Mass cytometry reagents and methods for signal amplification

Abstract

Described herein are reagents and methods for improving signal in imaging mass cytometry. Aspects include mass tags with a large number of labeling atoms, chemical modifications to the mass tags, and additional reagents to reduce background and / or preserve specific binding partner (SBP) binding of the mass label, as well as protocols for associating multiple mass tags with a single SBP. Thus, embodiments include any combination of one or more reagents and uses thereof. The reagents, kits, and methods herein are useful in mass cytometry, including imaging mass cytometry. In some aspects, the reagents, kits, or methods are useful for delivering a large number of radioisotopes to a target analyte, e.g., for therapeutic use or radioimaging. In certain aspects, only non-radioactive isotopes are used in mass cytometry.

Description

Background Technology

[0001] Mass spectrometry cytometry (including imaging mass spectrometry cytometry (IMC)) enables highly multiplexed detection of target analytes by detecting mass tags using mass spectrometry. Mass tags typically associate with the target analyte via a specific binding partner (SBP), such as an antibody. A mass tag may have one or more copies of a labeling atom (e.g., a single isotope, such as an enriched isotope), which is different from the mass-labeled atom of other mass tags. The labeling atom can be a non-cellular endogenous metal isotope.

[0002] Due to various considerations, such as space charge effects and the separation of labeled atoms from intrinsic atoms, most labeled atoms may be lost upstream of mass detection. Therefore, it may be necessary to associate multiple mass tags with SBP. However, polymer and / or nanoparticle mass tags present many unique challenges affecting background and SBP binding, challenges that are not a concern for other tags, such as fluorescence-based tags. Attached Figure Description

[0003] Figure 1 By comparing signals between cells that are positive and negative for the target analyte, background signals are revealed.

[0004] Figure 2 This is shown as nonspecific binding of edge effects in the IMC image. Detailed Implementation

[0005] This article describes reagents and methods for improving signal in imaging mass spectrometry cytology. Aspects include mass tags with a large number of labeled atoms, chemical modifications to the mass tags, and additional reagents to reduce background and / or maintain targeted binding of the mass tag's specific binding partner (SBP), as well as protocols for associating multiple mass tags with a single SBP. Therefore, implementation schemes include any combination of one or more reagents and their uses.

[0006] The reagents, kits, and methods described herein can be used in mass spectrometry cytometry, including imaging mass spectrometry cytometry. In some respects, the reagents, kits, or methods can be used to deliver large quantities of radioactive isotopes to a target analyte, such as for therapeutic purposes or radioactive detection. In other respects, only non-radioactive isotopes can be used in mass spectrometry cytometry.

[0007] Mass spectrometry cytology

[0008] As used herein, mass spectrometry is any method for detecting mass tags in biological samples, such as simultaneously detecting multiple distinguishable mass tags at single-cell resolution. Mass spectrometry includes suspension mass spectrometry and imaging mass spectrometry (IMC). Mass spectrometry can atomize and ionize mass tags in cellular samples using one or more of laser radiation, ion beam radiation, electron beam radiation, and / or inductively coupled plasma (ICP). Mass spectrometry can simultaneously detect different mass tags from a single cell, such as by time-of-flight (TOF) or magnetic sector mass spectrometry (MS). Examples of mass spectrometry include suspension mass spectrometry (where cells flow into an ICP-MS) and imaging mass spectrometry (where cellular samples (e.g., tissue sections) are sampled, for example, by laser ablation (LA-ICP-MS) or by a single ion beam (e.g., for SIMS).

[0009] Prior to elemental analysis, mass tags can be sampled, atomized, and ionized. For example, mass tags in biological samples can be sampled, atomized, and / or ionized by radiation such as laser beams, ion beams, or electron beams. Alternatively or additionally, mass tags can be atomized and ionized by plasma, such as inductively coupled plasma (ICP). In suspension mass spectrometry cytometry, whole cells including mass tags can flow into ICP-MS, such as ICP-TOF-MS. In imaging mass spectrometry cytometry, a form of radiation can remove (and optionally ionize and atomize) portions (e.g., pixels, regions of interest) of solid biological samples, such as tissue samples including mass tags. Examples of IMC include LA-ICP-MS and SIMS-MS of mass-tagged samples. In some respects, ion optics can deplete ions other than the isotopes of the mass tag. For example, ion optics can remove lighter ions (e.g., C, N, O), organic molecular ions. In ICP applications, ion optics can remove gases such as Ar and / or Xe, for example, through a high-pass quadrupole filter. In some respects, IMC can provide images with quality labels (e.g., targets associated with quality labels) at cellular or subcellular resolution.

[0010] Similar to fluorescence immunohistochemistry methods, mass spectrometry cytology (including imaging mass spectrometry cytology) workflows may include cell (e.g., tissue) fixation and / or permeabilization prior to staining with antibodies and / or other specific binding partners. Unlike fluorescence methods, in mass spectrometry cytology, mass tags (e.g., containing non-cellular endogenous heavy metals) associate with the target analyte via specific binding partners such as antibodies. Like fluorescence microscopy, imaging mass spectrometry cytology may include an antigen recovery step, where the sample is exposed to conditions such as heat to expose the target analyte for binding via SBP. Unbound SBP is typically washed away before mass spectrometry detection of the mass tag. It is worth noting that other detection methods, such as elemental analysis (e.g., emission spectroscopy or X-ray scattering spectroscopy), are also within the scope of this application.

[0011] Additional reagents for mass spectrometry cytometry include metal-containing biosensors (e.g., biosensors deposited or bound under conditions such as hypoxia, protein synthesis, cell cycle, and / or cell death) and / or metal-containing histochemical compounds that bind to structures (e.g., DNA, cell membrane, layers) based on their chemical properties. Furthermore, quality labels (e.g., the quality label of this application or other quality labels) can be combined to provide unique barcodes to mark specific samples or experimental conditions before they are aggregated with other samples or experimental conditions.

[0012] In IHC, tissue samples can be, for example, slices with a thickness ranging from 1 to 10 μm, such as 2 to 6 μm. In some cases, ultrathin slices with thicknesses less than 500 nm, 200 nm, 100 nm, or 50 nm can be used, for example, samples cut from resin-embedded tissue blocks. Techniques for preparing such slices are well-known in the IHC field, including the use of a microtome, and include dehydration steps, fixation, embedding, permeabilization, and sectioning. Thus, tissue can be chemically fixed, and then slices can be prepared on the desired plane. Cryosectioning or laser capture microdissection can also be used to prepare tissue samples. Samples can be permeabilized, for example, to allow reagents used to label intracellular targets. Even after antigen retrieval (e.g., by heating), the access of the SBP to the analyte may be spatially impeded. Therefore, smaller SBPs and certain quality tags can best allow the SBP to access its target analyte.

