• the plurality of metals comprises one or more enriched isotopes.
• the plurality of metals comprises at least 2 metals, at least 3 metals, or at least 4 metals.
• the amount of each metal of the plurality of metals is within about 20% or about 10% of the amount of another metal of the plurality of the metals. For example, this can be determined on a population of microbeads or a single microbead through mass cytometry.
• the metal can, for example, be a transition metal (i.e., a metal from groups 3-12 of the periodic table, or from the lanthanide or actinide series).
• the metal can, for example, be indium, bismuth, or a rare earth metal.
• the rare earth metal can for example be a lanthanide metal, yttrium, or mixtures thereof.
• the metal is indium, bismuth, a soft metal, or a rare earth metal.
• the soft metal can be cadmium, cobalt, copper, iron, zinc, nickel, tin, osmium, palladium, platinum, gold, thallium, mercury, lead, and isotopes thereof, as well as mixtures thereof.
• the metal comprises a rare earth metal that is selected from Y, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, distinct isotopes thereof, and mixtures thereof.
• the metal is selected from La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, distinct isotopes thereof, and mixtures thereof.
• the rare earth metal is selected from 89Y, 139La, 136Ce, 138Ce, 140Ce, 142Ce, 141 Pr, 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, 175L
• the metal can be distributed throughout the microbead substantially uniformly. It can also be compartmentalized, for example in the interior of the microbead.
• the microbead has a glass transition temperature (as measured, for example, by differential scanning calorimetry (DSC)) of about 60°C or above 60°C, optionally about 70°C or above 70°C, about 80°C or above 80°C, about 90°C or above 90°C, about 100°C or above 100°C, about 115°C or above 115 °C, about 125°C or above 125°C, or about 135°C or above 135°C.
• DSC differential scanning calorimetry
• the DSC result is obtained on the second or third heating scan at a scan rate of about 10°C/min to about 20°C/min.
• the microbead has a diameter of about 0.6 pm to about 20 pm, about 1 pm to about 15 pm, about 2 pm to about 10 pm, or about 2 pm to about 6 pm. In some embodiments, the microbeads of the present disclosure have a size suitable for mass cytometry.
• the microbead is colloidally stable in water.
• the microbeads of the present application are substantially stable upon storage in buffers and/or in physiological media.
• substantially stable in buffers and/or physiological media means there is no significant leakage or less than 1% leakage of metals upon storage in buffers and/or physiological media.
• a surface of the microbead comprises functionalization for attachment to a biomolecule.
• the functionalization can be introduced by adding a coating layer.
• the attachment is covalent attachment.
• the microbeads can be coated with silica, and functionalized with reactive functional groups, such as a carboxylic acid group.
• a biomolecule can be added to the microbead.
• a biomolecule may be classified as a protein, an oligonucleotide, a lipid, a carbohydrate, or a small molecule. Alternatively or in addition, a biomolecule may be classified by its functionality. The biomolecule is not particularly limited and different functionalizations can be used to conjugate the biomolecule to the microbead.
• an oligonucleotide may be a single stranded DNA molecule, optionally 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.
• a biomolecule may be an oligonucleotide that specifically hybridizes a target oligonucleotide, such as a target mRNA endogenous to a sample (e.g. hybridizes to the sample oligonucleotide).
• Hybridization may be of a sequence that is more than 8, more than 10, more than 15, or more than 20 nucleotides.
• a biomolecule may be classified by its functionality.
• a biomolecule may be an affinity reagent, an antigen (e.g., an analyte specifically bound by an affinity reagent), or an enzyme substrate.
• An affinity reagent may be an antibody (e.g., or fragment thereof), aptamer, receptor (e.g., or portion thereof), or any other biomolecule that specifically binds a target (e.g., an avidin, such as streptavidin, that specifically binds biotin).
• a bead may be associated with an antibody may be used to detect the presence of its target antigen in a sample, such as the presence of a cytokine, viral protein, cancer biomarker, or the like.
• a microbead may be functionalized with an avidin for attachment of another biomolecule functionalized with biotin (e.g., to allow a bead to be adapted to any of a number 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.
• a bead may be attached to a viral antigen (such as a viral protein sequence), and may be used to detect the presence of antibodies in the sample that specifically bind the viral antigen, as described further herein.
• An enzyme substrate may be any substrate that is acted on by a specific enzyme, such as by an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase.
• a 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, demethylases.
• Non-protein substrates include, for example, a double stranded oligonucleotide comprising a restriction sequence cleavable by a restriction enzyme or a site (such as a nick) for DNA repair, an oligonucleotide sequence comprising a sequence targeted by a DNA methyltransferase, or any non-protein substrate known to one of skill in the art.
• a bead may be attached to a substrate and exposed to a sample comprising an enzyme that modifies the substrate, and modification (or lack thereof) of the substrate may be detected (e.g., as described further herein).
• the affinity reagent is or comprises an antibody or a binding fragment thereof.
• the antibody can for example be a biotinylated antibody or binding fragment and can be added directly or indirectly to the microbead.
• NAv Neutravidin
• a biotinylated antibody was attached to the NAv-modified microbead surface through the strong biotin-avidin affinity.
• the affinity reagent can be avidin or related biotin binding molecules such as streptavidin, NeutrAvidin and CaptAvidin. Microbeads comprising such an affinity reagent can be customized using a biotinylated antibody specific for a target analyte of interest.
• the affinity reagent optionally the 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 factors.
• the antibody may also be specific for a pathogenic protein such as a viral, bacterial or fungal pathogen.
• a pathogenic protein such as a viral, bacterial or fungal pathogen.
• Such microbeads can be used to detect for the presence of such pathogens or their products in samples such as environmental or patient samples.
• the antigen is a viral antigen.
• Such microbeads comprising a viral antigen can be used for example to detect the presence of viral antibodies in patient samples.
• antigens such as those from other pathogens.
• the copolymer of the microbead further comprises a third monomer, the third monomer being present at least on the surface of the microbead and comprising at least one reactive functional group.
• the microbead is functionalized through the at least one reactive functional group comprised in the third monomer, for example through 3-stage dispersion polymerization.
• the third monomer is substituted or unsubstituted acrylic acid or methacrylic acid.
• the surface of the microbead can be functionalized with a reactive functional group.
• the reactive functional group is on the coating of silicon dioxide.
• the reactive functional group is selected from amine, thiol, alcohol, aldehyde, carboxylic acid, epoxide, vinyl, alkyne, maleimide, or a click chemistry moiety (e.g. such as dibenzocyclooctyne (DBCO), azide, trans-cyclooctenes (TCO), or tetrazine, or derivatives thereof) or mixtures thereof.
• DBCO dibenzocyclooctyne
• TCO trans-cyclooctenes
• tetrazine tetrazine
• the functionalization comprises a coating of silicon dioxide on the surface of the microbead, optionally the functionalization further comprises functionalizing the coating of silicon dioxide.
• the surface of the microbead is functionalized with a reactive functional group.
• the reactive functional group is on the coating of silicon dioxide.
• the reactive functional group can be selected from amine, thiol, alcohol, aldehyde, carboxylic acid, epoxide, vinyl, alkyne, maleimide, or mixtures thereof.
• the attachment to the biomolecule is non-covalent attachment.
• the surface of the microbead is functionalized with avidin, streptavidin, neutravidin, or mixtures thereof.
• the surface of the microbead is conjugated to the biomolecule.
• the metal provides a barcode that identifies the biomolecule.
• an interior structure of the microbead comprises the copolymer.
• the metal-chelating monomer chelates a single metal atom and not a plurality of metal atoms.
• the microbead comprises a polymer seed that does not comprise the metal-chelating monomer.
• 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.
• the polymer seed comprises a structural monomer; optionally wherein the structural monomer of the polymer seed is identical in structure to the structural monomer of the copolymer.
• the present disclosure includes a population of microbeads of the present disclosure.
• the population has a size distribution having a coefficient of variation (CV) of about 10% or less than 10%.
• the coefficient of variation is of less than 5%.
• each microbead comprises a plurality of metals
• the average amount across the population of microbeads of each metal of the plurality of metals is about 10% or within 10% of the average amount of another metal of the plurality of metals.
• the plurality of metals comprises one or more enriched isotopes.
• the amount of each metal of one microbead of the population of microbeads is about 20% or within 20%, or about 10% or within 10%, or about 5% or within 5% of the amount of the same metal of another microbead of the population of microbeads.
• the amount of each metal of the population of microbeads has a distribution of a coefficient of variation of about 20% or less than 20%.
• the amount of each metal of the population of microbeads has a distribution of a coefficient of variation of about 10% or less than 10%.
• the microbeads of the population of microbeads comprise the same metal in substantially the same amount.
• the same metal is a plurality of metals and the microbeads comprise each metal of the plurality of metals in substantially the same amount.
• a further aspect is a composition comprising the microbead or plurality of microbeads.
• the composition can for example be a buffered solution, for example buffered to a pH of about 7.
• the buffered solution can comprise ammonium acetate and like buffers.
• the composition may also comprise PVP.
• the composition is an aqueous colloidal suspension.
• composition can comprise one or more components selected from stabilizers, preservatives, buffers, and mixtures thereof.
• the present disclosure includes a kit comprising the microbead, the plurality of distinct populations of microbeads and/or compositions of the present disclosure.
• each population of microbeads is distinguishable from another population of microbeads based on the metal or the plurality of metals of the microbeads.
• the microbeads of at least one population (e.g., each population) of microbeads comprise a metal or a plurality of metals different from the metal or the plurality of metals of the microbeads of another population of microbeads.
• the microbeads of at least one population (e.g., each population) of microbeads comprise a plurality of metals at a ratio different from the microbeads of another population of microbeads.
• the microbeads of each population of microbeads are conjugated to a different biomolecule.
• the microbead can be used as a barcoding agent for the biomolecule attached to the microbead through the nature of the metal or plurality of metals in the microbead.
• the present disclosure includes a microbead prepared by a method of the present disclosure.
• the structural monomer and metal chelating monomer may have been copolymerized according to any of the methods described herein.
• the structural monomers and/or metal chelating monomers used in the methods described herein may have any of the aspects described in this section.
• a kit of the present disclosure may include any of the microbeads, or populations of microbeads, described herein. Microbeads, or kits thereof, can also include additional aspects or for mass spectrometry assays as described further herein. Aspects of the present disclosure also include additional methods, such as mass spectrometry assays, as described further herein.
• the present disclosure includes a method of preparing 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 chelator attached to at least one polymerizable end group to obtain a second mixture, wherein the chelator coordinates the metal at least at 3 sites and wherein the metalchelating monomer is polymerizable with the structural monomer; and polymerizing the second mixture to form a copolymer of the microbead; wherein the structural monomer does not comprise the chelator.
• the structural monomer and the metal-chelating monomer should be soluble in the reaction medium.
• a monomer is substituted for example with a substituent that may interfere with polymerization (e.g. halo, amine, alcohol)
• the substituent can be temporarily protected prior to and/or during polymerization using protective groups known in the art.
• the protective groups can be selective removed and the substituents selectively deprotected using methods known in the art.
• the metal is a plurality of metals.
• the structural monomer is polymerized in the nucleation stage to about 5% to about 20% completion based on the structural monomer.
• the polymerizing 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 monomer.
• the structural monomer is as defined herein.
• the metal-chelating monomer is as defined herein.
• the steric stabilizer is as defined herein.
• the metal is as defined herein.
• the method further comprises functionalizing the microbead.
• the functionalizing 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; and polymerizing the third mixture.
• the reactive functional group can be selected from amine, thiol, alcohol, aldehyde, carboxylic acid, epoxide, vinyl, alkyne, maleimide, or mixtures thereof. It can be appreciated that certain reactive functional groups may interfere with the polymerization process and can be protected prior and/or during polymerization using protective groups known in the art. For example, amines and thiols can be protected by protective groups. For example, monomers substituted with reaction functional groups can be used in a protected form such that the reactive functional groups would not interfere with the polymerization process. Optionally, protected reactive functional groups can be deprotected using methods known in the art.
• the third monomer is selected from optionally substituted acrylic acid, optionally substituted methacrylic acid, and mixtures thereof.
• the functionalizing of the microbead comprises coating the microbead with silicon dioxide.
• the functionalizing of the microbead further comprises functionalizing the coating of silicon dioxide.
• the functionalization of the coating of silicone dioxide can comprise reacting the coating of silicon dioxide with an organic silane.
• the organic silane can be selected from chlorosilane, alkoxysilane, derivatives thereof, and mixtures thereof.
• the reacting the coating of silicone dioxide with the organic silane is done in presence of a catalyst.
• the catalyst can be selected from ammonia, hydroxide, organic amine, or mixtures thereof.
• the organic silane comprises a reactive functional group.
• the reactive functional group is selected from amine, thiol, alcohol, aldehyde, carboxylic acid, epoxide, vinyl, alkyne, maleimide or mixtures thereof.
• the organic silane is APTES.
• the method further comprises conjugating the microbead to a biomolecule.
• the biomolecule is as defined herein.
• the microbead has a diameter of about 0.6 pm to about 20 pm, about 1 pm to about 15 pm, about 2 pm to about 10 pm, about 2 pm to about 6 pm.
• the microbead is a microbead of the present disclosure.
• an interior structure of the microbead comprises the copolymer.
• the present disclosure includes a method of preparing metal-encoded microbeads, the method comprising: providing an aqueous dispersion of swellable seed particles and an anionic surfactant; contacting the aqueous dispersion with monomers comprising a structural monomer and a metal-chelating monomer, wherein the metal-chelating monomer comprises a metal and a chelator attached to at least one polymerizable end, and wherein the chelator coordinates the metal at least at 3 sites; and allowing the monomers to diffuse into the seed particles to form an aqueous dispersion of swollen seed particles; and initiating polymerization of the monomers in the aqueous dispersion of swollen seed particles; wherein the structural monomer does not comprise the chelator.
• the present disclosure includes a method of preparing a metal-encoded microbead comprising providing an aqueous dispersion comprising a swellable polymer seed, an organic compound, an anionic surfactant, and optionally an organic solvent in which the organic compound is soluble; 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 microbead; wherein the organic compound has a molecular weight below 5000 Da and a water solubility at 25°C of less than 10 -2 g/L.
• the present disclosure includes method of preparing a metal-encoded microbead comprising preparing a swellable polymer seed by emulsion polymerization, wherein an anionic surfactant is used as emulsifier under substantially oxygen-free conditions; contacting the swellable polymer seed with an aqueous dispersion comprising an organic compound, the 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 microbead; wherein the organic compound has a molecular weight below 5000 Da and a water solubility at 25°C of less than 10 -2 g/L.