[0013] To detect RNA, cells in the biological samples discussed herein can be prepared using the methods and apparatus described herein for the analysis of RNA and protein content. In some respects, the cells are fixed and permeabilized prior to the hybridization step. Cells can be provided in fixed and / or permeabilized form. Cells can be fixed with cross-linking fixatives such as formaldehyde or glutaraldehyde. Alternatively or additionally, cells can be fixed with precipitation fixatives such as ethanol, methanol, or acetone. Cells can be permeabilized with detergents such as polyethylene glycol (e.g., Triton X-100), polyoxyethylene (20) sorbitol monolaurate (Tween-20), saponins (a group of amphoteric glycosides), or chemicals such as methanol or acetone. In some cases, the same reagents or groups of reagents can be used for both fixation and permeabilization. Jamur et al. discussed fixation and permeabilization techniques in "Permeabilization of Cell Membranes" (Methods Mol. Biol., 2010).

[0014] Signal amplification

[0015] As used herein in the context of mass spectrometry cytometry, signal amplification is the association (e.g., isotopically enriched) of more than 30, 50, 100, 200, or 500 labeled atoms with a target analyte (i.e., a single instance of a target analyte bound by a specific binding partner). In some aspects, the labeled atoms may be heavy metals, such as lanthanides or transition metals. In some aspects, signal amplification may be performed on more than 2, 5, 10, or 20 target analytes. In some aspects, signal amplification may include branching conjugation using mass tags with SBPs, highly sensitive polymers, large mass-tagged particles, mass-tagged nanoparticles, and / or hybridization schemes. In some aspects, signal amplification uses mass-tagged polymers.

[0016] As described herein, signal amplification can be achieved by using mass tags containing a large number of labeled atoms and / or by associating a large number of mass tags with a single target analyte (such as through hybridization-based signal amplification and / or by conjugating mass tags with SBPs via branched heterofunctional connectors). In some respects, a single mass tag can have more than 30, 50, 100, 200, 500, or 1000 labeled atoms. In some respects, the hydrodynamic diameter of the mass tag may be low, such as less than 20 nm, less than 15 nm, less than 10 nm, less than 5 nm, less than 3 nm, or less than 2 nm. The hydrodynamic diameter can be less than 1000 nm. 3 less than 500nm 3 less than 100nm 3 less than 50nm 3 less than 20nm 3 or less than 10nm3 Techniques such as EM can be used to identify size, and light scattering can be used to identify the hydrodynamic diameter of mass tags (such as the larger mass tags described herein). Furthermore, chromatographic methods, including size exclusion and ion exchange (e.g., anion exchange) chromatography, can be used to characterize mass tags, such as the smaller mass tags described herein.

[0017] Initial attempts at signal amplification resulted in nonspecific binding of the SBP to the target analyte (e.g., high background) and disrupted binding. Mass tags can cause nonspecific binding, which may be due to the low solubility of the polymer, nonspecific binding of the polymer to the biomolecule or slide surface, and / or steric hindrance of the SBP bound to the mass tag. This problem may be more pronounced for larger mass tags (e.g., with a large number of labeled atoms) and / or for mass tags with non-lanthanide labeled atoms. The reagents and methods described herein may be valuable for non-lanthanide mass tags, which may require novel polymers (e.g., polymers with different chelating agents for lanthanides) that may affect solubility and / or cause nonspecific binding. This paper reports signal amplification techniques that amplify the signal while maintaining SBP binding to the target analyte and / or low nonspecific binding. In some aspects, signal amplification may include maintaining a low background. For example, less than 20%, less than 10%, less than 5%, or less than 1% of the signal may originate from the background (e.g., cells and / or cellular regions that do not express the target analyte) and / or from features such as nonspecific points and / or edge effects (e.g., such as...). Figure 2 (As shown). In some respects, the difference between the high signal and the background can be greater than 100 counts or greater than 1000 counts. In some respects, more than 10%, more than 20%, more than 50%, or more than 80% of the target analyte can be combined using SBP in signal amplification methods.

[0018] Chelating agents used to load lanthanides and some non-lanthanide metals may include DOTA, DTPA, EDTA, PEPA and / or their derivatives. Alternatively, quality-labeled polymers may include chelating agents that preferentially chelate certain non-lanthanides, such as DFO and / or sarcophagine.

[0019] The inventors have discovered that, based on the design of the quality-labeled polymer, the selection of the SBP, and their combination, signal amplification and background are variable, allowing certain SBPs to be better paired with different quality labels. Furthermore, the general strategies described herein for providing signal amplification and reducing background can be combined to improve the signal-to-noise ratio.

[0020] For example, Figure 1The data shows the signal in the background improved by the subject-specific methods and reagents, including for non-lanthanide mass tags (top row) that typically have low signal and / or high background. This shows the concentration of the antibody based on the mass tag and the type of mass tag. The average signal per cell ranges from 20 to 2000 counts, while the background (signal from cells negative for the target analyte) remains close to 0 counts. Although the data shown are from suspension mass spectrometry cytology, similar analyses of imaging mass spectrometry cytology data are possible.

[0021] In another example, Figure 2 The edge effect of nonspecific mass-labeled polymer binding is shown in the left figure (right figure), which is reduced by the subject method and reagents.

[0022] Use a heterofunctional connector to attach the branch quality label to the SBP.

[0023] The development of quality-tagged polymers, particularly high-sensitivity polymers, has benefited from a careful balance between preserving antibody functionality and producing a high degree of labeling sensitivity. This can be achieved, for example, by linking low-molecular-weight metal-chelating polymers to multiple binding sites on biomolecules, such as SBPs (e.g., antibodies), or by linking larger metal-chelating polymers to fewer binding sites on biomolecules of interest. In some respects, multiple quality tags (e.g., quality-tagged polymers) are linked to the same binding site on an SBP via branched conjugates, such as through branched heterofunctional connectors as described herein.

[0024] In some respects, over-labeling antibodies with low-molecular-weight mass-tagged polymers via the amino group of the antibody can impair the activity of the conjugate. Over-modification of antibodies can also impair their specificity. However, the inventors have found that linking small amounts of high-molecular-weight polymer chains can also lead to decreased sensitivity; antibody-polymer conjugates that are too large may cause steric hindrance between them and epitopes in tissues. Branched heterofunctional linkers can allow the linking of large amounts of mass-tagged polymers to antibodies without impairing the antibody due to over-modification and without significant steric hindrance. The linker may contain a single reactive group at one end for linking to the biomolecule of interest and multiple reactive moieties at the other end for linking to the polymer. The linker molecule may contain solubility-enhancing groups (such as dendritic polymers or branched PEGs based on polyethylene glycol or poly(amidoamine)) as a core structure with reactive end groups for covalent bonding between the biomolecule and the metal-loaded polymer.

[0025] This strategy will allow us to generate conjugates with amplified signals, reduced steric hindrance, and site-specific conjugations. Previous mass tagging strategies may have allowed only one mass tag to be attached to each thiol group. SBP may provide only a limited number of conjugation sites for mass tag attachment. For example, antibodies or their derivatives may provide only a few thiol or amine groups to which the mass tag can be conjugated. Thiol- or amine-terminated oligonucleotides may provide only one such group.

[0026] In some respects, multiple mass tags (e.g., mass-tagged polymers) can be conjugated to a smaller number of linkage sites on the SBP. For example, polymers with multiple small-branched mass tags (e.g., binding or capable of binding fewer than 30 or fewer than 20 tag atoms) can be linked to a single linkage site via a branched heterofunctional connector. Such smaller polymers may be easier to synthesize (e.g., at a uniform size), and the size and shape of the branched heterofunctional connector bound to the mass-tagged polymer can be controlled by the heterofunctional chemistry of the connector.

[0027] Alternatively, as described herein, various highly sensitive polymers can be bound via a single branched heterofunctional connector. For example, six or more mass tags can bind to three or fewer linking sites, four or more mass tags can bind to two linking sites, or two or more mass tags can bind to a single linking site. For example, the heterofunctional connector may comprise multiple instances of a first group that binds (e.g., covalently binds) to the SBP linking site and a second group that binds (e.g., covalently binds) to different chemical linking sites on the mass tags. For example, the heterofunctional connector may branch into two, three, four, or more instances of the second group.

[0028] Branched heterofunctional connectors can be formed via a third type of chemistry (e.g., without a connection chemical reaction at either end of the connector). For example, branched heterofunctional connectors can be formed via aldehyde chemistry and can have a thiol reactive group for connection to the SBP, and can have multiple instances of click chemical groups (such as strain-promoted click chemical groups) for connection to multiple quality tags. Branched heterofunctional connectors can have 2, 3, 4, at least 2, at least 3, or at least 4 branches, each branch terminating in a second reactive group (for connection to the quality tag).

[0029] Heterofunctional connectors possess two distinct linkage mechanisms. The first linkage can be used to bind SBP linkage sites, such as thiols or amines. The second linkage mechanism can be used to bind mass tags, such as via click chemistry. Branched heterofunctional connectors can have multiple ends for the second linkage chemistry. The branching provided by branched heterofunctional connectors can reduce the need to synthesize polymers with large mass tags (e.g., having or being able to bind more than 30, 50, or 100 tag atoms). Binding can be covalent, such as via thiol reaction chemistry, amine reaction chemistry, or click chemistry (such as strain-promoted click chemistry). Binding can also be non-covalent, such as via hybridization or via affinity interactions (e.g., biotin-avidin or antibody or its derivative with a target analyte (such as a peptide sequence)).

[0030] One or both linkage mechanisms of the branched heterofunctional linker can be covalent. In some aspects, the heterofunctional linker may have multiple instances of a thiol-reactive first group (e.g., maleimide) and a click-chemically reactive second group (e.g., TCO or DBCO). The antibody can be reduced (e.g., by TCEP) to present a thiol group and react with the first group of the heterofunctional linker. In some aspects, TCEP reduction cleaves the antibody SBP into smaller fragments, thereby allowing smaller, less sterically hindered conjugates to better reach epitopes in tissues. Such antibody fragments bound to multiple mass tags may be easier to purify, such as by spin filtration, the opposite of FPLC. Multiple mass tags (such as mass tag polymers, each functionalized with a click-chemically reactive group (e.g., tetrazine or azide)) can be linked to the second group of the heterofunctional linker. This invention relates to site-specific linkage of antibodies with multiple DBCO moieties in the hinge region for azide-functionalized MCPs. The use of branched heterofunctional linkers may involve a two-step conjugation strategy, in which the disulfide bond at the antibody hinge region is partially reduced to generate a reactive thiol group, which then reacts with the maleimide functional group of the branched heterofunctional linker. Then, using a copper-free click chemistry procedure, an azide or tetrazine-functionalized polymer branched from the heterofunctional linker can be attached to the DBCO or TCO moiety. Similarly, other multifunctional groups and different conjugation chemistry can be designed for different applications requiring improved signal amplification and conjugation efficiency. Second- or third-generation dendritic polymers, as branched structures, can also be used to introduce multiple mass tag conjugation sites onto the branched heterofunctional linker.

[0031] In some respects, the ligation mechanism of branched heterofunctional adapters can be non-covalent. For example, the adapter can have branched oligonucleotides (e.g., single-stranded DNA with the same or similar sequences) covalently bound to the polymer backbone of the adapter, and quality-labeled oligonucleotides can hybridize directly or indirectly with the branched oligonucleotides. This hybridization can occur after the sample has been contacted with the SBP bound to the branched heterofunctional adapter, because the hybridization scheme can target different labeled atoms to different SBPs, as discussed further herein. In some respects, the non-covalent ligation of branched heterofunctional adapters to SBPs can be carried out via affinity reactions, such as biotin-avidin or the binding of secondary antibodies to primary antibody SBPs.

[0032] The heterofunctional linker may include a long linker (e.g., more than 5, 10, or 20 repeating subunits) between the first group and the branch point and / or between the branch point and the second group. The subunits may include solubility-enhancing (e.g., polar) subunits, such as polyethylene glycol (PEG).

[0033] In some respects, the SBP can be first attached to one or more heterofunctional connectors, and then a quality tag can be attached. Heterofunctional connectors have better access to the SBP attachment site when not hindered by the large number of tags that can be attached subsequently.

[0034] Quality label and / or connector fitting

[0035] Various suitable conjugation methods are known in the art. For example, quality tags can be conjugated to bioactive materials, such as through covalent bonding (e.g., amine chemistry, thiol chemistry, phosphate chemistry, enzymatic reactions, redox reactions (such as with metal halides), affinity intermediates (e.g., streptavidin or biotin), or click chemistry, such as strain-promoted click chemistry or metal-catalyzed click chemistry). In some aspects, the conjugation methods described herein can be used to conjugate oligonucleotides with SBPs, such as when hybridization protocols are used to indirectly associate quality-tagged oligonucleotides with SBP-oligonucleotide conjugates.

[0036] Quality tags can be conjugated to bioactive materials, such as through covalent bonding (e.g., amine chemistry, thiol chemistry, phosphate chemistry, enzymatic reactions, or click chemistry, such as strain-promoted click chemistry or metal-catalyzed click chemistry). Bioactive materials can be affinity reagents (such as antibodies or fragments thereof, aptamers, lectins, etc.) or oligonucleotide probes that hybridize with endogenous targets (e.g., DNA or RNA) or intermediates (e.g., antibody-oligonucleotide intermediates and / or oligonucleotide hybridization schemes). As described herein, suitable conjugation chemistry may include carboxyl-amine reaction chemistry (e.g., reactions with carbodiimide), amine reaction chemistry (e.g., reactions with NHS esters, imino esters, pentafluorophenyl esters, hydroxymethylphosphine, etc.), mercapto-hydrogen reaction chemistry (e.g., reactions with maleimide, haloacetyl (bromo- or iodo-), pyridyl disulfide, thiosulfonate, vinyl sulfone, etc.), aldehyde reaction chemistry (e.g., reactions with hydrazides, alkoxyamines, etc.), and hydroxyl reaction chemistry (e.g., reactions with isothiocyanates). Alternative linking methods include click chemistry, such as strain-promoted click chemistry (e.g., via DBCO-azides or TCO-tetraazines).