• the structural monomer is selected from the group consisting of acrylic monomers, methacrylate monomers and vinyl monomers selected from the group consisting of styrene, divinylbenzene (DVB), ethyl vinyl benzene, vinyl pyridine, amino- styrene, methyl-styrene, dimethylstyrene, ethyl styrene, ethyl-methyl-styrene, p-chlorostyrene and 2,4-dichlorostyrene.
• VVB divinylbenzene
• ethyl vinyl benzene vinyl pyridine
• amino- styrene methyl-styrene
• dimethylstyrene dimethylstyrene
• ethyl styrene ethyl-methyl-styrene
• p-chlorostyrene 2,4-dichlorostyrene
• the aqueous dispersion of swollen seed particles further comprises a steric stabilizer.
• the steric stabilizer is polyvinylpyrrolidone.
• the providing the aqueous dispersion of swellable seed particles comprises preparing monodisperse swellable seed particles by emulsion polymerization.
• the aqueous dispersion of swellable seed particles further comprises an organic compound with a molecular weight below 5000 Dalton and a water solubility at 25° C. of less than 10 2 g/L; and optionally an organic solvent in which said organic compound is soluble.
• the swellable seed particles are monodisperse swellable seed oligomer particles.
• the anionic surfactant can be an alkyl sulfate or an alkyl sulfonate.
• the anionic surfactant is a Cs-i 6 alkyl sulfate or sulfonate or salts thereof.
• the anionic surfactant can be decylsulfate, dodecylsulfate, decylsulfonate, dodecylsulfonate or salt thereof.
• the anionic surfactant is sodium dodecyl sulfate or sodium decylsulfate.
• the structural monomer is as defined herein.
• the metal-chelating monomer is as defined herein.
• the organic compound is a polymerization initiator.
• the polymerization initiator is a peroxide, an azo compound, or mixtures thereof.
• the organic solvent is a non-polymerizable solvent selected from alcohol, ether, ketone, dialkylsulfoxides (e.g. DMSO), dialkylformamides (e.g. DMF), or mixtures thereof.
• a non-polymerizable solvent selected from alcohol, ether, ketone, dialkylsulfoxides (e.g. DMSO), dialkylformamides (e.g. DMF), or mixtures thereof.
• the monomers of the present disclosure can be prepared by various synthetic processes. The choice of particular structural features and/or substituents may influence the selection of one process over another. The selection of a particular process to prepare a given monomer is within the purview of the person of skill in the art.
• Some starting materials for preparing compounds described in the present disclosure are available from commercial chemical sources. Other starting materials, for example as described below, are readily prepared from available precursors using straightforward transformations that are well known in the art. In the Schemes below showing the preparation of the second monomer of the application, all variables are as defined in the present description, unless otherwise stated.
• the compounds of Formula I are prepared as shown in Scheme A.
• the chelator or the ligand of Formula A is attached to one or more linkers with a polymerizable end group of Formula B to form a monomer capable of metal chelation of Formula C.
• a metal D can be chelated to the monomer of Formula C to form the metalchelating monomer of Formula I.
• the ligand of Formula A can comprise one or more carboxylic acid groups. Accordingly, step a in Scheme A can be accomplished according to Scheme B.
• the ligand of Formula E can be attached to one or more linkers with a polymerizable group of Formula B through an amide bond formation or an esterification to obtain the monomer capable of metal chelation of Formula F. It can be appreciated that amide bond formation can be carried out using methods known in the art for example through the formation of an activated ester. a
• the metal chelation step b can be carried our using methods known in the art.
• the monomer capable of metal chelation of Formula C or F and the metal of Formula I can each be dissolved into a suitable solvent.
• Suitable solvent can be selected by a person skilled in the art and can include water.
• Metal of Formula D can be salts of the metal, for example halide salts of the metal. Solutions of the monomer capable of metal chelation and of the metal can be mixed together.
• the pH of the resulting mixture can be monitored and adjusted to a suitable pH.
• a suitable pH includes a pH of about 5.5 to about 7.5, or about 6. The resulting mixture can be stirred until the metal-chelating monomer of Formula I forms.
• a transformation of a group or substituent into another group or substituent by chemical manipulation can be conducted on any intermediate or final product on the synthetic path toward the final product, in which the possible type of transformation is limited only by inherent incompatibility of other functionalities carried by the molecule at that stage to the conditions or reagents employed in the transformation.
• Such inherent incompatibilities, and ways to circumvent them by carrying out appropriate transformations and synthetic steps in a suitable order will be readily understood to one skilled in the art. Examples of transformations are given herein, and it is to be understood that the described transformations are not limited only to the generic groups or substituents for which the transformations are exemplified.
• microbeads and kits described above may comprise additional aspects for use in mass spectrometry assays. Described below are methods and kits for mass spectrometry assays, which may include or use any suitable microbeads or kits described elsewhere herein. Suitable assays include assays to detect a target or sample biomolecule, such as target oligonucleotides, proteins (e.g., cytokines, cancer biomarkers, or antibodies to a specific antigen), or enzymes (e.g., activity of enzymes such as kinases, phosphatases, proteases, or any other enzyme of interest that modifies a substrate biomolecule attached to a microbead).
• a target or sample biomolecule such as target oligonucleotides, proteins (e.g., cytokines, cancer biomarkers, or antibodies to a specific antigen), or enzymes (e.g., activity of enzymes such as kinases, phosphatases, proteases, or any other enzyme of interest that
• Assay may use hybridization and/or a sandwich ELISA format to detect a target sample biomolecule. Suitable assays also include analysis of cells (e.g., use of microbeads as a standard for calibration, normalization or quantitation in a mass cytometry assay).
• Microbeads (and kits or methods thereof) of the present disclosure may be used for optical detection (e.g., fluorescence-based detection). Alternatively, microbeads (and kits or methods thereof) of the present disclosure may be analyzed by elemental analysis (e.g., mass spectrometry).
• optical detection e.g., fluorescence-based detection
• elemental analysis e.g., mass spectrometry
• the microbeads may exhibit low fluorescence (e.g., may have a fluorescence quantum yield less than 0.2, less than 0.1 , less than 0.05, or less than 0.02, across the visible and/or UV range) but would still be suitable for analysis by mass spectrometry (e.g., atomic mass spectrometry such as by ICP-MS) or another form of elemental analysis (e.g., by ICP-OES, by x-ray dispersion spectroscopy).
• mass spectrometry e.g., atomic mass spectrometry such as by ICP-MS
• ICP-OES atomic mass spectrometry
• x-ray dispersion spectroscopy e.g., by x-ray dispersion spectroscopy
• samples include any biological sample, such as a cell sample (e.g., a suspension of cells or a tissue section) or a biological fluid (e.g., comprising sample biomolecules in suspension).
• a sample may be a cell line, cell culture supernatant, or may be harvested from an organism such as a human, rodent or other mammal.
• a sample may be a blood sample, such as whole blood, serum, plasma, or peripheral blood mononuclear cells (PBMCs).
• PBMCs peripheral blood mononuclear cells
• a sample may be a saliva sample, nasal swab, excision (e.g., biopsy of solid tissue).
• a sample may comprise whole cells or may be homogenized from (e.g., a lysate from) a sample containing whole cells.
• a sample may be a purified sample biomolecule that is characterized by an assay, or using a kit, described herein.
• an assay or kit may screen potential sample biomolecules that are drug candidates.
• additional steps may be performed in any method described herein, such to remove unbound sample biomolecules from microbeads, unbound reporters from microbeads, or unbound mass tagged antibodies from cells.
• a sample may be a tissue section that is labeled with mass tags (e.g., by mass tagged antibodies) for analysis by imaging mass spectrometry (e.g., LA-ICP-MS or SIMS).
• imaging mass spectrometry e.g., LA-ICP-MS or SIMS.
• particles in suspension such as cells and/or beads
• a mass cytometer e.g., an ICP-MS system
• Mass spectrometry of the subject application may be atomic mass spectrometry.
• a mass spectrometer of the subject application may simultaneously detect a plurality of mass channels. Different mass channels may correspond to a metal or isotope thereof from different mass tags.
• Such simultaneous mass spectrometers may be, for example, a time-of-flight mass spectrometer (TOF-MS) or a magnetic sector mass spectrometer.
• a mass spectrometer may atomize the sample for atomic mass spectrometry.
• ICP laser ablation ICP
• secondary ion e.g., for secondary ion mass spectrometry (SIMS)
• any suitable ionization source may be used to atomize a sample.
• a mass cytometer may specifically detect metals from mass tags and/or microbeads described herein, and may, for example, comprise ion optics for filtering out lighter atoms (e.g., endogenous elements such as C, N, O, and light metals, and optionally further plasma gas elements such as Argon and Argon dimer if the mass spectrometer is an ICP system).
• lighter atoms e.g., endogenous elements such as C, N, O, and light metals, and optionally further plasma gas elements such as Argon and Argon dimer if the mass spectrometer is an ICP system.
• Exemplary mass cytometers are described further in US patent publication numbers 20050218319 and 20160049283, which are incorporated herein by reference.
• a copolymer microbead of the present disclosure may comprise a barcode.
• the barcode may be a plurality of metals or enriched metal isotopes chelated by a metal chelating monomer.
• the barcode may be an assay barcode (i.e., that identifies a sample biomolecule the microbead is functionalized to bind to).
• the microbead may comprise a sample barcode used to identify a sample the microbead was, or will be, mixed with.
• the microbead may be attached to (e.g., covalently bound to) a biomolecule (e.g., a capture biomolecule) that specifically binds to sample biomolecule.
• a biomolecule e.g., a capture biomolecule
• the biomolecule may include, for example, an affinity reagent such as an antibody that specifically binds a sample antigen biomolecule of interest such as a viral particle, cytokine, cancer biomarker.
• the biomolecule may include, for example, an oligonucleotide such as a ssDNA oligonucleotide that has a sequence that specifically hybridizes to a sample oligonucleotide biomolecule of interest.
• a 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 mass tag.
• the methods of the present disclosure may include binding a sample biomolecule to a biomolecule (e.g. capture biomolecule) attached to a barcoded microbead, and may further include binding a mass tagged reporter to the sample biomolecule. The barcode and mass tag may then be detected by atomic mass cytometry as described herein.
• certain kits described herein may include barcoded microbeads optionally attached to a biomolecule that specifically binds a sample biomolecule, and the kit may further include a reporter that associates a mass tag with the sample biomolecule.
• kits includes a population of microbeads, or a plurality of distinct populations of microbeads, as described in any of the other embodiments herein.
• each population of microbeads may be distinguishable (e.g., by atomic mass spectrometry) from another population of microbeads based on the metal or the plurality of metals of the microbeads.
• the microbeads of at least one population (e.g., each population) of microbeads comprise a metal or a plurality of metals different from the metal or the plurality of metals of the microbeads of another population of microbeads, or may comprise a plurality of metals at a ratio different from the microbeads of another population of microbeads.
• the microbeads of each population of microbeads in a kit are conjugated to a different biomolecule.
• a kit may further include a reporter comprising a mass tag, such as a reporter that is capable of specifically binding a sample biomolecule specifically bound by at least one of the different biomolecules.
• a reporter comprising a mass tag, such as a reporter that is capable of specifically binding a sample biomolecule specifically bound by at least one of the different biomolecules.
• Different biomolecules attached to microbeads of the kit may specifically bind to different sample biomolecules.
• Sample biomolecules may be any biomolecule described herein that is present in a sample. Such sample biomolecules may be a protein (e.g., or peptide thereof), oligonucleotide, lipid, carbohydrate or small molecule.
• a protein may be, for example, an antibody or a cytokine.
• An 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.
• a sample biomolecule may include an oligonucleotide (e.g., an RNA), and at least one of the different biomolecules of the microbeads may be an oligonucleotide (e.g., a ssDNA) that specifically hybridizes to the sample biomolecule (e.g. target analyte).
• the reporter comprises a plurality of oligonucleotides that hybridize to indirectly bind a plurality of mass tagged oligonucleotides to the sample biomolecule.
• a reporter comprising a mass tag may directly hybridize to the sample biomolecule.
• a sample biomolecule may be a biomolecule other than an oligonucleotide, such as an antigen (e.g., a protein such as a cytokine), and the reporter may comprise an oligonucleotide conjugated to an antibody, where the antibody is able to bind the antigen and the oligonucleotide conjugated to the antibody is bound, directly or indirectly (e.g., through hybridization) to mass tag oligonucleotide of the reporter.
• Such indirect hybridization may allow for association of multiple mass tags with a sample biomolecule, such as through hairpin chain reaction, branched in- situ hybridization, or any other suitable method.
• a reporter as described herein may (or may not) include a system of separate biomolecules that together associate a mass tag with a sample biomolecule.
• At least one of the different biomolecules attached to a microbead is an antibody (e.g., or a fragment thereof, such as a nanobody).
• the sample biomolecule is a viral particle
• the at least one of the different biomolecules is a first antibody that specifically binds the viral particle
• the reporter comprises a second antibody that specifically binds the viral particle.
• At least one sample biomolecule is a cytokine, for example wherein at least one of the different biomolecules attached to a microbead is a first antibody that specifically binds the cytokine, and wherein the reporter comprises a second antibody that specifically binds the cytokine.
• the cytokines are selected from IL-18, IL-1F4, TNFa, IL-6, IFNy, IL-4, CD163, CXCL-9/MIG, IL-10, IL-1 b, and combinations thereof.
• At least one sample biomolecule is a cancer biomarker (for example a prostate specific antigen), wherein the 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.
• a cancer biomarker for example a prostate specific antigen
• the at least one of the different biomolecules includes a viral antigen, wherein the sample biomolecule is an antibody that specifically binds the viral antigen, and wherein the reporter comprises a secondary antibody that binds to the sample biomolecule.
• the methods and kits of the present disclosure may include a plurality of different reporters, wherein each of the different reporters is capable of binding a sample biomolecule specifically bound by a different biomolecule.
• a plurality of different reporters may each comprise the same mass tag (detected in the same mass channel), or a different mass tag (detected in different mass channels).
• the mass tag may include a metal nanoparticle, such as a metal nanocrystal (e.g., a nanogold particle), a quantum dot, a polymer nanoparticle, or the like.
• a nanoparticle may be at or less than 100nm, less than 50nm, less than 20nm, less than 10 nm in diameter, such as between 2nm and 100nm or between 5 and 50 nm.
• the mass tag may include a metal chelating polymer, such as a linear or branched polymer comprising metal binding (e.g., metal chelating) pendant groups.
• Other mass tags may be suitable, such as an organotellurium polymer mass tag in which a tellurium atom is covalently bound to carbon atoms of the polymer, are also within the scope of the present disclosure.
• a mass tag may have one or more atoms of a metal element or enriched isotope thereof.
• Mass tags may be conjugated to a biomolecule (e.g., a biomolecule of a reporter that binds to a sample biomolecule) through any suitable conjugation means described herein or known to one of skill in the art.
• a biomolecule attached to a microbead is an enzyme substrate for a sample biomolecule.
• a method or kit of the present disclosure may further include a reporter that specifically binds (e.g. is capable of specifically binding) to the enzyme substrate when it has been modified by the sample biomolecule or that specifically binds (e.g. is capable of specifically binding) to the enzyme substrate when it has not been modified by the sample biomolecule.
• the enzyme substrate may include a kinase substrate
• the sample biomolecule e.g. target analyte
• the reporter may be a phosphorylation specific antibody that binds the phosphorylated substrate.
• the enzyme substrate includes a mass tag that is removed when the enzyme substrate is modified by the sample biomolecule, such as a peptide sequence that is cleaved by a sample biomolecule that is a protease.
• a mass tag that is removed when the enzyme substrate is modified by the sample biomolecule, such as a peptide sequence that is cleaved by a sample biomolecule that is a protease.