[0037] Polymers can be functionalized to bind bioactive materials. In some respects, polymers can be functionalized via thiol reaction chemistry, amine reaction chemistry, or click chemistry. For example, polymers can be functionalized for thiol reactivity (e.g., by linking a maleimide group to a thiol group on the Fc moiety of an antibody, for example, reduced via TCEP). The type of conjugation and conjugation conditions (e.g., the concentration of the reducing agent) can vary depending on the type of SBP to maintain the integrity of the SBP.

[0038] For example, polymer quality tags (e.g., containing multiple metal-binding groups, such as metal chelate side groups) can be functionalized with thiol-reactive groups (such as maleimide). In some aspects, the SBP may contain cysteine, which can be reduced (e.g., by TCEP reduction) to provide a thiol for conjugation with the polymer. However, cysteine ​​on the SBP may be inaccessible, and the destruction of cysteine ​​may reduce the affinity of the SBP, or the reduction step may reduce the affinity of the SBP. In this case, other functional groups on the SBP can be thiolated prior to conjugation, even on SBPs that already contain thiols or cysteine. For example, recombinant antibodies may be designed to be smaller (e.g., to reduce steric hindrance and thus improve binding), and therefore may not have accessible cysteine ​​on the Fc region. In this case, the amine can be indirectly thiolated, such as by reacting with acetylthioacetate succinimide followed by removal of the acetyl group with 50 mM hydroxylamine or hydrazine. In another example, the amine can be indirectly thiolated by reacting with succinimide 3-(2-pyridyldithio)propionate followed by reduction of the 3-(2-pyridyldithio)propionyl conjugate using DTT or TCEP. Reduction releases a 2-pyridinium chromophore, which can be used to determine the degree of thiolation. Alternatively, the thiol can be incorporated at the carboxylic acid group via an EDAC-mediated reaction with cystamine, followed by reduction of the disulfide using DTT or TCEP. Finally, tryptophan residues in thiol-free proteins can be oxidized to thiotryptophan residues, which can then be conjugated to a mass tag containing iodoacetamide or maleimide. In some aspects, the reduction step described for thiolation can be omitted, or the required step of thiolation with the reduced cysteine ​​can be less stringent, such that the maleimide-functionalized mass tag polymer is conjugated to the thiolated portion rather than at the reduced cysteine ​​of the SBP. In some respects, non-peptide-based SBPs (such as oligonucleotides) may be more flexible, and conjugation can include reduction at concentrations of TCEP or DTT equal to or greater than 25 mM or 50 mM. In some respects, conjugation of non-peptide-based SBPs can include more demanding temperatures, such as denaturation by heating or freezing.

[0039] In some respects, SBPs (such as oligonucleotides or peptides) can be very small, such as within 50% of the polymer mass tag size. This can provide better tissue penetration and / or reduced steric hindrance, but may complicate the purification of the mass-tagged SBP in the filtration step. Therefore, the mass tag can be modified to present an epitope that allows affinity-based purification. In some respects,

[0040] According to certain aspects of this disclosure, a variety of different metal catalysts can be used for free-click chemical reactions, such as strain-promoted reactions.

[0041] Reaction of alkynes with azides

[0042] The first example is the reaction between strained alkynes and azides, such as the strain-promoted azide-alkyne cycloaddition between cyclooctyne derivatives and azides.

[0043] Here, the reaction of cyclooctyne and azide covalently links the R1 and R2 groups. Given the cyclostrain of alkynes, the reaction of an organoazide with a cyclic alkyne (typically a cycloalkyne) is generally referred to as stress-promoted azide-alkyne cycloaddition (SPAAC). In some aspects of this disclosure, R1 may be an SBP, and R2 may be a mass tag. Alternatively, R2 may be an SBP, and R1 may be a mass tag. Therefore, in some embodiments of this disclosure, the method includes conjugating an SBP to a mass tag using click chemistry, wherein the click chemistry is a reaction of an azide with an alkyne.

[0044] The reaction of cyclooctyne with azides is characterized by relatively slow reaction kinetics and requires large amounts of excess reagent and long incubation times. Therefore, cycloalkyne derivatives with higher reactivity can be used without compromising reactivity. These include monofluorinated cyclooctyne (MOFO), difluorocyclooctyne (DIFO), dimethoxyazinocyclooctyne (DIMAC), dibenzocyclooctyne (DIBO), dibenzoazinocyclooctyne (DIBAC), diarylazinocyclooctane (BARAC), bicyclic nonyne (BCN), 2,3,6,7-tetramethoxy-DIBO (TMDIBO), sulfonated DIBO (S-DIBO), carboxymethyl monobenzocyclooctyne (COMBO), and pyrrolocyclooctyne (PYRROC). It is well known that the enhanced reactivity of (ii) benzocyclooctynes ​​is due to the increased ring strain imparted by multiple sp2 hybridized carbons. Many types of dibenzocyclooctynyl (DBCO) derivatives can be used in this invention, such as those derived from NHS esters (directly or via a linker), such as those used for conjugation with amines on SBPs (e.g., the N-terminal amino group of the amino group of the side chain of lysine, arginine, and histidine in proteins (e.g., antibodies and lectins), conjugation with amino-modified oligonucleotide probes (commercially available from IDT (IL, USA), Sigma Aldrich (MO, USA), Bio-Synthesis, Inc. (TX, USA), etc.), amino sugar derivatives, etc. Alternatively, DBCO derivatives can be derived with maleimides (e.g., dibenzocyclooctynyl-maleimide; Sigma Aldrich catalog number 760668, or dibenzocyclooctynyl-PEG4-maleimide; Sigma Aldrich catalog number 760676) (directly or via a linker). The maleimide functional group can be used to couple DBCO to the mercapto group of the quality label.

[0045] Therefore, in some embodiments, the alkyne is a cyclic alkyne, for example, wherein the cyclic alkyne is an 8-membered ring portion. The cyclic alkyne can be varied. Sometimes, the cyclic alkyne is a portion of a polycyclic structure comprising three or more rings, optionally wherein the polycyclic structure comprises at least two benzene rings. In some embodiments, the cyclic alkyne is dibenzocyclooctyne (DBCO).

[0046] Azides can also be made from NHS esters (such as azide-dPEG8-NHS ester, azide-dPEG...) 12 -NHS esters, etc., can be coupled to SBPs (directly or via a linker) (available from Sigma Aldrich; catalog numbers QBD10503 and QBD10505, respectively). Through NHS esters, azides can be coupled to amines of the SBP (e.g., the N-terminal amino group of the amino group of the side chain of lysine, arginine, and histidine in proteins (e.g., antibodies and lectins), conjugated with amino-modified oligonucleotide probes (commercially available from IDT (IL, USA), Sigma Aldrich (MO, USA), Bio-Synthesis, Inc. (TX, USA), etc.), amino sugar derivatives, etc. Alternatively, azide-modified oligonucleotides / sugars can be synthesized directly (i.e., without the need for a separate NHS ester reaction to link the azide functional group to the amino modifier). Azide components can also be coupled to quality labels. For example, azo polymerization initiators can be used in conventional polymerization reactions. Azide-terminated polymethacrylates are also available from Sigma Aldrich.

[0047] Therefore, certain aspects of this disclosure provide a series of methods for generating SBPs with mass labels, including conjugating a mass label and an SBP via a SPAAC reaction. In some embodiments, the method includes providing an alkyne-functionalized SBP and an azide-functionalized mass label, and reacting the alkyne-functionalized SBP with the azide-functionalized mass label, such as wherein the alkyne-functionalized SBP is functionalized with a strained cycloalkyne (e.g., DBCO). In some embodiments, the method includes providing an azide-functionalized SBP and an alkyne-functionalized mass label, and reacting the azide-functionalized SBP with the alkyne-functionalized mass label, such as wherein the alkyne-functionalized mass label is functionalized with a strained cycloalkyne (e.g., DBCO). As described below, a spacer group may be present between the SBP and the alkyne or azide and / or the mass label and the azide or alkyne.

[0048] Some aspects of this disclosure also provide a mass-labeled SBP, wherein the SBP and the mass label are joined by a connector comprising a reaction product of an alkyne and an azide, such as strained cycloalkynes and azides, for example, DBCO and an azide. In some cases, the reaction product is the product of a metal-free click chemistry reaction, such as a copper-free click chemistry reaction. Therefore, some aspects of this disclosure provide a mass-labeled SBP, wherein the SBP and the mass label are joined by a connector comprising a triazole. The triazole group in the connector may be part of a polycyclic structure. In some aspects, the polycyclic structure may comprise four or more rings. For example, the polycyclic structure may be a 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered ring, etc. In some aspects, the polycyclic structure may comprise two or more benzene rings. Specifically, the polycyclic structure may comprise a dibenzocyclooctene group. The triazole group and the dibenzocyclooctene group may be in any orientation. For example, the triazole group can be separated from the SBP by the dibenzocyclooctene group (therefore meaning the dibenzocyclooctene group will be separated from the quality label by the triazole group). Alternatively, the dibenzocyclooctene group can be separated from the SBP by the triazole group (therefore meaning the triazole group will be separated from the quality label by the dibenzocyclooctene group).

[0049] As described below, a mass tag contains one or more labeled atoms, which allows for the simple identification of the SBP's target in a mass detector. However, it is not always the case that the SBP will be conjugated with a mass tag whose labeled elements are already in the tag. Sometimes, the SBP will be conjugated with a metal chelate moiety before one or more metal labeled atoms are loaded onto it to form the mass tag. As explained in more detail below, the metal chelate moiety can be a single metal chelate group, or it can be a polymer in which the metal chelate group has been attached to two or more subunits.

[0050] Therefore, certain aspects of this disclosure provide a series of methods for generating SBPs conjugated with metal chelate moieties, including conjugating the metal chelate moieties and SBPs via a SPAAC reaction. In some embodiments, the method includes providing an alkyne-functionalized SBP and an azide-functionalized metal chelate moieties, and reacting the alkyne-functionalized SBP with the azide-functionalized metal chelate moieties, such as wherein the alkyne-functionalized SBP is functionalized with a strained cycloalkyne (e.g., DBCO). In some embodiments, the method includes providing an azide-functionalized SBP and an alkyne-functionalized metal chelate moieties, and reacting the azide-functionalized SBP with the alkyne-functionalized metal chelate moieties, such as wherein the alkyne-functionalized metal chelate moieties are functionalized with a strained cycloalkyne (e.g., DBCO). As described below, spacer groups may be present between the SBP and the alkyne or azide and / or between the metal chelate moieties and the azide or alkyne.

[0051] The SPAAC reaction can be carried out under physiological conditions and is compatible with large biomolecules such as proteins (including antibodies, ligands, receptors, nucleic acids, etc., as described below and included in the sections related to SBP). Physiological conditions may include isotonic solutions or biological buffers such as saline, phosphate-buffered saline (PBS), etc. Physiological conditions may alternatively or additionally include temperatures ranging from 1°C to 42°C, 4°C to 37°C, 4°C to 25°C, 10°C to 37°C, 25°C to 37°C, etc. Physiological conditions may include neutral pH, pH 5.5 to 8.5, pH 6 to 8, pH 6.5 to 7.5, etc. Because the reaction between DBCO and azides can be a slow process, a relatively long incubation time may be preferred. For example, the fusion in step b) can be performed at 4°C for 4 to 48 hours, at 4°C for 10 to 24 hours, at 4°C for 18 to 20 hours, at room temperature for 10 to 10 hours, at room temperature for 30 to 5 hours, at room temperature for 30 to 3 hours, at 37°C for 5 to 5 hours, at 37°C for 10 to 2 hours, etc.

[0052] Some aspects of this disclosure also provide a method for preparing a quality-labeled SBP, the method comprising performing the method described in the preceding paragraph to produce an SBP conjugated with a metal chelate portion, and further comprising the step of loading a metal onto the metal chelate portion (e.g., a polymer).

[0053] Some aspects of this disclosure also provide SBP-metal chelate moieties, wherein the SBP and the metal chelate moiety are joined via a linker comprising a reaction product of an alkyne and an azide, such as strained cycloalkynes and azides, for example, DBCO and an azide. In some cases, the reaction product is a product of a metal-free click chemistry, such as a copper-free click chemistry. Therefore, some aspects of this disclosure provide SBP-metal chelate moieties, wherein the SBP and the metal chelate moiety are joined via a linker comprising a triazole. The triazole group in the linker may be part of a polycyclic structure. In some aspects, the polycyclic structure may comprise four or more rings. For example, the polycyclic structure may be a 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered ring, etc. In some aspects, the polycyclic structure may comprise two or more benzene rings. Specifically, the polycyclic structure may comprise a dibenzocyclooctene group. The triazole group and the dibenzocyclooctene group may be in any orientation. For example, the triazole group can be separated from the SBP by the dibenzocyclooctene group (therefore meaning that the dibenzocyclooctene group can be separated from the metal chelate by the triazole group). Alternatively, the dibenzocyclooctene group can be separated from the SBP by the triazole group (therefore meaning that the triazole group can be separated from the metal chelate by the dibenzocyclooctene group).

[0054] SBPs containing a triazole group as a quality label can be stable in solution. For example, SBP-quality labels can be stable in solution for up to 1 week, 1 month, 6 months, 2 years, 3 years, 5 years, etc. SBP-quality labels can be stable in solutions at -20°C (e.g., where the solution contains glycerol), below freezing point, 4°C, 10°C, or room temperature. When the SBP is an antibody, stability can be measured by antibody affinity.

[0055] In some cases, click chemistry is carried out without a metal catalyst, particularly in the absence of copper or iron. In other cases, click chemistry is carried out under physiological conditions, optionally at a pH of 6 to 8, for example in a buffer solution, such as an isotonic buffer solution.

[0056] Sometimes, the alkyne is attached to the SBP, and the azide is attached to the mass label or the metal chelate portion. Sometimes, the azide is attached to the SBP, and the alkyne is attached to the mass label or the metal chelate portion. Sometimes, when the alkyne is attached to the SBP, it is attached via a linker component (optionally via a spacer group). Sometimes, when the alkyne is attached to the mass label or the metal chelate portion, it is attached via a linker component (optionally via a spacer group). Sometimes, when the azide is attached to the SBP, it is attached via a linker component (optionally via a spacer group). Sometimes, when the azide is attached to the mass label or the metal chelate portion, it is attached via a linker component (optionally via a spacer group).