• the absence of a signal of a mass tag may indicate the presence of a sample biomolecule enzyme. Examples of enzyme assays are described in US patent publication number 20070190588, which is incorporated herein by reference.
• microbeads from a plurality of different populations are in admixture (in a first mixture of microbeads).
• aspects may further include a second mixture of microbeads comprising the same biomolecules 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 barcode is for example on the interior of the microbeads (e.g., may be a subset of metals chelated by the metal chelating monomer of the microbeads of the first and second mixture).
• sample barcode may be on the surface of the microbeads of the first and second mixture.
• Methods or kits may include a plurality of sample barcodes in separate partitions that are functionalized to bind to the surface of microbeads from a plurality of different populations (e.g., where the sample barcodes of each partition are applied to the microbeads of a different mixture).
• Sample barcoded microbeads may be mixed together (e.g., after mixing with their respective samples but optionally before any mixture with a reporter), then analyzed by mass spectrometry. The mass spectrum from the sample barcode of each microbead may thereby be used to identify the sample it came from.
• kits or methods may further include a panel of mass-tagged antibodies in admixture with one another, wherein at least some antibodies of the panel are specific for cell surface markers (e.g., are used to bind cell surface proteins in a cellular sample).
• the plurality of antibodies may in admixture with microbeads of the kit, e.g., in a buffered solution or in a lyophilized form (e.g., less than 5% or less than 1% moisture by volume).
• a plurality of (e.g., each of) the mass-tagged antibodies comprise metals identical to metals chelated by metal chelating monomers of microbeads of the kit.
• cells as described herein may be distinguishable by atomic mass spectrometry.
• cells as described herein may include one or more metals (or enriched isotopes thereof) that no naturally occurring cells usually comprise, such as an iridium intercalatorthat binds to DNA of the cells.
• Microbeads may be analyzed alongside cells as a mass standard and/or to detect biomolecules (e.g., cytokines, antibodies, cancer biomarkers) in a sample solution (e.g., a cell culture supernatant or a serum).
• biomolecules e.g., cytokines, antibodies, cancer biomarkers
• a kit comprising one or more populations of microbeads as described herein further includes a steric stabilizer (e.g., in admixture with microbeads of the kit).
• the steric stabilizer may be polyvinylpyrrolidone (PVP), such as at more than 0.05%, 0.1%, 0.2%, 0.5%, or 1% by weight, such as at or between about 0.05% and about 10%, about 0.1% and about 5%, about 0.2% and about 2% by weight.
• PVP polyvinylpyrrolidone
• microbeads of the kit may be in a solution buffered at a pH at or between about 4 and about 10, a pH at about or between about 5 and about 9, a pH at about or between about 6 and about 8, a pH greater than about 3, a pH greater than about 4, or a pH less than about 10.
• the microbeads of a kit of the present disclosure are lyophilized.
• the microbeads of the kit are at 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% moisture by weight.
• microbeads may be fused to a solid support, such as for calibration (e.g. as reference particles), normalization or quantitation in imaging mass spectrometry (or imaging mass cytometry) as described further herein.
• the solid support may be any suitable support such a slide (e.g., a microscope slide, such as a transparent glass or quartz slide) or an adhesive film (e.g., for application to a microscope slide in any of the subject methods).
• the solid support may further comprise a biological sample.
• 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 fused microbeads.
• the microbeads may all be the same, or the microbeads may differ in the metals or amounts thereof. When different microbeads are used, there will typically be multiple of each population of microbead.
• the microbeads may be dispersed on the solid support such that substantially all of the microbeads are located individually (i.e. discretely) on the solid support, such that each fused microbead can be individually identified and sampled.
• the solid support may further comprise some fused microbeads that have agglomerated on the sample carrier and thus these agglomerates may be unsuitable 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 may be agglomerated on the solid support.
• At least about 80% of the microbeads may be individually isolated, such as at least about 85%, at least about 90%, at least about 92%, or at least about 95%.
• Optical interrogation may identify which locations of the solid support have discrete microbeads, and can guide the acquisition by imaging mass spectrometer.
• the step of fusing the at least one microbead to the solid support may comprise heating the solid support.
• 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.
• the fusing of the at least one microbead to the sample carrier or solid support can occur by vitrification.
• the sample carrier or solid support is heated to a maximum of up to 300 °C, for example up to 275 °C, up to 250 °C, up to 225 °C or up to 200 °C
• a kit of any embodiments described herein may further include one or more of a buffer (e.g., PBS, red blood cell lysis buffer, wash buffer, staining buffers, and/or buffers for reconstituting lyophilized reagents such as lyophilized microbeads, lyophilized reporters, lyophilized antibodies), anticoagulant (e.g., for processing a blood sample), fixation reagents, permeabilization reagents, or any reagents for performing the methods or examples described herein.
• a buffer e.g., PBS, red blood cell lysis buffer, wash buffer, staining buffers, and/or buffers for reconstituting lyophilized reagents such as lyophilized microbeads, lyophilized reporters, lyophilized antibodies
• anticoagulant e.g., for processing a blood sample
• fixation reagents e.g., for processing a blood sample
• permeabilization reagents e
• the microbeads may be an element standard used for calibrating a mass spectrometer and/or to normalization (e.g., normalization of a mass spectrum obtained from mass tags) or quantitation (e.g., quantitation of the number of antibodies in a cell or pixel) as described further herein.
• normalization e.g., normalization of a mass spectrum obtained from mass tags
• quantitation e.g., quantitation of the number of antibodies in a cell or pixel
• a method of mass spectrometry analysis includes: mixing the distinct populations of microbeads of the present disclosure with a sample, wherein microbeads of each population of microbeads are bound to a different biomolecule and wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or the plurality of metals of the microbeads; binding different sample biomolecules of the sample to the different biomolecules of distinct populations of microbeads; binding, directly or indirectly, a reporter to each of the different sample biomolecules, wherein the reporter bound to each of the different sample biomolecules comprises a mass tag; and detecting the metal and the mass tag of individual microbeads by mass spectrometry.
• Such a method may further include attaching the different biomolecules to the microbeads of the distinct populations of microbeads prior to the step of mixing the distinct populations of microbeads with the sample.
• One or more of the different sample biomolecules may include oligonucleotides, antibodies (or another affinity reagent), cytokines, cancer biomarkers (such as one or more prostate specific antigens) etc.
• a method of mass spectrometry analysis uses a kit of any of the embodiments described herein, and further includes the steps of: mixing distinct populations of microbeads with a sample, wherein microbeads of each population of microbeads are bound to a different biomolecule and wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or the plurality of metals of the microbeads; binding different sample biomolecules of the sample to the different biomolecules of distinct populations of microbeads; binding, directly or indirectly, a reporter to each of the different sample biomolecules, wherein the reporter bound to each of the different sample biomolecules comprises a mass tag; and detecting the metal and the mass tag of individual microbeads by mass spectrometry.
• a kit or method of mass spectrometry analysis of a cellular sample using the microbeads of any embodiment described herein includes the steps of: providing a plurality of mass-tagged antibodies, wherein each antibody of the mass tagged antibody is conjugated to a polymer mass tag chelating multiple atoms of a metal or enriched isotope thereof; contacting a sample with the plurality of mass-tagged antibodies; detecting the mass- tagged antibodies bound to the sample and the microbeads by mass spectrometry.
• Microbeads and the cellular sample may be combined prior to analysis by mass spectrometry, or may be analyzed in separate sample runs.
• the microbeads may be used to assay one or more sample biomolecules. Alternatively or in addition, microbeads may be an element standard used for calibration, normalization and/or quantitation as described further herein.
• the microbeads may be of a single population having a consistent size and/or amount of one or more metals as described herein.
• the microbeads may be of distinct populations each characterized by a different set of metals, combination of metals, and/or amount of metals.
• a set of microbead populations that together have metals from more than 10 distinct elements (e.g., more than 30 distinct isotope masses).
• the microbeads of the subject application may together provide signal 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 alongside cells in a suspension mass cytometry workflow or may be provided on the same support as a tissue section or cell smear in an imaging mass cytometry workflow. Microbeads may be used in an assay as described herein, or may be a standard (e.g., that provides a signal in most or all mass channels that a mass tag detected by mass cytometry is detected in). The mass spectra of microbeads that have different amounts of the same metal (e.g., or enriched isotope thereof) may be used to create a curve (e.g., of a signal intensity to a known amount of metal) used in calibration, normalization or quantitation as described further herein.
• a curve e.g., of a signal intensity to a known amount of metal
• Microbeads may be used for calibrating the mass spectrometer used in the step of detecting of any method described herein.
• Calibration may be of 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 mass and may be based in part of the shape of a peak from a specific isotope. A mass resolution above a certain value may indicate a pass.
• Mass Calibration may include auto-tuning that checks the values of one or more mass channels (e.g., from a metal of microbead standard) and then calculates the TOF values for additional mass channels and/or may include aligning the correct ions to the detection channel so that the entire signal for each ion is collected.
• Dual Count Calibration may determine dual count coefficients (dual slopes to correlate pulse count and intensity (the dual count coefficient converts analog signal to ion count signal). This correlation may be important when ion concentrations increase and pulses overlap, for example, during a cell or microbead event.
• XY optimization is the process by which the optimal alignment of the torch with the vacuum interface is determined to provide the maximum signal for a mass channel (e.g., from a metal of microbead standard). Optimizing the alignment of the system is important for maximum transmission of ions into the vacuum interface.
• Detector voltage calibration uses the dual count calibration to determine the detector voltage that provides the best signal while ensuring the longevity of the detector. The optimum detector voltage may be achieved when the dual count coefficient is 0.03 ⁇ 0.003. The detector voltage should not be more positive than -1 ,100 V.
• Gases and/or current calibration may optimize the nebulizer gas flow and the makeup gas flow (and optionally additional gas flows) using the maximum mass channel signal (e.g., from a metal of microbead standard) that can be achieved by varying the makeup gas flow and nebulizer gas flow while controlling oxide formation. This may ensure that the plasma temperature is optimal in the system and minimal metal oxides are formed.
• the current that is applied at the vacuum interface is increased in increments to drive the transfer of the ion cloud through the interface. The value that provides the highest signal may then be selected. Calibration may be performed during a sample run (e.g., to account for sensitivity drift).
• Microbeads may be used as a standard for normalizing a mass spectrometry signal obtained from the mass tags (e.g., mass tags of a sample as described herein) based on a mass spectrometry signal obtained from the microbeads (e.g., obtained from microbeads comprising the one or more of the same metals as at least one of the mass tags, or similar mass spectrum, or standard curve created from a plurality of microbead populations that comprise different amounts of a metal).
• Each of the tags may provide a signal in one or more mass channels.
• Such normalization of mass tag signal may be for individual cell or assay microbead events (e.g., in suspension mass cytometry) or individual cells, assay microbeads or pixels (e.g., in imaging mass cytometry).
• individual cells of a cell smear may be detected by IMC, or cells of a solid tissue section segmented algorithmically based on a membrane stain, and the mass tag signal across the single cell may be normalized or quantified as described herein.
• the mass tag signals of a cell or pixel may be normalized to signal from microbeads that are 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 when the cell or pixel was detected.
• 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 when the cell or pixel was detected.
• normalization of a mass tag signal may be based on one or more microbeads whose metals are detected in the same mass channel as the mass tag.
• microbeads used as a standard include microbeads of distinct populations that have different amounts of the metal
• normalization of a signal from a mass tag comprising the same metal may be based on one or more microbeads whose that provide a similar signal intensity (e.g., at or less than a ten fold difference, at or less than a five fold difference, at or less than a two fold difference, etc.) for the same mass channel.
• a metal may be an enriched isotope.
• a microbead standard comprising population of microbeads having different metals and/or amounts of metals may be used.
• Such normalization may be similar to the use of EQ4TM beads provided by Fluidigm to normalize mass cytometry data (e.g., normalize data of FCS files obtained by mass cytometry).
• the microbeads of the present disclosure may together provide signal 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 used as a standard for quantitating the amount of one or more mass- tagged antibodies (or other mass-tagged biomolecules), such as when a known (or estimated) number of metal atoms from the mass tag are associated with the antibody (or other biomolecule).
• the number of metal atoms associated with a mass-tagged antibody (or other biomolecule) that may have more than one instance of the mass tag may be determined by: starting from a known number of metals per mass tag (e.g., on a polymer mass tag) and UV/visible spectrum or ICP-MS signal characteristic; and further analyzing fractions of the mass-tagged antibody (or other biomolecule) by UV or Visible spectrum or ICP-MS.
• Quantifying the mass-tagged antibodies may therefore be based on the average number of metal atoms for each of the mass-tagged antibodies and the detected mass spectrometry signal from the mass-tagged antibodies and the microbeads.
• the microbeads may have a known number of metal atoms (e.g., determined as described herein).
• the microbeads and the mass-tagged antibodies (or other biomolecule) may include the same metals.
• quantifying the mass-tagged antibodies may be calculated as the number of a metal in a microbead, times the ratio of a signal from a mass-tagged antibody (or other biomolecule) to the signal of the metal from the microbead, and divided by the average number of metal atoms per antibody (or other biomolecule). Quantitation may be performed for individual cells, assay microbeads, or pixels.
• Embodiments of the present disclosure include a computer readable medium configured to perform one or more of calibration, normalization, or quantitation as described herein.
• a step of detecting may include inductively coupled plasma mass spectrometry (ICP-MS), such as for suspension mass cytometry or imaging mass spectrometry.
• ICP-MS inductively coupled plasma mass spectrometry
• the mass spectrometry may be by a simultaneous mass spectrometry, such as time-of-flight mass spectrometry (TOF-MS) or magnetic sector mass spectrometry.
• TOF-MS time-of-flight mass spectrometry
• magnetic sector mass spectrometry magnetic sector mass spectrometry
• detecting the microbeads may be by imaging mass spectrometry (e.g., imaging mass cytometry).
• the microbeads e.g., a microbead standard
• Imaging mass spectrometry may be, for example, by laser ablation ICP-MS or by secondary ion mass spectrometry (SIMS).
• a biological sample can include any sample of a biological nature that requires analysis.
• samples may include biological molecules, 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, blood cells (e.g., PBMCs), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.
• Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
• Another typical source of biological samples are viruses and cell cultures of animal, plant, bacteria, fungi where gene expression states can be manipulated to explore the relationship among genes.