[0057] Reaction of alkenes with tetrazines

[0058] The second example is the reaction between strained alkenes and tetrazines, such as the tetrazine-olefin cycloaddition between strain-promoted trans-cyclooctene derivatives and tetrazines.

[0059] Here, the reaction of trans-cyclooctene and tetrazine covalently links the R1 and R2 groups. This is due to the alkyne ring strain, specifically the reaction of an organotetrazine with a cyclic alkene (typically trans-cyclooctene) in an electron-demanding Diels-Alder cycloaddition (iEDDA). In some aspects of this disclosure, R1 may be an SBP, and R2 may be a mass tag. Alternatively, R2 may be an SBP, and R1 may be a mass tag.

[0060] Of the three different possible tetrazine isomers, 1,2,4,5-tetrazine was used in the iEDDA reaction. The completion of the reaction releases N2 gas as the only byproduct, making the iEDDA reaction irreversible and more suitable for biolabeling than the conventional reversible Diels-Alder reaction.

[0061] Trans-cyclooctene (TCO) is one of the most reactive cyclic alkenes known to be used as a reagent in this reaction. Many kinds of derivatives can be used in certain aspects of this disclosure, such as those derived with NHS esters (directly or via a linker), for example, for conjugation with amines (e.g., lysine) on SBPs. Alternatively, TCO derivatives can be derived with maleimides (e.g., the N-terminal amino group of the amino group on the side chain of lysine, arginine, and histidine in proteins (e.g., antibodies and lectins) (directly or via a linker) and conjugated with amino-modified oligonucleotide probes (commercially available from IDT (IL, USA), Sigma-Aldrich (MO, USA), Bio-Synthesis, Inc. (TX, USA), etc.), amino sugar derivatives, etc. The maleimide functional group can be used to couple TCO to the mercapto group of the mass tag. Trans-bicyclic [6.1.0]nonene derivatives can also be used, wherein substitution occurs on the cyclopropyl ring. Although methylcyclopropene, bicyclo[6.1.0]nonyne, cyclooctyne and norbornene are not as fast as TCO, they can also react with tetrazine.

[0062] Tetraazines can also be coupled to SBPs directly or via linkers using NHS esters (such as those available from Sigma Aldrich). Through NHS esters, tetraazines can be coupled to amines on the SBP (e.g., the N-terminal amino group of the amino group on the side chain of lysine, arginine, and histidine in proteins such as antibodies and lectins), conjugated to amino-modified oligonucleotide probes (commercially available from IDT (IL, USA), Sigma Aldrich (MO, USA), Bio-Synthesis, Inc. (TX, USA), etc.), amino sugar derivatives, etc. Tetraazine components can also be coupled to mass tags, for example, where the component also contains maleimide functional groups. Two main types of tetraazines are widely used: 6-methyl-substituted tetraazines and 6-hydro-substituted tetraazines. Methyl-substituted tetraazines exhibit high stability even when dissolved in aqueous media, while still providing faster reaction kinetics with TCO derivatives (approximately 1000 MΩ) than any other bioorthogonal reaction pair. -1 s -1 Furthermore, it is tolerant of a wide range of reaction conditions, making it a preferred choice for applications such as protein tagging. On the other hand, hydrogen-substituted tetrazines exhibit lower stability and less tolerance to harsh reaction conditions, but offer extremely fast reaction kinetics (up to 30,000 M) for applications such as in vivo imaging. -1 s -1 Tetraazine can be 3-(benzylamino)-tetraazine.

[0063] Therefore, certain aspects of this disclosure provide a series of methods for generating mass-tagged SBPs, comprising conjugating a mass tag and an SBP via a reverse electron-demanding Diels-Alder cycloaddition reaction between a strained olefin and a tetrazine (followed by a reverse Diels-Alder reaction eliminating N2). In some embodiments, the method includes the steps of providing an olefin-functionalized SBP and a tetrazine-functionalized mass tag, and reacting the olefin-functionalized SBP with the tetrazine-functionalized mass tag, such as wherein the olefin-functionalized SBP is functionalized with a strained cycloolefin (e.g., TCO). In some embodiments, the method includes the steps of providing a tetrazine-functionalized SBP and an olefin-functionalized mass tag, and reacting the tetrazine-functionalized SBP with the olefin-functionalized mass tag, such as wherein the olefin-functionalized mass tag is functionalized with a strained cycloolefin (e.g., TCO). As described below, a spacer group may be present between the SBP and the olefin or tetrazine and / or the mass tag and the tetrazine or olefin.

[0064] Some aspects of this disclosure also provide a mass-labeled SBP, wherein the SBP and the mass label are connected by a connector comprising a reaction product of an olefin and a tetrazine, such as a strained cycloolefin and a tetrazine, for example, TCO and a tetrazine. In some cases, the reaction product is the product of a metal-free click chemistry reaction, such as a copper-free click chemistry reaction. Therefore, some aspects of this disclosure provide a mass-labeled SBP, wherein the SBP and the mass label are joined by a connector comprising a pyridazine. The pyridazine group in the connector may be part of a polycyclic structure. In some aspects, the polycyclic structure may comprise two or more rings. For example, the polycyclic structure may be a 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered ring, etc. In some aspects, the polycyclic structure may comprise 6-membered and 8-membered rings. Specifically, the polycyclic structure may comprise a cyclooctyl group. The pyridazine group and the cyclooctyl group can be in any orientation. For example, the pyridazine group may be separated from the SBP by the cyclooctyl group (therefore meaning the cyclooctyl group will be separated from the mass label by the pyridazine group). Alternatively, the cyclooctyl group can be separated from the SBP by a pyridazine group (therefore meaning the pyridazine group will be separated from the quality label by the cyclooctyl group).

[0065] SBP and a small part of SBP

[0066] As used herein, SBP is a specific binding partner (or specific binding pair). SBPs can non-covalently bind to their target analytes, such as through affinity (e.g., tertiary structure) or hybridization. Therefore, certain aspects of this disclosure also provide kits for labeling SBPs, and kits for mass-labeled SBPs generated by the click chemistry disclosed herein, optionally including mass-labeled SBPs and other reagents containing labeled atoms (e.g., DNA intercalators). Similarly, certain aspects of this disclosure also provide methods for labeling samples using the mass-labeled SBPs of this disclosure, optionally using a variety of such mass-labeled SBPs, for example, where the mass-labeled SBPs include different types of SBPs, such as antibody SBPs (including a variety of antibody SBPs), nucleic acid SBPs (including a variety of nucleic acid SBPs), lectins (including a variety of lectins), sugars (including a variety of sugars), and DNA intercalators (including a variety of DNA intercalators). Similarly, certain aspects of this disclosure include labeling samples using the quality-labeled SBPs of this disclosure, such as labeling samples using a variety of such quality-labeled SBPs, for example, wherein the quality-labeled SBPs include different types of SBPs, such as antibody SBPs (including a variety of antibody SBPs), nucleic acid SBPs (including a variety of nucleic acid SBPs), lectins (including a variety of lectins), sugars (including a variety of sugars), and DNA intercalators (including a variety of DNA intercalators). Therefore, certain aspects of this disclosure also provide samples labeled according to this disclosure, such as samples labeled with the quality-labeled SBPs of this disclosure, optionally samples labeled with a variety of such quality-labeled SBPs, for example, wherein the quality-labeled SBPs include different types of SBPs, such as antibody SBPs (including a variety of antibody SBPs), nucleic acid SBPs (including a variety of nucleic acid SBPs), lectins (including a variety of lectins), sugars (including a variety of sugars), and DNA intercalators (including a variety of DNA intercalators).