• other samples can be interrogated, such as artificial samples. Certain aspects of the present disclosure are especially useful when interrogating samples of a human origin, and especially useful when interrogating samples of human peripheral blood.
• Mass tagged oligonucleotides may be hybridized, directly or indirectly, to a target oligonucleotide.
• one or more intermediate oligonucleotides may provide a scaffold on which a plurality of mass tagged oligonucleotides can hybridize, thereby amplifying signal.
• Aspects of the subject application therefore include oligonucleotides for hybridization based signal amplification.
• a sample biomolecule may be a target oligonucleotide, such as a DNA or RNA molecule (such as coding RNA, small interfering RNA, or micro RNA) of a cell or bead.
• the target oligonucleotide may be single stranded.
• the target oligonucleotide may have a known specific sequence (or homology to a known specific sequence).
• a reporter may include a non-oligonucleotide biomolecule, such as an antibody or derivative thereof, which may be conjugated to an oligonucleotide, such as to a synthetic single stranded DNA oligonucleotide comprising a known sequence.
• an oligonucleotide such as to a synthetic single stranded DNA oligonucleotide comprising a known sequence.
• both the antibody and the oligonucleotides may be referred to as a part of a reporter.
• the signal from a target oligonucleotide, or a reporter comprising a non-oligonucleotide biomolecule conjugated to an oligonucleotide may be amplified through a hybridization scheme.
• the hybridization may be branched or linear.
• a polymerase may extend the first oligonucleotide along a template to provide additional sites for attachment of an element tag (such as additional hybridization sites for an element tagged oligonucleotide).
• Mass tagged oligonucleotides may include a single labeling atom, or may include a polymer comprising multiple labeling atoms, and may be referred to as a reporter.
• Mass tagged oligonucleotides may include a labeling atom, such as a heavy metal atom, in the chemical structure of the oligonucleotide itself.
• a labeling atom such as a heavy metal atom
• Signal amplification may uniquely benefit bead-based assays, in which the same reporter tag (labeling metal element or isotope) can be amplified and used across different beads and their target analytes.
• an element tagged reporter oligonucleotide may hybridize to another portion of the target RNA or DNA, thereby providing a signal when the target RNA or DNA is bound to the bead.
• an element tagged reporter affinity reagent e.g., reporter antibody
• an element tagged reporter affinity reagent may bind to another epitope on the analyte, thereby providing a signal when the target analyte is bound to the bead.
• the analyte may further be bound by a reporter, such as an element tagged reporter antibody or oligonucleotide.
• the reporter may comprise a high sensitivity (e.g., intensity) element tag that provides a highly abundant isotope (e.g., more than 50, 100, 200, 500, 1000 copies of a single isotope), thereby enabling detection of a smaller number of a target analyte bound to an assay bead.
• a high sensitivity element tag may include a nanoparticle (e.g., comprising a metal nanocrystal surface functionalized to bind biomolecule such as an antibody or oligonucleotide) or a hyperbranched polymer.
• a nanoparticle tag e.g., gold nanoparticle
• a reporter probe through a biotin-avidin (e.g., biotin-streptavidin) interaction.
• the nanoparticle e.g., gold nanoparticle
• streptavidin e.g., streptavidin
• the reporter may also comprise a low sensitivity element tag that provides a low abundance isotope (e.g., less than 100, 50, 30, 20, 10, or 5 copies of an isotope) that is distinct from the highly abundant isotope, thereby allowing detection/quantitation of the amount of an analyte that is such high abundance that the highly abundant isotope would saturate the detector.
• the high and low abundance isotopes 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.
• Reporters for different analytes may comprise the same isotopes or combination of isotopes, since the analytes will be distinguished by the unique assay barcodes of the beads.
• a reporter may include a reporter system that provides signal amplification through association of a plurality of instances of an element tag with a single instance of the target analyte (e.g. sample biomolecule).
• Signal amplification may be by enzymatic deposition, hybridization (e.g., branched hybridization, chain hybridization, and/or hybridization of a plurality of reporter oligonucleotides to a single long intermediate oligonucleotide), extension (e.g., a single extension, rolling circle extension), and/or a series of branched conjugations.
• a plurality (e.g., all) of the analytes detected with the assay beads may be detected with the same reporter system.
• a signal amplification reporter system may have a high sensitivity element tag.
• an element tag comprising an enzyme substrate moiety may be deposited from solution onto the bead (or a molecule attached to the bead) by an enzyme attached to a reporter biomolecule.
• Such a reaction may be covalent binding by a tyramide element tag acted on by horse radish peroxidase bound to a reporter biomolecule.
• aspects include a hybridization scheme, such that a plurality of element tagged oligonucleotides hybridize indirectly (through one or more oligonucleotide intermediates) to a single oligonucleotide target.
• 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.
• mass cytometry may enable enough detection channels (mass channels) to detect both a sample and assay barcode in beads while allowing an additional channel for a reporter (e.g., for detecting the assay target).
• the bead assays described herein may be sample and/or assay barcoded for use in mass cytometry.
• a plurality of different conditions e.g., drug candidates such as enzymes or an agonist or antagonist of one or more enzymes
• Individual conditions may be identified with a sample barcode shared across different assay beads exposed to the same conditions.
• Assay barcoded beads may be combined prior to analysis, such as before exposure to a condition. Sample barcoded beads may be combined prior to analysis.
• the enzyme may be a protease, kinase, phosphatase, or a DNA modifying protein such as a DNA methyltransferase.
• the target may be the substrate acted on by the enzyme, and the reporter (e.g., a reporter biomolecule as described herein) may only bind the target (e.g. sample biomolecule) before or after it is acted on by the enzyme.
• the reporter e.g., a reporter biomolecule as described herein
• a phospho-specific antibody that detects the phosphorylated form of a protein target, which may be increase in abundance when acted on by a kinase enzyme or decreased in abundance when acted on by a phosphatase.
• Sample barcode may be used to indicate which of a number of enzymes (or agonists/antagonists thereof) was tested in a particular assay.
• the candidate enzyme, agonist, antagonist may be added to a biological fluid such as a cell lysate, after which the sample is contacted with assay barcoded beads to detect activity of the enzyme.
• the candidate may be administered to cells (e.g., directly or by genetic engineering) or to an organism such as a patient or mammalian test subject, and a sample taken from that source may be contacted with the assay beads.
• Sample barcoding allows many such candidates to be screened in parallel.
• the sample barcode can be added as described for beads herein to identify the candidate. For example, more than 10, more than 20, more than 50, more than 100, more than 500, or more that 1000 distinct samples can be barcoded.
• 12 distinct isotopes in unique combinations of 6 provides 924 distinct combinations (e.g., for barcoding of up to 924 samples). Another 12 distinct isotopes could be used to barcode close to 1000 assays.
• more than 10, more than 20, more than 50, more than 100, more than 500, or more that 1000 distinct assay beads can be barcoded (e.g., beads detecting amount of a different substrate acted on by the candidate). At least one channel would be left for detection of the substrate by a reporter, as described herein. This may allow unprecedented screening with an immediate readout by mass cytometry.
• Post-translational modifications of proteins are carried out by enzymes within living cells.
• Known post-translational modifications include protein phosphorylation and dephosphorylation as well as methylation, prenylation, sulfation, and ubiquitination.
• the presence or absence of the phosphate group on proteins, especially enzymes, is known to play a regulatory role in many biochemical pathways and signal transduction pathways.
• a kinase function is to transfer phosphate groups (phosphorylation) from high- energy donor molecules, such as ATP, to specific target molecules (substrates).
• An enzyme that removes phosphate groups from targets is known as a phosphatase.
• the largest group of kinases are protein kinases, which act on and modify the activity of specific proteins.
• Various other kinases act on small molecules (lipids, carbohydrates, amino acids, nucleotides and more) often named after their substrates and include: Adenylate kinase, Creatine kinase, Pyruvate kinase, Hexokinase, Nucleotide diphosphate kinase, Thymidine kinase.
• Protein kinases catalyze the transfer of phosphate from adenosine triphosphate (ATP) to the targeted peptide or protein substrate at a serine, threonine, or tyrosine residue. Protein kinases are distinguished by their ability to phosphorylate substrates on discrete sequences. Commercially available kinases can be in the active form (phosphorylated by supplier) or in the inactive form and require phosphorylation by another kinase.
• ATP adenosine triphosphate
• a protein phosphatase hydrolyses phosphoric acid monoesters at phosphoserine, phosphothreonine, or phosphotyrosine residue into a phosphate ion and a protein or peptide molecule with a free hydroxy group. This action is directly opposite to that of the protein kinase. Examples include: the protein tyrosine phosphatases, which hydrolyse phospho tyrosine residues, alkaline phosphatase, the serine/threonine phosphatases and inositol monophosphatase.
• Another aspect of the present disclosure is to provide a kit for the detection and measurement of elements in a sample, where the measured elements include an element tag attached to a phosphorylated substrate, an element of a metal ion coordination complex, and elements of uniquely labeled supports, comprising: an element tag for directly tagging phosphorylated substrate; a multitude of phosphorylated substrates; uniquely labeled supports; metal ion coordination complex; and optionally, phosphatase, phosphatase buffer and ADP.
• Another aspect of the subject disclosure is to provide a method for a kinase assay, comprising: incubating ATP, at least one kinase, a free metal ion coordination complex, and a multitude of non-phosphorylated substrates immobilized on element labeled supports in such manner that a single type of non-phosphorylated substrate is attached to a single type of element labeled support, in conditions to enable the kinase to phosphorylate the substrates; separating the multitude of phosphorylated substrates immobilized on element labeled supports having attached metal ion coordination complex from the free metal ion coordination complexes and the multitude of immobilized non-phosphorylated substrates; and measuring the multitude of phosphorylated substrate immobilized on element labeled supports having attached metal ion coordination complex by elemental analysis.
• Another aspect of the subject disclosure is to provide a kit for the detection and measurement of elements in a sample, where the measured elements include an element tag attached to a non-phosphorylated substrate and a metal ion coordination complex, comprising: an element tag for directly tagging non-phosphorylated substrate; non- phosphorylated substrate; a solid support; a metal ion coordination complex; and optionally, kinase; kinase buffer; and ATP.
• Proteases are a subclass of protein-degrading enzymes that have recently been shown to play a vital role in signaling pathways, the dysregulation of which can result in cancer, cardiovascular disease, and neurological disorders.
• One aspect of the invention is a method for detecting protease activity in a biological fluid.
• the method may include attaching a coded bead to a first amino acid of a peptide substrate to form an immobilized peptide substrate, the peptide substrate comprising a first amino acid and a last amino acid and being a substrate for a protease enzyme: attaching an element tag to the last amino acid of the peptide substrate to form a tagged peptide substrate: incubating the immobilized, tagged peptide substrate with the biological fluid: and detecting the element tag and the coded bead in the biological fluid by elemental analysis.
• An encoded microbead may be both assay and sample barcoded, as discussed herein.
• a protease assay kit may include an assay coded bead attached to a first amino acid of a peptide substrate (an immobilized peptide substrate), the peptide substrate may include a first amino acid and a last amino acid and may be a substrate for a protease enzyme.
• An element tag may be attached at or near the last amino acid of the peptide substrate to form a tagged peptide substrate.
• the coded bead may be both assay and sample barcoded, as discussed herein.
• sample barcodes may distinguish assay barcode beads and/or cells from at least 5, 10, 20, 50, or 100 or more different samples.
• Sample barcoding reagents for cells may include an element-tagged antibody or antibodies (that bind across a plurality of cell types or majority of cells in the sample), an element tag functionalized to bind non-specifically to cells (e.g., through a covalent interaction), and/or metal in solution.
• Sample barcoding reagents for cells may further include a reagent for bringing the sample barcode into the cell (e.g., DMSO, cell permeabilization reagents such as a detergent or alcohol, etc.).
• Sample barcoding reagents for assay barcoded beads may be present within the beads, on the surface of the beads, or may be applied to the beads. If for application to beads, the sample barcoding reagent may comprise functional groups as described herein to bind to the surface of the bead (e.g., to bind to functional groups presented by the bead or to a blocking reagent present on the bead surface).
• Sample barcoding reagents for a given sample may comprise a unique combination of isotopes specific to that sample.
• cells and assay barcoded beads from the same sample may be labeled with the same assay barcode.
• the same assay barcode used for labeling of cells and beads may comprise the same combination of isotopes and/or same means of attachment (e.g., functional group).
• Sample barcoding reagents may be provided in admixture or alongside with an antibody panel, such as a lyophilized antibody panel. Sample barcoding reagents may be provided in admixture or alongside with assay barcoded beads. Assay barcoded beads may be provided in admixture or alongside an antibody panel. Assay barcoded beads and sample barcoding reagents may be provided in admixture with or alongside a lyophilized antibody panel (e.g., where the sample barcoding reagents bind both assay barcoded beads and cells in the sample). In certain embodiments above, sample barcoding reagents may be in, on, or provided alongside sample and/or assay barcoded beads.
• barcoding reagents can be provided in a pre-configured form by preparing the barcoding reagents with a number of unique combinations of assay barcodes and sample barcodes.
• each unique barcoding reagent can be stored in distinct containers, such as distinct wells of a well plate.
• a well plate can be established such that all wells along a particular column (or row) share the same assay barcode, whereas all wells along a particular row (or column) share the same sample barcode.
• a well plate can be established such that each filled well contains barcoding reagents with various combinations of a particular unique sample barcode and numerous assay barcodes.
• a first well may contain barcoding reagents all having a first sample barcode but each having different assay barcodes
• a second well may contain barcoding reagents all having a second barcode but each having different assay barcodes.
• pre-configured barcoding reagents can require the manufacture of thousands of groups of unique beads.
• barcoding reagents e.g., beads
• each group of barcoding reagents can be coupled to a biomolecule (e.g., antibody) having a targeting function associated with the assay that is associated with the assay barcode of that group of barcoding reagents.
• the sample barcodes can be bound to the barcoding reagents before combining the barcoding reagents with samples.
• the different barcoding reagents can be mixed together and then placed across a set of containers (e.g., wells in a well plate). Then, unique sample barcodes can be added to each of the containers, the result of which can be mixed with a unique sample to perform assay- barcode-identifiable assays on that sample and simultaneously tag that sample with the sample barcode.
• the sample barcodes can be bound to the barcoding reagents after combining the barcoding reagents with samples.
• the semi-configured barcoding reagents can be provided together or otherwise mixed together. Then, the barcoding reagents can be added to each of a set of samples. Separately, before or after the barcoding reagents are added, a unique sample barcode can be mixed with each of the set of samples.
• the sample barcode can tag the barcoding reagents and/or the cells or particles of the sample.
• barcoding reagents can include assay barcoded beads functionalized with poly dopamine for the attachment of capture antibodies. Another molecule (e.g., avidin) can be added alongside the capture antibodies. After capture antibodies are added to the assay barcoded beads, the beads could be mixed and split into an aliquot for each sample. Forthe sample barcode, a unique combination of element tags functionalized (e.g., with biotin) to bind the molecule can be added.