[0067] In some respects, SBPs can be antibodies (such as antibody fragments or synthetic antibodies), nucleic acid aptamers, and non-immunoglobulin proteins (such as avidin), peptides (such as binding domains that match or are derived from proteins, such as zinc fingers that bind nucleic acids or receptor-binding domains that bind small peptides or molecules), or derivatives of their corresponding analytes. In this case, the SBP can be a smaller fraction than a conventional antibody. For example, the molecular weight of a small fraction of SBP can be less than 50 kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa, or less than 5 kDa. Small fractions of SBP can allow for larger mass tags without the disadvantages discussed herein. Small fractions of SBP can penetrate cells or tissues better, for example, allowing for deeper staining of tissues.

[0068] Different SBPs may be affected differently by conjugation methods and quality tags. Therefore, aspects of this invention include using different signal amplification methods for different SBPs in the analysis of the same sample. Different SBPs can be different types of SBPs (e.g., oligonucleotides compared to antibodies), different antibody isoforms (IgM, and different isoforms such as IgG1, IgG2a, and IgG2b), or the same SBP type but with different target analytes. When conjugating SBPs to quality tags using thiol reactive chemistry, different conjugation methods include different reduction strictness. Different quality tags include different high-sensitivity polymers (e.g., different compositions with different chelating groups, polymer sizes, polymer shapes, and / or solubility-enhancing groups). For example, higher-strictness conjugation (e.g., reduction) can be used for SBPs presenting fewer linkage sites (e.g., thiol groups). The kits of this application include a variety of different SBPs conjugated to quality tags with different polymeric structures (e.g., besides having different labeled atoms).

[0069] In some respects, different SBPs (including different antibody immunoglobulin classes) can be conjugated with different quality tags, or with chemically identical or similar quality tags under different conditions. For example, some antibodies may respond differently to reduction reactions (e.g., via TCEP) when using thiol-reactive chemistry. Therefore, multiple SBPs can be conjugated with the same quality-tagged polymer structure (possibly loaded with different isotopes) through different conjugation schemes.

[0070] A small fraction of the mass-tagged SBP can be purified by methods other than FPLC, such as spin filtration. In some respects, the mass tag bound to the SBP (or the total amount of the mass tag) can be at least 20%, 30%, 50%, or 80% of the size of the SBP itself, which can allow spin filtration. In some respects, the mass-tagged antibody can be purified by spin filtration.

[0071] Including high-sensitivity polymers and quality-labeled polymers

[0072] The quality tags of this application include polymers containing multiple labeled atoms, such as those loaded on metal chelating side groups or incorporated into the polymer backbone. In some aspects, the quality-tagged polymers may be provided separately from elemental or isotopic compositions (e.g., those loaded onto or already loaded onto a chelating agent of the quality-tagged polymer). Quality-tagged polymers linked to specific binding partners (SBPs) such as antibodies or fragments thereof may be provided. In some aspects, the quality-tagged polymers may have or be able to (e.g., by chelation) bind more than 10, more than 20, more than 30, more than 50, more than 100, or more than 200 labeled atoms (e.g., labeled atoms of a single isotope, such as enriched isotopes). High-sensitivity polymers may have or be able to bind (e.g., on average) more than 30, more than 50, more than 100, or more than 200 labeled atoms.

[0073] The highly sensitive polymer can be linear or branched. Branched polymers can be dendritic polymers (e.g., containing at least second-, third-, or fourth-generation branches) or star-shaped polymers (e.g., containing at least three linear polymers extending from a central core).

[0074] In some respects, in addition to metal chelating side groups, highly sensitive polymers may include solubility-enhancing side groups that do not have chelating agents (e.g., those with polar groups, such as PEG).

[0075] Highly sensitive mass-tagged polymers (i.e., highly sensitive polymers) with a large number (e.g., more than 30) of labeled atoms can present numerous challenges, including steric hindrance in SBP binding to the target, poor connectivity with SBP, low solubility, and non-specific binding to the sample or underlying substrate. In some respects, mass-tagged polymers with a large number of labeled atoms can be chemically modified to reduce or eliminate one or more of these trade-offs.

[0076] In some respects, the density of labeled atoms in a mass-tagged polymer can be increased by coupling multiple metal chelate groups to a single side group of the polymer.

[0077] In some respects, the hydrodynamic diameter of highly sensitive polymers may be relatively low, for example, less than 20 nm, less than 15 nm, less than 10 nm, less than 5 nm, less than 3 nm, or less than 2 nm. The hydrodynamic diameter can be less than 1000 nm. 3 less than 500nm 3 less than 100nm 3 less than 50nm 3 less than 20nm 3 or less than 10nm 3 .

[0078] To reduce steric hindrance, highly sensitive polymers can be separated from SBPs by long connectors, such as connectors containing more than 10, 20, 30, or 50 chemical bonds along a linear chain (without labeled atoms or metal chelate side groups).

[0079] The polymer structure may include solubility-enhancing groups (e.g., nonionic polar groups, such as PEG groups), for example, such that the quality-labeled polymer has more than 5, more than 10, or more than 20 such groups. The solubility-enhancing groups may be organized along the linear chain, such as at the joint, the polymer backbone, and / or on the side groups (e.g., containing or not containing metal chelators).

[0080] The chelating agent itself can be modified to affect its overall charge balance (e.g., unloading or loading at approximately neutral pH), such as by adding acid or base groups, coordination (e.g., to have at least 6, 7, or 8 coordination sites), and / or incorporating solubility-enhancing groups within or near the chelating group (e.g., having fewer than 10, 5, or 3 bonds).

[0081] The size of the quality-labeled polymer can be uniform. For example, the polymer can have low polydispersity, such as a polydispersity index of less than 1.5, 1.2, or 1.1. Therefore, the size of the nanoparticles can be uniform (e.g., they can have a polydispersity index of less than 1.5, 1.2, or 1.1).