• element tags functionalized e.g., with biotin
• elemental analysis can be conducted on an individual particle basis, known as particle elemental analysis.
• Particle elemental analysis includes determining the 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 make use of particle elemental analysis on a cell-by-cell basis, which can be known as cytometric elemental analysis.
• elemental analysis can be conducted on a bulk basis, known as bulk elemental analysis or solution elemental analysis. Bulk elemental analysis includes determining the elemental composition of the entire volume of a sample.
• Elemental analysis can be used to interrogate a sample, such as a biological sample. If the sample is labelled with a known element tag, detection of the element tag during elemental analysis can be indicative of characteristics of the sample associated with the element tag.
• mass cytometry is any method of detecting element tags (mass tags) in a biological sample, such as simultaneously detecting a plurality of distinguishable mass tags with single cell resolution.
• Mass cytometry may include analysis of mass tagged beads, separate from or in addition to cells. Any of the subject kits and methods may include or be adapted to mass cytometry.
• Mass cytometry includes suspension mass cytometry and imaging mass cytometry (IMC).
• Suspension mass cytometry includes analysis of suspended element tagged cells and/or beads by mass spectrometry (e.g., by atomic mass spectrometry), and is described in US patent publications including US20050218319, US20150183895, US20150122991 , all of which are incorporated by reference herein.
• Imaging mass cytometry includes any imaging mass spectrometry (e.g., imaging atomic mass spectrometry) of element tagged biological sample, such as a tissue section or cell smear.
• IMC may atomize and ionize mass tags of a cellular sample by one or more of laser radiation, ion beam radiation, electron beam radiation, and/or inductively coupled plasma (ICP).
• Mass cytometry may simultaneously detect distinct mass tags from single cells, such as by time of flight (TOF) or magnetic sector mass spectrometry (MS).
• TOF time of flight
• MS magnetic sector mass spectrometry
• mass cytometry examples include suspension mass cytometry where cells are flowed into and ICP-MS and imaging mass cytometry where a cellular sample (e.g., tissue section) is sampled, for example by laser ablation (LA-ICP-MS) or by a primary ion beam (e.g., for SIMS).
• LA-ICP-MS laser ablation
• primary ion beam e.g., for SIMS
• Laser based IMC is described in US patent publications US20160131635, US20170148619, US20180306695, and US20180306695 all of which are incorporated by reference herein.
• the sample when the sample is a cell smear for analysis by IMC, the cells may be processed as described herein such as by staining with a lyophilized panel, sample barcoding, and/or assay barcoding.
• assay beads described herein may be analyzed separately or in mixture with cells, by IMC.
• Mass tags may be sampled, atomized and ionized prior to elemental analysis.
• mass tags in a biological sample may be sampled, atomized and/or ionized by radiation such as a laser beam, ion beam or electron beam.
• mass tags may be atomized and ionized by a plasma, such as an inductively coupled plasma (ICP).
• ICP inductively coupled plasma
• whole cells including mass tags may be flowed into an ICP- MS, such as an ICP-TOF-MS.
• a form of radiation may remove (and optionally ionize and atomize) portion (e.g., pixels, region of interest) of a solid biological sample, such as a tissue sample, including mass tags.
• a solid biological sample such as a tissue sample
• mass tags include LA-ICP-MS and SIMS-MS of mass tagged sample.
• ion optics may deplete ions other than the isotope of the mass tags. For example, ion optics may remove lighter ions (e.g., C, N, O), organic molecular ions. In ICP applications, ion optics may remove gas such as Ar and/or Xe, such as through a high-pass quadrupole filter.
• IMC may provide an image of mass tags (e.g., targets associated with mass tags) with cellular or subcellular resolution.
• Embodiment 1 A metal-encoded microbead comprising: a copolymer comprising: a structural monomer, and a metal-chelating monomer comprising a metal and a chelator; wherein the chelator coordinates the metal at least at 3 sites; and wherein the structural monomer does not comprise the chelator.
• Embodiment 2 The microbead of embodiment 1 , wherein the structural monomer is selected from 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 microbead of embodiment 1 or 2, wherein the metal-chelating monomer has a structure of Formula I prior to polymerization
• Ligand is the chelator
• L is a linker
• X is a polymerizable end group
• M is the metal
• n is 1 or an integer greater than 1 , wherein the metal-chelating monomer is neutral in charge prior to polymerization.
• Embodiment 4 The microbead of embodiment 3, wherein L is selected from a bond,
• Embodiment 5 The microbead of embodiment 3 or 4, wherein L is attached to the chelator through an amide or an ester.
• Embodiment 6 The microbead of any one of embodiments 3 to 5, wherein the polymerizable end group is selected from arylvinyl, styrene, alpha-methylstyrene, acrylate ester, methacrylate ester, acrylamide, 2-methylacrylamide, and mixtures thereof, optionally the polymerizable end group is arylvinyl or styrene.
• Embodiment 7 The microbead of any one of embodiments 1 to 6, wherein the chelator is tetradentate, pentadentate, hexadentate, heptadentate, or octadentate, optionally the chelator is hexadentate or octadentate.
• Embodiment 8 The microbead of any one of embodiments 1 to 7, wherein the chelator comprises an aminopolyacid moiety, or a derivative thereof.
• Embodiment 9 The microbead of embodiment 8, wherein the aminopolyacid moiety is selected from aminopolycarboxylic acid, aminopolyphosphonic acid, or combinations thereof.
• Embodiment 10 The microbead of embodiment 8 or 9, wherein the aminopolyacid moiety is a substituted oligomer of one or more of ethylene imine, propylene amine, or mixtures thereof, the oligomer being substituted with two or more carboxylic acids and/or phosphonic acids, optionally the oligomer is a crown ether or an aza-crown ether.
• Embodiment 11 The microbead of embodiment 10, wherein the oligomer is further substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, akylaryl, alkylheteroaryl, C3- C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN, or mixtures thereof.
• substituents selected from C1-C6 alkyl, C1-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, amide, ester, aryl, heteroaryl, akylaryl, alkylheteroaryl, C3- C8 cycloalkylaryl, C3-C8 cycloalkylheteroaryl, CN
• Embodiment 12 The microbead of any one of embodiments 1 to 11, wherein the chelator is selected from DFO, EDTA, DTPA, EGTA, EDDS, EDDHA, BAPTA, H4neunpa, H6phospa, H4CHXoctapa, H4octapa, H2CHXdedpa, H5decapa, Cy-DTPA, Ph-DTPA, a TACN-type chelator, a TACD-type chelator, a cyclen-type chelator, a cyclam-type chelator, a (13)aneN4-type chelator, a 1 ,7-diaza-12-crown-4-type chelator, a 1 ,10-diaza-18-crown-6-type chelator, or derivatives thereof.
• the chelator is selected from DFO, EDTA, DTPA, EGTA, EDDS, EDDHA, BAPTA, H4neun
• Embodiment 13 The microbead of embodiment 12, wherein the TACN-type chelator is selected from NOTA, NOPO, TRAP, or derivatives thereof.
• Embodiment 14 The microbead of embodiment 12, wherein the cyclen-type chelator is DOTA or derivatives thereof.
• Embodiment 15 The microbead of embodiment 12, wherein the cyclam-type chelator is selected from TETA, cross bridged-TETA, DiAmSar, or derivatives thereof.
• Embodiment 16 The microbead of embodiment 12, wherein the (13)aneN4-type chelator is selected from TRITA or derivatives thereof.
• Embodiment 17 The microbead of embodiment 12, wherein the 1 ,10-diaza-18- crown-6-type chelator is selected from MACROPA, or derivatives thereof.
• Embodiment 18 The microbead of embodiment 12, wherein the chelator is selected from DTPA, Cy-DTPA, Ph-DTPA, or derivatives thereof.
• Embodiment 19 The microbead of embodiment 18, wherein the derivative of DTPA comprises DTPA where two adjacent carbon atoms are joined together with atoms therebetween to form a 5- membered or 6-membered ring, optionally a cycloalkyl ring, an aryl or a heteroaryl ring.
• Embodiment 20 The microbead of embodiment 18, wherein prior to polymerization, the metal-chelating monomer is wherein L and X are as defined in any one of embodiments 4 to 6.
• Embodiment 21 The microbead of embodiment 20, wherein the metal-chelating monomer is selected from
• Embodiment 22 The microbead of any one of embodiments 1 to 9, wherein the chelator comprises porphyrin or phthalocyanine.
• Embodiment 23 The microbead of embodiment 22, wherein the chelator is substituted or unsubstituted porphyrin.
• Embodiment 24 The microbead of embodiment 22 or 23, wherein the metalchelating monomer prior to polymerization is selected from integer from 1 to 4.
• Embodiment 25 The microbead of embodiment 24, wherein L is aniline.
• Embodiment 26 The microbead of embodiment 24 or 25, wherein n is at least 2.
• Embodiment 27 The microbead of embodiment 3, wherein the metal-chelating monomer is selected from
• Embodiment 28 The microbead of any one of embodiments 1 to 27 further comprising a steric stabilizer, optionally the steric stabilizer is selected from PVP, polyvinyl alcohol, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, polyacrylic acid, water soluble homopolymers of acrylic acid esters, water soluble homopolymers of methacrylic acid esters, water soluble homopolymers of acrylamide, homopolymers of methacrylamide, water soluble copolymer steric stabilizers, or mixtures thereof.
• PVP polyvinyl alcohol
• hydroxymethylcellulose hydroxyethylcellulose
• hydroxypropylcellulose polyacrylic acid
• water soluble homopolymers of acrylic acid esters water soluble homopolymers of methacrylic acid esters
• water soluble homopolymers of acrylamide homopolymers of methacrylamide
• water soluble copolymer steric stabilizers or mixtures thereof.
• Embodiment 29 The microbead of embodiment 28, wherein the water soluble copolymer steric stabilizer is selected from copolymers of acrylic acid ester, methacrylic acid ester, acrylamides or methacrylamide with methyl acrylate and/or ethyl acrylate, or mixtures thereof.
• Embodiment 30 The microbead of any one of embodiments 1 to 29, wherein the copolymer is crosslinked.
• Embodiment 31 The microbead of any one of embodiments 1 to 30, wherein the metal is a plurality of metals.
• Embodiment 32 The microbead of embodiment 31 , wherein the plurality of metals comprises one or more enriched isotopes.
• Embodiment 33 The microbead of embodiment 31 or 32, wherein the plurality of metals comprises at least 2 metals, at least 3 metals, or at least 4 metals.
• Embodiment 34 The microbead of any one of embodiments 31 to 33, wherein the amount of each metal of the plurality of metals is within about 20% or about 10% of the amount of another metal of the plurality of the metals.
• Embodiment 35 The microbead of any one of embodiments 1 to 34, wherein the metal comprises indium, bismuth, or a rare earth metal, optionally the rare earth metal is selected from lanthanide metal, yttrium, or mixtures thereof.
• Embodiment 36 The microbead of embodiment 35, wherein the metal comprises a rare earth metal that is selected from Y, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, distinct isotopes thereof, and mixtures thereof.
• the metal comprises a rare earth metal that is selected from Y, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, distinct isotopes thereof, and mixtures thereof.
• Embodiment 37 The microbead of embodiment 36, wherein the rare earth metal is selected from 89Y, 139La, 136Ce, 138Ce, 140Ce, 142Ce, 141 Pr, 142Nd, 143Nd, 145Nd, 146Nd, 148Nd, 145Pm, 144Sm, 149Sm, 150Sm, 152Sm, 154Sm, 151Eu, 153Eu, 154Gd, 155Gd, 156Gd, 157Gd, 158Gd, 160Gd, 152Gd, 159Tb, 156Dy, 158Dy, 160Dy, 161 Dy, 162Dy, 163Dy, 164Dy, 165Ho, 162Er, 164Er, 166Er, 167Er, 168Er, 170Er, 169Tm, 168Yb, 171Yb, 172Yb, 173Yb, 174
• Embodiment 38 The microbead of any one of embodiments 1 to 37, wherein the metal is distributed throughout the microbead.
• Embodiment 39 The microbead of any one of embodiments 1 to 38, wherein the microbead has a glass transition temperature of about 60°C or above 60°C, optionally about 70°C or above 70°C, about 80°C or above 80°C, about 90°C or above 90°C, about 100°C or above 100°C, about 115°C or above 115 °C, about 125°C or above 125°C, or about 135°C or above 135°C.
• Embodiment 40 The microbead of any one of embodiments 1 to 39, wherein the microbead has a diameter of about 0.6 pm to about 20 pm, about 1 pm to about 15 pm, about 2 pm to about 10 pm, about 2 pm to about 6 pm.
• Embodiment 41 The microbead of any one of embodiments 1 to 40, wherein the microbead is colloidally stable in water.
• Embodiment 42 The microbead of any one of embodiments 1 to 41 , wherein a surface of the microbead comprises functionalization for attachment to a biomolecule.
• Embodiment 43 The microbead of embodiment 42, wherein the attachment is covalent attachment.
• Embodiment 44 The microbead of embodiment 42 to 43, wherein the biomolecule is selected from a protein, an oligonucleotide, a small molecule, a lipid, a carbohydrate, or a mixture thereof.
• Embodiment 45 The microbead of embodiment 42 to 43, wherein the biomolecule is an affinity reagent, optionally wherein the affinity reagent is an antibody.
• Embodiment 46 The microbead of embodiment 45, wherein the 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 factors.
• cytokine optionally a chemokine, an interferon, a lymphokine, a monokine, an interleukin, such as IL-1-36, tumor necrosis factor and colony stimulating factors.
• Embodiment 47 The microbead of embodiment 44, wherein the antigen is a viral antigen.
• Embodiment 48 The microbead of any one of embodiments 42 to 47, wherein the functionalization comprises a coating of silicon dioxide on the surface of the microbead, optionally the functionalization further comprises functionalizing the coating of silicon dioxide.
• Embodiment 49 The microbead of embodiment 42, wherein the attachment to the biomolecule is non-covalent attachment.
• Embodiment 50 The microbead of any one of embodiments 42 to 49, wherein the surface of the microbead is functionalized with avidin, streptavidin, neutravidin, or mixtures thereof.
• Embodiment 51 The microbead of any one of embodiments 42 to 50, wherein the surface of the microbead is conjugated to the biomolecule.
• Embodiment 52 The microbead of any one of embodiments 42 to 51, wherein the metal provides a barcode that identifies the biomolecule.
• Embodiment 53 A population of microbeads as defined in any one of embodiments 1 to 52.
• Embodiment 54 The population of microbeads of embodiment 53, wherein the population has a size distribution having a coefficient of variation (CV) of about 10% or less than 10%.
• CV coefficient of variation
• Embodiment 55 The population of microbeads of embodiment 54, wherein the coefficient of variation is of less than 5%.
• Embodiment 56 The population of microbeads of any one of embodiments 53 to 55, wherein each microbead comprises a plurality of metals, the average amount across the population of microbeads of each metal of the plurality of metals is about 10% or within 10% of the average amount of another metal of the plurality of metals.
• Embodiment 57 The population of microbeads of embodiment 56, wherein the plurality of metals comprises one or more enriched isotopes.
• Embodiment 58 The population of microbeads of any one of embodiments 53 to 57, wherein the amount of each metal of one microbead of the population of microbeads is about 20% or within 20%, or about 10% or within 10%, or about 5% or within 5% of the amount of the same metal of another microbead of the population of microbeads.
• Embodiment 59 The population of microbeads of any one of embodiments 53 to 57, wherein the amount of each metal of the population of microbeads has a distribution of 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 of the population of microbeads has a distribution of a coefficient of variation of about 10% or less than 10%.
• Embodiment 61 The population of microbeads of any one of embodiments 58 to 60, wherein the microbeads of the population of microbeads comprise the same metal in substantially the same amount.
• Embodiment 62 The population of microbeads of embodiment 61 , wherein the same metal is a plurality of metals and the microbeads comprise each metal of the plurality of metals in substantially the same amount.
• Embodiment 63 A kit comprising a plurality of distinct populations of microbeads as defined in any one of embodiments 53 to 62.
• Embodiment 64 The kit of embodiment 63, wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or the plurality of metals of the microbeads.
• Embodiment 65 The kit of embodiment 63 or 64, wherein the microbeads of at least one population of microbeads comprise a metal or a plurality of metals different from the metal or the plurality of metals of the microbeads of another population of microbeads.
• Embodiment 66 The kit of embodiment 63 or 64, wherein the microbeads of at least one population of microbeads comprise a plurality of metals at a ratio different from the microbeads of another population of microbeads.
• Embodiment 67 The kit of any one of embodiments 64 to 66, wherein the microbeads of each population of microbeads are conjugated to a different biomolecule.
• Embodiment 68 Method of preparing 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 chelator attached to at least one polymerizable end group to obtain a second mixture, wherein the chelator coordinates the metal at least at 3 sites and wherein the metalchelating monomer is polymerizable with the structural monomer; and polymerizing the second mixture to form a copolymer of the microbead; wherein the structural monomer does not comprise the chelator.
• Embodiment 69 The method of embodiment 68, wherein the metal is a plurality of metals.
• Embodiment 70 The method of embodiment 68 or 69, wherein the structural monomer is polymerized in the nucleation stage to about 5% to about 20% completion based on the structural monomer.
• Embodiment 71 The method of any one of embodiments 68 to 70, wherein the polymerizing 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 monomer.
• 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 metalchelating monomer is as defined in any one of embodiments 2, and 4 to 26.
• Embodiment 74 The method of any one of embodiments 68 to 73, wherein the steric stabilizer is as defined in embodiment 27.
• Embodiment 75 The method of any one of embodiments 68 to 74, wherein the metal is as defined in any one of embodiments 29 to 35
• Embodiment 76 The method of any one of embodiments 68 to 75, the method further comprising functionalizing the microbead.
• Embodiment 77 The method of embodiment 76, wherein the functionalizing 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; and polymerizing the third mixture.
• Embodiment 78 The method of embodiment 77, wherein the reactive functional group is selected from alcohol, aldehyde, carboxylic acid, epoxide, vinyl, alkyne, maleimide, or mixtures thereof.
• Embodiment 79 The method of embodiment 76, wherein the functionalizing of the microbead comprises coating the microbead with silicon dioxide.
• Embodiment 80 The method of embodiment 76, wherein the functionalizing of the microbead further comprises functionalizing the coating of silicon dioxide.
• Embodiment 81 The method of any one of embodiments 68 to 80, the method further comprising conjugating the microbead 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 microbead has a diameter of about 0.6 pm to about 20 pm, about 1 pm to about 15 pm, about 2 pm to about 10 pm, about 2 pm to about 6 pm.
• Embodiment 84 The method of any one of embodiments 68 to 83 wherein the microbead is as defined in any one of embodiments 1 to 52.
• Embodiment 85 A microbead prepared by the method of any one of embodiments 68 to 83.
• Embodiment 86 The microbead of any one of embodiments 1 to 52, wherein an interior structure of the microbead comprises the copolymer.
• Embodiment 87 The microbead of any one of embodiments 1 to 52, and 86, wherein the metal-chelating monomer chelates a single metal atom and not a plurality of metal atoms.
• Embodiment 88 The microbead of any one of embodiments 1 to 52, 86 and 87, wherein the microbead comprises a polymer seed that does not comprise the metal-chelating monomer.
• Embodiment 89 The microbead of embodiment 88, wherein the polymer seed comprises a structural monomer; optionally wherein the structural monomer of the polymer seed is identical in structure to the structural monomer of the copolymer.
• Embodiment 90 The method of embodiment 68, wherein an interior structure of the microbead comprises the copolymer.
• Embodiment 91 A method of preparing metal-encoded microbeads, the method comprising: providing an aqueous dispersion of swellable seed particles and an anionic surfactant; contacting the aqueous dispersion with monomers comprising a structural monomer and a metal-chelating monomer, wherein the metal-chelating monomer comprises a metal and a chelator attached to at least one polymerizable end, wherein the chelator coordinates the metal at least at 3 sites, and wherein the structural monomer does not comprise the chelator; allowing the monomers to diffuse into the seed particles to form an aqueous dispersion of swollen seed particles; and initiating polymerization of the monomers in the aqueous dispersion of swollen seed particles.
• Embodiment 92 The method of embodiment 91 , wherein the structural monomer is selected from the group consisting of acrylic monomers, methacrylate monomers and vinyl monomers selected from the group consisting of styrene, divinylbenzene (DVB), ethyl vinyl benzene, vinyl pyridine, amino-styrene, methyl-styrene, dimethylstyrene, ethyl styrene, 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 the providing the 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 with a molecular weight below 5000 Dalton and a water solubility at 25° C. of less than 10— 2g/L; and optionally an organic solvent in which said organic compound is soluble.
• Embodiment 97 The method of any one of embodiments 91 to 96, wherein the swellable seed particles are monodisperse swellable seed oligomer particles.
• Embodiment 98 The method of any one of embodiments 91 to 97, wherein the anionic surfactant is sodium dodecyl sulfate.
• Embodiment 99 The method of any one of embodiments 91 to 98, wherein the structural monomer is as defined in embodiment 2.
• Embodiment 100 The method of any one of embodiments 91 to 99, wherein the metalchelating monomer is as defined in any one of embodiments 1 , and 3 to 27.
• Embodiment 101 The kit of embodiment 67, further comprising a reporter comprising a mass tag, wherein the reporter specifically binds a sample biomolecule 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 the 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 tagged oligonucleotides to the sample biomolecule.
• Embodiment 104 The kit of any one of embodiments 101 to 103, wherein the 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 the 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 the 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 the 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.
• Embodiment 108 The kit of any one of embodiments 101 to 107, wherein the 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 secondary antibody that binds to 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 specifically bound by a different biomolecule.
• Embodiment 110 The kit of embodiment 109, wherein the plurality of different reporters each comprise the same mass tag.
• Embodiment 111 The kit of any one of embodiments 101 to 110, wherein the mass tag comprises a metal nanoparticle.
• Embodiment 112. The kit of any one of embodiments 101 to 110, wherein the mass tag comprises a metal chelating polymer.
• Embodiment 113 The kit of embodiment 67, wherein at least one of the biomolecules is an enzyme substrate for a sample biomolecule.
• Embodiment 114 The kit of embodiment 113, further comprising a reporter that specifically binds to the enzyme substrate when it 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 it 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 to 116, wherein the microbeads from a plurality of different populations are in a first mixture of microbeads.
• Embodiment 118 The kit of embodiment 117, further comprising a second mixture of microbeads comprising the same biomolecules as the first mixture of microbeads, wherein the microbeads of the first mixture comprise a sample barcode that is different from the microbeads of the second mixture.
• Embodiment 119 The kit of embodiment 118, wherein the sample barcode is on the interior of the microbeads.
• Embodiment 120 The kit of embodiment 119, wherein the sample barcode is a subset of metals chelated by the metal chelating monomer of the microbeads of the first and 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 mixture.
• Embodiment 122 The kit of embodiment 118, further comprising a plurality of sample barcodes in separate partitions that are functionalized to bind to the surface 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 panel of mass-tagged antibodies in admixture with one another, wherein at least some antibodies of the panel are specific for cell surface markers.
• Embodiment 124 The kit of embodiment 123, wherein the plurality of antibodies is in admixture with microbeads of the kit.
• Embodiment 125 The kit of embodiment 123 or 124, wherein at least some of the mass-tagged antibodies comprise metals identical to metals chelated by metal chelating monomers of microbeads of the kit.
• Embodiment 126 The kit of any one of embodiments 101 to 125, further comprising a steric stabilizer in admixture 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 solution buffered at a pH at 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 to 132, further comprising one or more of a buffer, anticoagulant, fixation reagents, and permeabilization reagents.
• Embodiment 134 A method comprising detecting the population of microbeads of any one of embodiments 53 to 62 by mass spectrometry.
• Embodiment 135. The method of embodiment 134, further comprising calibrating a mass spectrometer used to detect the microbeads based on a mass spectrum obtained from the microbeads.
• Embodiment 136 The method of embodiment 134, further comprising normalizing a mass spectrometry signal obtained from a plurality of mass tags based on a mass spectrum obtained from the microbeads.
• Embodiment 137 A method of mass spectrometry analysis, comprising: mixing the distinct populations of microbeads of any one of embodiment 53 to 62 with a sample, wherein microbeads of each population of microbeads are bound to a different biomolecule and wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or the plurality of metals of the microbeads; binding different sample biomolecules of the sample to the different biomolecules of distinct populations of microbeads; binding, directly or indirectly, a reporter to each of the different sample biomolecules, wherein the reporter bound to each of the different sample biomolecules comprises a mass tag; and detecting the metal and the mass tag of individual microbeads by mass spectrometry.
• Embodiment 138 The method of embodiment 137, further comprising attaching the different biomolecules to the microbeads of the distinct populations of microbeads prior to the step of mixing the distinct populations of microbeads with the sample.
• Embodiment 139 The method of embodiment 137 or 138, wherein the different sample biomolecules comprise oligonucleotides.
• Embodiment 140 The method of any one of embodiments 137 to 139, wherein the different sample biomolecules comprise antibodies.
• Embodiment 141 The method of any one of embodiments 137 to 140, wherein the different sample biomolecules comprise cytokines.
• Embodiment 142 The method of any one of embodiments 137 to 141 , wherein the different sample biomolecules comprise cancer biomarkers.
• Embodiment 143 A method of mass spectrometry analysis using the kit of any one of embodiments 101 to 133 comprising: mixing distinct populations of microbeads with a sample, wherein microbeads of each population of microbeads are bound to a different biomolecule and wherein each population of microbeads is distinguishable from another population of microbeads based on the metal or the plurality of metals of the microbeads; binding different sample biomolecules of the sample to the different biomolecules of distinct populations of microbeads; binding, directly or indirectly, a reporter to each of the different sample biomolecules, wherein the reporter bound to each of the different sample biomolecules comprises a mass tag; and detecting the metal and the mass tag of individual microbeads by mass spectrometry.
• Embodiment 144 A method of mass spectrometry analysis of a cellular sample using the microbeads of any one of embodiments 63 to 74 comprising: providing a plurality of mass-tagged antibodies, wherein each antibody of the mass tagged antibody is conjugated to a polymer mass tag chelating multiple atoms of a metal or enriched isotope thereof; contacting a sample with the plurality of mass-tagged antibodies; detecting the mass-tagged 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 step of detecting, wherein the calibrating is based a mass spectrometry signal obtained from the microbeads.
• Embodiment 146 The method of embodiment 144, further comprising normalizing a mass spectrometry signal obtained from the mass tags based on a mass spectrometry signal obtained from the microbeads.
• Embodiment 147 The method of embodiment 144, further comprising quantifying the mass-tagged antibodies based on the average number of metal atoms for each of the mass- tagged antibodies and the detected mass spectrometry signal from the mass-tagged antibodies and the microbeads.
• Embodiment 148 The method of embodiment 146 or 147, wherein the microbeads and the mass-tagged antibodies comprise the same metals.
• Embodiment 149 The method of any one of embodiments 134 to 148, wherein the step of detecting comprises imaging mass cytometry.
• Embodiment 150 The method of embodiment 149, further comprising quantifying or normalizing the mass-tagged antibodies at individual pixels or bound to individual cells 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 step of detecting.
• Embodiment 152 The method of any one of embodiments 134 to 151 , wherein the step of detecting comprises suspension mass cytometry.
• Embodiment 153 The method of any one of embodiments 134 to 152, wherein the step of detecting comprises inductively coupled plasma mass spectrometry (ICP-MS).
• ICP-MS inductively coupled plasma mass spectrometry
• Embodiment 154 The method of any one of embodiments 149 to 151 , wherein the step of detecting comprises laser ablation ICP-MS or secondary ion mass spectrometry (SIMS).
• ICP-MS laser ablation ICP-MS or secondary ion mass spectrometry (SIMS).
• Embodiment 155 The method of any one of embodiments 134 to 154, wherein the mass spectrometry is time-of-flight mass spectrometry (TOF-MS).
• TOF-MS time-of-flight mass spectrometry
• PS Polystyrene (PS) microbeads with one or more Lanthanides chelated by DTPA
• Metal-encoded PS microbeads were synthesized by introducing polymerizable metal- DTPA complexes into dispersion polymerization reactions of styrene in ethanol as second stage aliquots. Synthesis of the components and microbeads and the materials used are described.
• DTPA dianhydride Diethylenetriaminepentaacetic dianhydride (DTPA dianhydride, 98%), 2,2’-azobis(2- methylpropionitrile) (AIBN, 98%), polyvinylpyrrolidone (PVP, Mw ⁇ 55 kDa), Triton-X305 (TX305, 70% solution in water), 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 (H2O2, 30% in H2O), and metal salts with the purity > 99.99% (trace metals basis), including yttrium(lll) chloride hexahydrate (YCI 3 -6H 2 0), cerium(lll) chloride
• VBA 4-Vinylbenzylamine
• EtOH Absolute ethanol
• Nitric acid tracemetal grade, 68-69%
• sulfuric acid tracemetal grade
• sodium hydroxide sodium hydroxide
• phosphate buffered saline (1x PBS solution, Fisher BioReagents) were purchased from Fisher Scientific. All of the above chemicals were used without further purification.
• Styrene St, Sigma-Aldrich, >99%
• R-NH 2 VBA, BA, ALA or APMAm
• the synthesis method was developed from the protocols reported by Zhang et al. 16 for DTPA-bis(vinyl benzylamide) (DTPA-VBAm 2 ), Aime et al. 17 for DTPA- bis(benzylamide) (DTPA-BAm 2 ), and Shuhendler et al. 18 for DTPA-bis(allylamide) (DTPA- ALAm 2 ) with some modifications.
• Two-stage dispersion polymerization (2-stage DisP) was used to prepare metal-encoded polystyrene (PS) microbeads in presence of polyvinylpyrrolidone (PVP) as a steric stabilizer and DTPA derivative metal complexes as metal ligands.
• PVP polyvinylpyrrolidone
• DTPA derivative metal complexes metal ligands.
• a warm solution of the desired amount of DTPA metal complexes in ethanol was introduced as the second stage aliquots at 2 h.
• the reaction was terminated 24 h after the initiation, with the styrene conversion above 90%.
• Table 1 describes a typical recipe of this 2-stage DisP used to prepare microbeads.
• Table 1 A typical recipe for the two-stage dispersion polymerization of styrene for bead synthesis
• DTPA derivative-metal complexes -- c a The reaction was initiated by immersing the flask into a 70°C oil bath. Prior to the initiation, reaction solution was purged with nitrogen gas for 30 min. b. Second stage aliquot was introduced to the reaction 2 h after the initiation. c. A desired amount of DTPA derivative-metal complexes (M(DTPA-R 2 )) dissolved in ethanol was introduced to the aliquot. Details of the metal addition are described in Table 2.
• Example 1 Twelve batches of bead synthesis were prepared using the methods and materials of Example 1 and various amounts of the M(DTPA-R 2 ) complex as the feed in the second stage. 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 complexes (M(DTPA-R 2 )) feed in the second stage aliquots of a series of 2-stage DisP.
• VBAm stands for vinylbenzylamide
• BAm stands for benzylamide
• ALAm stands for allyamide
• AmPMAm stands for amidopropyl methacrylamide
• Smaller numbers of Ho and Lu complexes were added to these syntheses to avoid the saturation of MC detector by resulting microbeads.
• the numbers of metal complex addition were designed to achieve microbeads generating similar levels of intensities among all five isotopes.
• microbead dispersions were washed by sedimentation- redispersion cycles twice with absolute ethanol and four times with water to remove free stabilizers, unreacted monomers, and any smaller diameter particles. Dispersions of these washed microbeads were used for MC characterization, and aliquots were freeze dried to measure the solids content.
• Example 3
• Microbeads were coated with a silica shell and conjugated to a secondary antibody.
• TEOS Tetraethyl orthosilicate
• APTES (3-Aminopropyl)triethoxysilane
• DMSO anhydrous dimethyl sulfoxide
• Ammonia solution 25% (NH OH), MES buffer (0.5 M pH 5.5), phosphate buffered saline (1x PBS, pH 7.4), /V-hydroxysuccinimide (NHS), A/-(3-Dimethylaminopropyl)- /V-ethylcarbodiimide hydrochloride (EDC), NeutraAvidin (NAv), biotin-xx-goat anti-mouse (H+L IgG) and bovine serum albumin (BSA) were ordered from ThermoFisher Scientific. MaxPar® 175 Lu-labeled mouse anti-TNFa (human) (clone MAb11), cell staining buffer and cell acquisition solution were kindly provided by Fluidigm Canada.
• reaction solution was quickly switched to 1x PBS buffer (100 mI_; pH 7.4) by 2 cycles of centrifugal washings and followed by the addition of NAv solution (50 mI_, 2 mg/ml_).
• NAv solution 50 mI_, 2 mg/ml_.
• the NAv conjugation reaction was incubated for 4 hours followed by removing the unattached NAv by 4 cycles of centrifugal washing with PBS buffer (100 mI_).
• GAM Goat anti-mouse secondary antibody was attached to Eu-1 microbeads by incubating NAv-modified Eu-1 beads (ca. 2 million beads) in a BSA-PBS solution (100 mI_, 0.5% wt. BSA in PBS) containing biotin-xx-GAM (20 pg) for 2 hours. The excess GAM were then removed by four cycles of centrifugal washing with BSA-PBS solution (100 mI_).
• Microbead dispersions were digested with sulfuric acid and H2O2 prior to ICP-MS measurements.
• sulfuric acid 500 pl_
• microbead dispersion 100 mI_, 0.5-2% solids content
• the microbead dispersion in sulfuric acid was then heated to 250°C on a hotplate and held for 40 min with magnetic stirring, followed with an addition of 30% H 2 0 2 solution (50 mI_).
• the digestion solution was subsequently diluted with 2% HN0 3 .
• ICP-MS Inductively coupled plasma-mass spectrometry
• ICP-MS iCAP-Q, Thermo Scientific
• KED kinetic energy discrimination
• the elemental standard solutions were sequentially diluted with 2% HNO3 to a series of concentrations of 40, 20, 10, 1, and 0.1 ppb as the calibration solutions. Based on the calibration fitting curve, the metal content in each solution sample was determined. The detection limits for elements of interests were estimated to be below 10 ppt.
• Mass cytometry The metal content of microbead was characterized bead-by-bead by a mass cytometry system (Helios® CyTOF, Fluidigm).
• the mixture consisting of 7E1 reference beads and microbead samples was then introduced to the MC system at a speed of 30 pL/min, and the beads were individually but stochastically introduced into the ICP.
• singlet events were identified and gated on the dot-plot generated by MC.
• the means, medians, robust standard deviations (RSD) and robust coefficient of variations ( RCV) of singlet signals were reported as unnormalized raw data.
• Robust statistics provide an alternative approach to classical statistical estimators such as mean, SD, and CV.
• DTPA was modified with functional groups that can react with styrene during the DisP.
• two of five carboxylates on DTPA were substituted with functional groups so the remaining three carboxylates in the functionalized DTPA derivatives could form charge-neutral complexes with lanthanide(lll) ions, which promotes the binding stability and the ethanol solubility of their metal complexes in DisP.
• Fig. 1 presents the 1 H-NMR spectrum of Na 3 (DTPA-VBAm 2 ), showing the distinct chemical shifts of protons in the vinyl groups at 5.3 (b, 2H), 5.8 (a, 2H), and 6.8 (c, 2H).
• the chemical shift at 4.4 ppm (f, 4H) indicates the formation of the amide linkage.
• the integration of protons from the vinyl, benzyl, and DTPA moieties confirms the bis-substitution of vinyl benzyl groups in each DTPA molecule.
• the 1 H-NMR spectra shown in Fig. 8A and B match the spectra of DTPA-BAm 2 and DTPA-ALAm 2 , respectively, reported in the literature 18 1930 and verify the successful synthesis of these two DTPA-bis(amide) derivatives.
• Ce complexes of Ce(DTPA-VBAm 2 ), Ce(DTPA- BAm 2 ), and Ce(DTPA-ALAm 2 ) were prepared.
• Metal complexes were synthesized by adding an aqueous solution of CeCI 3 to DTPA-R 2 in water at pH 5.0 ⁇ 6.0 in 1:1 molar ratio.
• the Ce(DTPA-R 2 ) complexes were precipitated by addition of acetone, and characterized by 1 H- NMR to verify the metal chelation.
• the 1 H-NMR spectra of these Ce complexes are shown in Fig. 9 A to C.
• Metal ion incorporation in the reaction was determined by ICP-MS.
• the total metal ion content in the reaction was determined after digestion of the sample with H 2 SC> 4 + H 2 0 2 .
• the microbeads in the reaction stock were separated by centrifugal sedimentation.
• the metal content in supernatant was also analyzed by ICP-MS to quantify the metal ions remaining in solution. As described in Liu et al, 2020 15 , the metal incorporation efficiency of bead synthesis was calculated based on the difference between unincorporated metal content in the supernatant and the total metal content in the reaction.
• This metal incorporation efficiency reflects the Ce complex incorporation into PS beads, since Ce is fed to the reactions as Ce- DTPA complexes and Ce ions in all three Ce-complexes are expected to remain chelated with DTPA derivatives during the bead synthesis due to their relatively high binding stability.
• Ce-1 , Ce-2 and Ce-3 microbeads were examined by MC on a bead-by-bead basis, using calibration microbeads containing a known amount of 140 Ce. as a quantification standard.
• the Ce content in sample beads was evaluated by comparing the signal intensities between the sample beads and the calibration beads, assuming that the MC signal intensities from different microbeads were proportional to the metal content in the same measurement.
• Table 3 summarizes the Ce content of these three batches of microbeads as determined by MC.
• Ce-1 microbeads generated a sharp and strong 140 Ce signal (see Fig. 11) with a median intensity at 6000 counts per bead and an RCV of 6.2% (see Fig. 11A).
• the Ce content in Ce-1 microbeads was estimated to be 5.44x10 7 Ce ions per bead (see Table 3).
• Ce-2 and Ce-3 microbeads showed much weaker 140 Ce signal intensities (Fig. 11 B and C respectively).
• the Ce content for the Ce-2 and Ce-3 microbead samples was essentially identical at 1.6x10 ® and 1.6x10 ® Ce ions/bead.
• Ce(DTPA-AmPMAm 2 ) was prepared, employing methacrylamide as the functional group that can copolymerize with styrene.
• the Ce(DTPA-AmPMAm 2 ) complex was synthesized by first reacting DTPA dianhydride with APMAm in DMSO and then chelating Ce 3+ ions in water at pH 5 ⁇ 6.
• the 1 H-NMR spectra in Figure 8 (c) and Figure 9 (d) confirm the structure of Na 3 (DTPA-AmPMAm 2 ) and Ce(DTPA-AmPMAm 2 ), respectively.
• Ce(DTPA-AmPMAm 2 ) seemed more hydrophilic than Ce(DTPA-VBAm 2 ), because Ce(DTPA-AmPMAm 2 ) was fully soluble in the chelation solution without addition of ethanol.
• Table 3 The characteristics of four batches of Ce-encoded microbeads prepared by 2-stage DisP. of styrene with Ce complexes of different DTPA derivatives (Ce(DTPA-VBAm 2 ), Ce(DTPA-BAm 2 ), Ce(DTPA-ALAm 2 ) and Ce(DTPA- AmPMAm 2 )) as the metal chelators.
• Ce-4 DTPA-AmPMAm 2 2.9 1.3 35.7 ( ⁇ 3.5) 51 a.
• the mean diameter (d) and Coefficient of Variation (CV) of the microbeads were characterized by SEM images of the microbead sample after sedimentation-redispersion washing;
• the metal content per microbead were measured by MC using calibration beads as a standard;
• the mean and standard deviation of microbeads’ metal content were evaluated based on the median and robust standard deviation of their MC signal intensities;
• each of these different M(DTPA-VBAm2) complexes were used to prepare 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, as described in Table 2.
• the microbeads obtained from Y-1, Eu-1 , Ho-1 and Lu-1 syntheses were uniform and similar in size (see Table 4).
• the MC signal intensities of the encoded elements from these microbeads were strong and narrowly distributed, as presented in Fig. 12.
• the signal intensities of 140 Ce, 151 ⁇ 153 Eu, 165 Ho and 175 Lu were in the optimal MC sensitivity range (> 300 counts per bead) with narrow signal distributions ( RCV ⁇ 10%).
• the signal intensity of 89 Y from 5E1 beads was less than 100 counts per beads, due to the low transmission coefficient of the 89 amu channel in MC. 14
• the metal content per 5E1 bead was calculated. The results are presented in Fig. 4B.
• the content of Ce, Eu, Ho and Lu in 5E1 beads were in a similar range, 3.4 ⁇ 4.0x10 6 ions per bead.
• the Y content is slightly higher (5.7x10 ® ions/bead) as a consequence of there being more Y atoms in 5 mg Y(DTPA-VBAm2) than in 5 mg of the other Ln(DTPA-VBAm 2 ) samples.
• Fig. 3A summaries the metal incorporation efficiency results in the batches of bead syntheses (Y-1 , Ce-1 , Eu-1 , Ho-1 , Lu-1 and 5E1) described in Table 2 using M(DTPA-VBAm 2 ) metal complexes as the ligands.
• the x-axis in Fig. 3 represents the ionic radius of the incorporated metal ions.
• the incorporation efficiency of different metals in these batches of beads overlapped in a very close range of 62 ⁇ 74%, even though the metal feed in these bead syntheses varied. It appears that the reactivity of the 4-vinylbenzylamide group is independent of the metal ion bound to the chelator. As a result, the metal incorporation efficiencies in the bead syntheses were consistent when M(DTPA-VBAm2) metal complexes were employed as the ligands.
• Y(DTPA-VBAm 2 ) (70 pmol) takes longer to dissolve in ethanol (15 g) possibly due to its high polarity. Accordingly, ions other than Y ions were used in these reactions.
• Y can be a suitable metal for the microbeads of the present disclosure.
• Y can be used with a different metal-chelating monomer.
• Y can be used at a different concentration.
• Three samples of four-element PS microbeads containing Ce, Eu, Ho, and Lu were synthesized at concentration levels that differed by a factor of three. These samples are denoted as 4E1 , 4E2 and 4E3 as shown in Table 2.
• the aim was to obtain microbeads that produce MC signal intensity levels of 140 Ce, 151 Eu, 153 Eu, 165 Ho and 175 Lu that are at 0.2, 0.6 and 1.8 times, respectively, to the MC intensity levels of 7-element-encoded microbeads.
• microbeads obtained from the syntheses of 4E1 , 4E2 and 4E3 were colloidally stable and free of coagulum in the reaction stock.
• the average diameters of these microbeads after washing by sedimentation-redispersion were in the range of 2.8 ⁇ 3.0 pm with narrow size distributions ( CV ⁇ 1.5%), as shown in Table 4.
• Fig. 3B the incorporation efficiencies of different metal ions in these three bead syntheses were consistent and close to the levels observed in the syntheses 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 the panels sorted by element.
• the average MC signal intensities of 140 Ce, 151 Eu, 153 Eu, 165 Ho and 175 Lu from 4E3 beads were the highest among these three batches of beads and at similar levels, that were 2560, 2053, 2590, 2540 and 2530 counts per bead, respectively.
• the signal intensity distributions of 4E3 beads were narrow with the RCV values less than 9%, indicating the narrow distributions of encoded lanthanide elements in the beads.
• 4E1 and 4E2 beads also generated sharp and narrowly distributed MC signals with intensities at around 200 and 700 counts per beads, respectively, for all the encoded isotopes.
• the histograms in Fig. 6 show clear baseline resolution among these three batches of microbeads.
• the signal intensities of these three batches of beads increased ca. three times from 4E1 to 4E3 beads.
• the metal content in these three microbeads was evaluated. The values were plotted against the metal feed in the bead synthesis as open symbols in Fig. 5. These open symbol data points followed the linear trend line observed from previous bead syntheses presented in Fig. 5.
• metal complexes of the structure M(DTPA-VBAm 2 ) copolymerize effectively with styrene in two-stage dispersion polymerization reactions in ethanol in the presence of polyvinylpyrollidone.
• This reaction leads to PS microbeads with diameters on the order of 2 pm and very narrow size distributions (CV 1 - 2%).
• Metal complex incorporation efficiency is on the order of 60 to 70%, slightly increasing in this range for the lanthanide metal ions with the larger ionic radius. This efficiency appears to be independent of the amount of metal complex introduced into the reaction for the range of concentrations examined. This feature of the reaction allows one to dial in a particular metal content for a sample of PS microbeads.
• microbeads prepared according to Examples 1 and 2 were assessed under several experimental conditions.
• the sample 4E3 beads prepared by 2-stage DisP with M(DTPA-VBAm 2 ) was used for this test.
• Samples of washed microbeads (0.5% solids) were suspended in 30 mL of three different buffers that also contained 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°C. At various time intervals, each sample was first vortexed and then 2 mL aliquots were taken.
• Metal-encoded microbeads used in MC have to be stable against metal ions 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 °C. In parallel, another set of microbead samples were tested in the same buffers, but these beads were prepared by 2-stage DisP using acrylic acid (AA) as the ligands to incorporate metal ions, as reported in Abdelrahman et al., 2009. 9 The leakage of each element from 4E3 beads was monitored into these solutions as a function of time by ICP-MS.
• AA acrylic acid
• Fig. 13 presents the results of metal leaching experiments, where the solid symbols refer to DTPA-beads (4E3) and open symbols refer to the AA-beads as a comparison.
• Very minimum detectable leakage ( ⁇ 0.06%) of any incorporated element was observed from both microbeads in the pH 7 buffer over more than 100 days as described in Fig. 13B.
• Microbeads aged in acidic (pH 3) and basic (pH 10.5) buffers showed higher levels of ion loss.
• the leakage of elemental ions reached plateau values in 20 days, with no further loss over the next 80 days.
• the loss of Eu 3+ , Ho 3+ and Lu 3+ from DTPA-beads were small ( ⁇ 0.2%) in pH 3 buffer, whereas it was higher for Ce 3+ (ca. 1.5%) presumably due to the weaker binding stability between Ce 3+ and DTPA chelator.
• DTPA-beads show less metal ion leakage and stronger stability under four tested conditions.
• the mean diameter ( d) and Coefficient of Variation ( CV) of the microbeads were characterized by SEM images of the microbead sample after sedimentation-redispersion washing; b.
• the metal content per microbead were measured by MC using calibration beads as a standard.
• M(DTPA-VBAm 2 )-encoded beads functionalized with a goat anti-mouse (GAM) secondary antibody (Ab) as described in Example 3 were examined for the specific binding between GAM-modified Eu-1 microbeads using a 175 Lu-labeled Ab reporter by MC.
• GAM goat anti-mouse
• Fig. 14 illustrates the strategy used to surface functionalize the microbead sample and subsequently attach Abs to the microbead surface.
• the microbeads were first coated with a thin silica shell followed by introduction of amino groups in a two-step silica sol-gel reaction as reported by Abdelrahman 33 .
• TEOS and APTES were employed in the silica coating process.
• NH 2 -modified Eu-1 microbeads were obtained with silica shell ca. 10 nm thick and surface amino groups.
• the amino groups on microbead surface were then converted to carboxyl groups (COOH) by reacting with succinic anhydride in DMSO.
• the carboxylate groups serve as functionality for attaching bioaffinity agents and help reduce non-specific binding of reporters to the microbead surface.
• Neutravidin (NAv) was covalently conjugated to the COOH groups on the microbead surface by EDC/NHS coupling.
• biotinylated GAM was attached to the NAv-modified microbead surface through the strong biotin-avidin affinity.
• a cell staining solution 50 mI_
• 175 Lu-labeled mouse IgG 6.25 pg
• cell acquisition solution 100 mI_
• the sample was examined by MC.
• the signal obtained for a sample of Eu-1/NAv microbeads without GAM attached gave a signal that was statistically minimized to zero.
• Example 7 Encoding microbeads with CE/DTPA using a methacrylamide derivative Ce(DTPA-
• a methacrylamide-functionalized DTPA chelator N-(3-aminopropyl)- methacrylamide hydrochloride (APMAm) (2 mmol) was dissolved in anhydrous DMSO (5.0 mL), followed with the addition of triethylamine (TEA, 5.5 mmol) to deprotonate the amine hydrochloride.
• DTPA dianhydride (1 mmol) was then introduced to the APMAm-DMSO solution and the reaction was stirred overnight at room temperature.
• NaOH (1M, 3 eqv) in ethanol was added to the reaction to form the trisodium salt of DTPA-AmPMAm2, followed by filtration with a syringe filter (0.2 pm).
• the filtrate was diluted with 45 mL 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 under reduced pressure at room temperature overnight to remove the residual solvent. 1 H-NMR was employed to confirm the structure of the product.
• a typical two-stage dispersion polymerization was employed to prepare polystyrene (PS) microbeads encoded with Ce(DTPA-AmPMAm 2 ) complex.
• PVP-55 (1.00 g)
• AIBN (0.25 g)
• Triton X305 (0.35 g) were added to a flask and fully dissolved in a mixture of styrene (6.25 g) and ethanol (18.75 g).
• the solution was sealed in the flask and stirred with an overhead mixer. After 30 min nitrogen purging at room temperature, the polymerization reaction was initiated by immersing the flask in an oil bath at 70°C.
• the Ce content in microbeads measured by mass cytometry was ca. 2.7x10 7 ions per bead. Assessed by ICP-MS, 49% Ce added in the reaction remained in the solution after reaction termination. Therefore, the incorporation efficiency for Ce(DTPA-AmPMAm 2 ) complex in microbeads was estimated to be 51%.
• DTPA-R 2 DTPA-VBAn3 ⁇ 4 DTPA-BAn ⁇ DTPA-ALArr ⁇ DTPA-AmPMAn ⁇
• M Ln ion such as Y, Ce, Eu, Ho and Lu
• nucleic acid or oligonucleotide can be conjugated to the microbead using methods available in the art.
• the 3’-hydroxyl group can be conjugated to carboxylic acid (such as succinic acid) functional group on a functionalized microbead.
• carboxylic acid such as succinic acid
• a phosphoramidite derivative of a nucleotide can be used for the conjugation.
• Oligonucleotides may be functionalized for conjugation (e.g., to a microbead or biomolecule described herein) may easily be obtained commercially, such as from Integrated DNA Technologies (IDT).
• IDTT Integrated DNA Technologies
• oligonucleotides functionalized with biotin e.g., a destionbiotin
• amine e.g., a destionbiotin
• alkyne modifier e.g., a thiol modifier
• Acrydite e.g., a thiol modifier
• NHS ester such as azide
• Example 10 The synthesis and characterization of metal-encoded microbeads employed in this example are presented in Example 10. Buffers used in this example were purchased from Life Technologies. Antibodies (Abs), biotinylated Abs, and cytokine standards were purchased from Biolegend and R&D Systems (see Table 5) Streptavidin conjugated gold nanoparticles (AuNP, 10 nm, 10 OD) were bought from Abeam. Frozen human peripheral blood mononuclear cells (PBMC) were purchased from Immunospot (CTL, LP-188 HHU20130715).
• PBMC peripheral blood mononuclear cells
• Nanogold®-Streptavidin (Nanoprobe), EQTMFour Element Calibration Beads (EQ4) and Cell- IDTM Pd Barcoding Kits were kindly provided by Fluidigm Canada.
• TNFa 570102 b MAb1 502801 b
• CD163 1607-CD 215930 MAB16071 a 215901 BAM16072 a
• a frozen commercial human PBMC sample (CTL Immunospot) was thawed and added to a pre-warmed RPMI serum-free media (10 mL) containing 0.5 mL of CTL anti-aggregate wash supplement (20x).
• the PBMCs in the sample were spun down by centrifugation (8 min, 300 rpm). The supernatant was aspirated after centrifugation.
• the PBMCs were then resuspended with a warm serum-containing complete RPMI (10 mL).
• a series of four-plex assays were carried out to analyze IL-4, IL-6, TNFa and IFNy in 10 standard samples.
• four types of capture Ab-coated metal-encoded classifier microbeads were first mixed in approximately equal numbers in BSA solution (0.5% BSA in PBS). The classifier bead dispersion was then transferred to 10 wells of a 96-well filter plate (filter cut-off: 0.45 pm). Each well containing ca. 2 million beads in the dispersion (50 pL) can analyze one standard sample.
• a series of 10 standard solutions (50 pL each) consisting of four analytes at 0, 0.31 , 1 .22, 4.88, 19.5, 78.1, 313, 1250, 5000, 20000 pg/mL were added to each well, respectively.
• the mixture of standard solution and classifier beads in each well was first agitated with a pipette and then incubated on a microplate shaker (1200 rpm for 30 s, then 900 rpm for 2 h) at room temperature. After the incubation, the solution in the mixture was removed by reduced pressure filtration through the build-in filter at the bottom of the well.
• the classifier beads with analytes captured on their surface were washed by two redispersion- filtration cycles with washing buffer (200 pL, 0.025% TWEEN® 20 in PBS) to remove possible uncaptured analytes. Once the washing buffer was removed from the well by filtration, a detection Ab cocktail (100 mI_, 0.5% BSA in PBS) containing four types of biotinylated Abs (2.5 pg/mL for each Ab) was transferred to each well. The classifier beads were redispersed with detection Abs by pipette agitation. The mixture of classifier beads and detection Abs was incubated at room temperature for 1 h on a microplate shaker (900 rpm).
• the mixture was filtered and washed by two redispersion-filtration cycles with washing buffer to remove unbound detection Abs on the classifier beads.
• the classifier beads on the filter were then redispersed with a dispersion (100 pl_) of streptavidin-conjugated gold nanoparticles (AuNPs) as the reporter.
• the reporter dispersion was prepared by diluting the AuNP dispersion from the vendor (e.g. 200-times) with 0.5% BSA buffer.
• the mixture of classifier beads and AuNP reporter was incubated on a shaker (900 rpm) for 1 h and washed by two filtration-redispersion cycles with washing solution and two cycles with PBS buffer (100 mI_).
• the assay sample (100 mI_) in each well was stained with a unique palladium barcoding solution (40 mI_, 3* dilution of a stock solution in Cell-IDTM Pd Barcoding Kit).
• the barcoding staining reaction was incubated for 30 min with agitation and quenched by two filtration-redispersion cycles with 0.5% BSA solution (200 mI_) and two cycles with water (100 mI_).
• a total of 10 barcoded assay samples were combined in one test tube and examined by MC employing EQ4 beads as a calibration standard.
• Mass cytometry The microbead samples were characterized bead-by-bead by mass cytometry (HeliosTM a CyTOF® system, Fluidigm).
• HeliosTM a CyTOF® system, Fluidigm
• barcoded immunoassay samples and EQ4 beads as an internal standard were pooled into a test tube and introduced to the MC system at a speed of 30 pL/min.
• the MC signals were normalized using signals from the EQ4 beads and debarcoded to separate the results from the different assays.
• the singlets of all the classifier beads included in the assay were first identified and gated on the 140 Ce- 142 Ce dot-plot. Each type of classier beads was then gated based on their signature signals of metal encoding.
• the median signal intensities of the reporter, e.g. 197 Au for AuNPs are reported as the results.
• the immunoassay resulted in a variable 197 Au signal intensity of the Au NP reporter, proportional to the amount of analyte bound to the surface of each microbead. Since MC quantifies the content of different heavy metal isotopes in each microbead, the pool of microbeads could be separated into individual bead set with the median 197 Au signal intensity of NP reporter for each bead set. Due to this feature, many assays could be carried out simultaneously, allowing for the multiplexed quantification of multiple analytes in a single measurement. In addition, the concentration of the analytes in the sample could be determined by extrapolation from an internal standard.
• PS polystyrene
• DisP dispersion polymerization
• Example 10 The bead synthesis and characterization are described in Example 10.
• a scanning electron microscopy image of bead sample C-1 (see Table 6) is presented in panel (a) of Fig. 22. It shows that these microbeads were uniform with a mean diameter of 3.0 pm and a CV of 1%.
• the median intensity levels of MC signals of the encoded isotopes were in the range of 1000 ⁇ 1200 counts per bead.
• Table provides a summary of the beads prepared, their mean diameters (and CV values) as well as the labeling pattern and signal intensities in MC detection.
• These 11 types of microbeads were uniform (CV ⁇ 1 %) and shared a similar size with mean diameters in the range of 2.8 to 3.0 pm.
• the beads were coated with antibodies, and the Abs attached to the respective classifier beads are listed in Table .
• Table 6 Summary of the particle size, median intensities of MC signals, and target analytes of classifier microbeads prepared by two-stage DisP of styrene with M(DTPA-VBAni2) metal complexes. _
• microbead samples were evaluated by measuring the diameters of at least 300 microbeads in their SEM images; b. The median signal intensities of microbeads were measured by MC using EQ4 beads as a calibration standard. The robust coefficient of variation ( RCV) of the signal intensities for each type of these microbeads was in the range of 7 ⁇ 9% (no shown in the table); c. To capture the target analytes in assay samples, capture Abs were covalently coupled to the surface of microbeads
• microbeads can be surface modified with bioaffinity reagents for the capture of analytes.
• the metal ions incorporated in these microbeads generated MC signals at a similar intensity level of 800 ⁇ 1200 counts per bead. Since all the microbeads (C1 to C11) carried Ce 3+ that produced Ce signals at a similar level in MC, a mixture of all the beads were analyzed in a single MC measurement and isolated the singlets of 11 types of microbeads in one gating from the 140 Ce- 142 Ce dot-plot [see Fig. 17 (a)] The dot-plots in Fig. 17 (b-k) demonstrate the gating strategy to individually identify each type of classifier microbeads based on their signature signals of metal encodings.
• microbeads in this set of classifier beads were uniform with MC signal intensities at similar levels, a gating template was created in FlowJo software based on this microbead gating strategy to simplify the data analysis process of multiplex assays.
• the biotinylated Abs in the cocktail can recognize the analyte molecules captured on the classifier bead surface. These classifier beads in the assay were then washed on the filter to eliminate unbound detection Abs. Next, a streptavidin-conjugated Au mass tag dispersion (100 mI_) was applied to the assay as a reporter. These mass tags were able to attach to the biotinylated detection Abs on the classifier beads by the streptavidin-biotin interaction. 47 After washing off the unattached reporter particles, the assay sample was examined by MC for the metal content in individual microbead event. The exemplary design of this bead-based sandwich immunoassay is illustrated in Fig. 16.
• Fig. 17 demonstrates the gating strategy employed in the study.
• the singlet events of all classifier beads were first isolated in the panel (a) of Fig. 17.
• Each classifier beads in the gated singlet events was then individually identified through a series of gating steps in the dot-plot diagrams shown in the panel (b-k) of Fig. 17.
• the 197 Au signal in each classifier event was then examined and reported as the assay signal.
• the histograms shown in Fig. 18 demonstrate the signal intensities of reporters on IL-4- classifier beads in the four-plex assays.
• AuNP was employed as the reporter in the assays shown in Fig. 18 (a-c).
• a few positive 197 Au signals were recorded in Fig. 18 (a) when IL-4 was absent in the blank control solution.
• the median value of all the events in this plot was reported as a background noise control to reflect the non-specific binding of reporter NPs to the classifier beads.
• the intensity peaks of 197 Au signals on the IL-4-classifier beads were shifted upfield in the panel (b) and (c) of Fig. 18. as the IL-4 concentration in the assays increased from 1.2 to 20 pg/mL.
• NanoGold® was employed as the reporter to analyze standard solutions containing IL-4 at concentrations of 0, 1.2, and 20 pg/mL, respectively. Much weaker intensities of the NanoGold® reporter signals for 197 Au on the IL-4-classifier beads were observed, compared with the results of assays using AuNPs as the reporter. More events with signal intensities of ⁇ 0 were present in the histograms shown in panel (e) compared with panel (b) of Fig. 18.
• a typical log-log standard curve of a bead-based assay starts with a low and flat region when the analyte concentration is low. The curve then rises with the increase of the analyte concentration, followed with a plateau at a higher concentration. 41 43 48 In this Example, the detection range of an assay was estimated based on the slope region of the standard curve. Within this slope region, analyte concentration is generally measurable.
• Fig. 19 summarizes the median MC signal intensities of different types of NPs attached to classifier beads at different analyte concentrations.
• the signal intensities of both types of reporters were low ( ⁇ 10 counts per bead) and insensitive to the increase of analyte concentration.
• the signal intensities of these reporters increased significantly with the increase of analyte concentration.
• the median signal intensities in the assays that employed low concentrations (0.5 and 1.0 pg/mL) of biotinylated anti-CD163 and anti-CXCL-9 Abs were significantly lower than those obtained employing higher concentrations (2.0 and 2.5 pg/mL) of detection Abs.
• the signal intensity levels and assay sensitivities were similar under all of the experimental conditions.
• PBMC sample from a healthy donor
• RPMI cell culture media
• PMA/ionomycin was stimulated with PMA/ionomycin, and analyzed for the extracellular release of cytokines in the supernatant of the stimulated PBMC suspension by the nine-plex assays using the above optimized assay conditions.
• PMA has a structure analogous to diacylglycerol and can diffuse through the cell membrane into the cytoplasm. In the cytoplasm, PMA activates protein kinase C.
• a calcium ionophore that triggers calcium release When used in combination with ionomycin, a calcium ionophore that triggers calcium release, a moderate level of cytokine is released from cells.
• An unstimulated sample was collected from the supernatant of an unstimulated PBMC suspension and analyzed as a control in this experiment. Prior to the assays, the stimulated sample and unstimulated sample were diluted 2x 4x, 16x, 64x and 256x in the assays to vary the analyte concentrations in the measurements, so that some of the assays produce MC intensity values within the range of the standard curves.
• the median 197 Au signal intensities of AuNP attached to the classifier beads in these assays are presented in Fig. 21.
• sample solution 50 pL
• sample solution 50 pL
• concentrations of anti-CD163 and anti-CXCL9 in the assays were 0.5 pg/mL and 1.0 pg/mL, while the concentrations of other detection Abs were 2.5 pg/mL.
• Substantially higher 197 Au signals were detected from the IL-4, IFNY and TNFa-classifier beads in the assays of the stimulated samples at all dilutions than from the unstimulated samples.
• a set of 11 types of lanthanide-encoded microbeads was synthesized by two-stage DisP.
• the metal content of the six metals in these microbeads was finely controlled by varying the feed of the metal complexes in the second stage of the DisP to produce microbeads generating signals in MC with median intensities of ca. 1000 counts per bead.
• These microbeads are uniform in size (C1 ⁇ 4iameter ⁇ 2%) and in metal content ( RCV ⁇ 15%), which makes them good candidates for classifier beads in bead-based assays.
• VBA 4- Vinylbenzylamine
• EtOH Absolute ethanol
• ICP-MS inductively coupled plasma mass spectrometry
• DTPA-VBAm 2 Metal complexes of DTPA-b/s-vinylbenzyl amide (DTPA-VBAm 2 ) were employed as a chelator for incorporating different types of metal ions into polystyrene (PS) microbeads.
• PS polystyrene
• La(DTPA- VBAm 2 ), Ce(DTPA-VBAm 2 ) , Pr(DTPA-VBAm 2 ) , Tb(DTPA-VBAm 2 ) and Tm(DTPA-VBAm 2 ) were prepared in aqueous solution by mixing DTPA-VBAm 2 with l_aCI 3 , CeCI 3 , Pr(OAc) 3 , TbCI 3 and TmCI 3 , respectively.
• These metal complexes were characterized by 1 H NMR.
• Microbeads as classifier beads for bead-based assays were synthesized by a series of two-stage dispersion polymerization (DisP) as described in Table 7.
• DisP dispersion polymerization
• the first stage of the polymerization of styrene (6.25 g) in absolute ethanol (18.75 g) was initiated by AIBN (0.25 g) at 70 °C in the presence of PVP (1 g) and TX305 (0.35 g) as stabilizers.
• the reaction was protected with N 2 purging (3 mL/min) controlled by a gas mass controller (OMEGA).
• the diameters and diameter distributions of microbeads were characterized from their SEM images using a Hitachi S-5200 microscope. Typically, 2 pl_ of a diluted bead dispersion was dropped on a 300 mesh Formvar/carbon coated copper grid and allowed to dry. The diameters of microbeads were manually measured from multiple SEM images using ImageJ software. The mean average diameter, standard deviation (SD) and the coefficient of variation (CV) were calculated based on at least 300 measurements.
• SD standard deviation
• CV coefficient of variation
• the digestion solution was subsequently diluted with 2% HN0 3 .
• the reaction stock dispersion was filtered through a syringe filter (0.2 pm, Nylon) to remove the microbeads and collected the filtrate for ICP-MS analysis.
• the metal incorporation efficiency in the C-1 reaction was estimated by comparing the total metal content with free metal content in the reaction mixture as described above in Example 4.
• ICP-MS Inductively coupled plasma-mass spectrometry
• metal-encoded microbeads were prepared by two-stage DisP employing polymerizable metal complexes, M(DTPA-VBArri2), to incorporate the metal ions into the PS microbeads.
• M(DTPA-VBArri2) polymerizable metal complexes
• the synthesis of DTPA-VBAm 2 chelator was carried out in anhydrous DMSO by reacting DTPA dianhydride with 4-vinylbenzyl amine. La 3+ , Ce 3+ , Pr 3+ , Tb 3+ , Ho 3+ and Tm 3+ were loaded on the DTPA-VBAm 2 chelator in aqueous solution at pH 5 ⁇ 6. The products of these syntheses were characterized by 1 H-NMR. Details of the chelator synthesis and the metal load procedures are described in Example 4.
• the step was to optimize the feed of M(DTPA-VBAm 2 ) in the second stage aliquot to prepare microbeads producing signals of 1 39 La, 140 Ce, 141 Pr, 159 Tb, 165 Ho and 169 Tm in MC with intensity levels at 800 ⁇ 1000 counts per bead for each of these isotopic channels. Based on the linear relationship between the metal feed and metal content is discussed herein in Example 4, some trial syntheses were carried out to design the feed of six metal complexes. Based on the feed recipes developed in the trial experiments, C-1 sample was prepared as a set of classifier beads encoded with six types of metal ions.
• microbeads prepared by DisP varies from batch to batch, possibly due to the sensitivity of particle nucleation to the reaction conditions.
• 11 types of microbeads (C-1 to C-11) were prepared that were uniform ( CV ⁇ 2 %) and shared a similar size with their mean diameters in the range of 2.8 to 3.0 pm.
• Table 7 A typical recipe for the two-stage dispersion polymerization of styrene with metal complex of DTPA-VBAm 2 derivatives materials (g) 1 st stage a 2 nd stage b styrene 6.25
• DTPA-VBAm -metal complexes — c a The reaction was initiated by immersing the flask in a 70°C oil bath. Prior to the initiation, the reaction solution was purged with nitrogen gas for 30 min. b. The second-stage aliquot was introduced into the reaction mixture 2 h after the initiation c. A desired amount of DTPA-VBAm 2 -metal complexes [M(DTPA-VBAm 2 )] dissolved in ethanol was introduced into the aliquot. Details of the metal addition are described in Table 8.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Physics & Mathematics (AREA)
• Molecular Biology (AREA)
• Analytical Chemistry (AREA)
• Immunology (AREA)
• Medicinal Chemistry (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Polymers & Plastics (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Bioinformatics & Computational Biology (AREA)
• Food Science & Technology (AREA)
• Biotechnology (AREA)
• Cell Biology (AREA)
• Microbiology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biophysics (AREA)
• Nanotechnology (AREA)
• Spectroscopy & Molecular Physics (AREA)
• Dispersion Chemistry (AREA)
• Plasma & Fusion (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
• Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Processes Of Treating Macromolecular Substances (AREA)

Priority Applications (5)

Applications Claiming Priority (4)

Publications (1)

Family

ID=83322157

Family Applications (1)

Country Status (5)

Cited By (2)

Citations (12)

Family Cites Families (3)

• 2022
  • 2022-03-15 JP JP2023555572A patent/JP2024511743A/ja active Pending
  • 2022-03-15 CA CA3211376A patent/CA3211376A1/en active Pending
  • 2022-03-15 US US18/282,095 patent/US20250163239A1/en active Pending
  • 2022-03-15 WO PCT/CA2022/050386 patent/WO2022193004A1/en not_active Ceased
  • 2022-03-15 EP EP22770125.7A patent/EP4308647A4/en active Pending

Patent Citations (13)

Non-Patent Citations (62)

Also Published As

Similar Documents

Legal Events