[0082] The polymer provided may include polymerizing side groups via living polymerization. In living polymerization, chain termination and chain transfer reactions may be absent or minimal, and the rate of chain initiation may be faster than the rate of chain growth. The resulting polymer chains can grow at a more constant rate than conventional chain polymerization, and the polymer length can remain consistent (i.e., it has a low polydispersity index as described herein). The living polymerization used to prepare the polymer of this application may include one or more of the following: anionic polymerization, controlled radical polymerization (such as catalytic chain transfer polymerization, initiator-mediated polymerization, stable radical-mediated polymerization (SFRP), atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, and iodine transfer polymerization), cationic polymerization, and / or ring-opening polymerization. The side groups of the polymer may include chelating agents, solubilizing groups, or both. A single side group of the polymer may include a chelating agent, a solubilizing group, or both. The side groups of the polymer may be functionalized to allow the addition of chelating agents and / or solubilizing groups after polymerization. Alternatively or additionally, at least some side groups may include chelating agents and / or solubilizing groups prior to polymerization.

[0083] Polymers may include side groups that contribute to (e.g., increase) the solubility of the polymer, such as polyethylene glycolated side groups. For example, a polymer may be modified to include side groups that contribute to the solubility of the polymer before and / or after loading a metal isotope. These side groups may include hydrophilic groups that contribute to the solubility of the polymer before and after loading a metal isotope onto the side group. Therefore, one or more side groups of a polymer may include repeating chains of hydrophilic groups (e.g., which contribute to the solubility of the polymer). For example, coordinating side groups may include hydrophilic groups and / or be spaced apart from side groups that include hydrophilic groups. Repeating chains of hydrophilic groups may not affect the coordination chemistry of the coordinating side groups of the polymer. Hydrophilic groups may include PEG groups. The auxiliary (e.g., increased) solubility of the polymer can contribute to (e.g., increase) the loading of a metal isotope in solution.

[0084] In some respects, side groups (e.g., having chelating agents and / or solubilizing groups) can be incorporated during main-chain polymerization. Alternatively or additionally, side groups, solubilizing groups (e.g., chains), or both, can be linked to functional groups provided by the polymer backbone, such as by any linking chemistry known in the art. For example, a ratio of chelating agents to solubilizing groups can be added to the polymer to obtain a ratio of side groups having chelating agents to side groups having solubilizing groups (without chelating agents). Suitable linking chemistry may include carboxyl-amine reaction chemistry (e.g., such as reactions with carbodiimides), amine reaction chemistry (e.g., such as reactions with NHS esters, imino esters, pentafluorophenyl esters, hydroxymethylphosphine, etc.), mercapto-hydrogen reaction chemistry (e.g., such as reactions with maleimides, haloacetyl (bromo- or iodo-), pyridyl disulfides, thiosulfonates, vinyl sulfones, etc.), aldehyde reaction chemistry (e.g., such as reactions with hydrazides, alkoxyamines, etc.), and hydroxyl reaction chemistry (e.g., such as reactions with isothiocyanates). Alternative linking methods include click chemistry, such as strain-promoted click chemistry (e.g., via DBCO-azides or TCO-tetraazines).

[0085] The polymer may include solubilizing groups, at least some of which may be organized into chains. Solubilizing groups, as used herein, may not be coordinated with metal atoms. The polymer may be polyethylene glycolated to facilitate (e.g., increase) solubility. For example, the polymer may include at least 50, at least 100, at least 200, or at least 500 PEG units (e.g., PEG groups). PEG units may be distributed on multiple side groups such that multiple side groups of the polymer may be polyethylene glycolated. For example, at least some side groups may include more than 5, more than 10, more than 20, more than 30, or more than 40 PEG units (e.g., organized into chains). The number of PEG units on the polymer may facilitate (e.g., increase) the loading of metal isotopes on the polymer. In some aspects, less than 50% of all side groups on the polymer chelate zirconium and / or hafnium, and more than 50% of all side groups on the polymer include multiple PEG units. For example, less than 60% but more than 30%, such as less than 50% but more than 40%, of the side groups on the polymer may include chelating agents.

[0086] In some aspects, PEGylation of a polymer may include attaching PEG unit chains to side groups of the polymer. These chains may include 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more PEG units. The PEGylation side groups may include chelating agents, or may be spaced apart from side groups including chelating agents. The amount, distribution, and / or ratio of chelating agents and solubilizing groups (e.g., PEG) can contribute to the loading of the isotopic composition onto the polymer. For example, the amount, distribution, and / or ratio of chelating agents and solubilizing groups (e.g., PEG) can maximize the amount of isotopic composition (e.g., enriched isotopes of the composition) that can be loaded onto the polymer (e.g., within 80%, 90%, or 95% of the maximum value). The loading of the polymer will be discussed further herein.

[0087] Polymers (e.g., before loading, after loading, and / or after conjugation with bioactive materials) may not aggregate (e.g., may not readily aggregate). For example, more than 90%, more than 95%, more than 98%, more than 99%, or substantially all of the polymers may not aggregate. Polymers may be unloaded, may be loaded with isotopic compositions, and / or may be conjugated with the bioactive materials described herein. As described herein, polymers may be in solution. For example, more than 90%, more than 95%, more than 98%, more than 99%, or substantially all of the polymers may not aggregate. Polymers provided in the kit (e.g., with additional components described herein) may be stable for at least 1 month, at least 3 months, at least 6 months, or at least 1 year.

[0088] The polymer of this application may include any suitable number of side groups (e.g., repeating units attached to the polymer backbone), such as more than 2, 5, 10, 20, 30, 40, 50, or 100 side groups. For example, the polymer may include 2 to 100, 5 to 80, 10 to 50, or 20 to 40 side groups.

[0089] Methods and kits may include a metal loading buffer for loading an isotopic composition onto a polymer. The metal loading buffer may be mixed with the isotopic composition in solution prior to loading onto the polymer of this application. The metal loading buffer may be an acidic solution (e.g., including one or more strong acids such as nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, perchloric acid, hydrochloric acid, and chloric acid). The isotopic composition may be provided in a form suitable for loading onto the polymer of this application. Alternatively or additionally, the loading buffer may include acetates (e.g., alkali metal acetates), such as ammonium acetate, sodium acetate, and / or acetates paired with another base, such as carbonates or bicarbonates. In some aspects, metals may be loaded to unsaturate all chelating groups of the polymer, for example to improve polymer solubility and / or reduce background or influence on SBP binding.

[0090] Polymer chelating agents

[0091] The term "chelating agent" as used herein refers to a group of ligands that coordinate (e.g., stably coordinate) with a metal atom. Chelating agents can be present on the side groups of a polymer and / or incorporated into the polymer backbone. In some respects, chelating agents are included in the side groups of the polymer.

[0092] In some aspects, the polymer may include one or more side groups, said side groups comprising ligands such as hydroxamic acid salts (which may be used interchangeably with hydroxamic acid herein), azacyclic compounds, phenoxyamines, salophene, cyclam, and / or derivatives thereof. The polymer may include chelating agents known in the art or derivatives thereof, including hydroxamic acid salts, azacyclic compounds, phenoxyamines, salophene, or cyclam. In some aspects, the chelating agents of this application may be coordinated to six or more, more than six, or eight sites on zirconium or hafnium atoms. For example, the chelating agent may form an eight-coordinate complex with at least one of zirconium or hafnium. For example, at least one of zirconium and hafnium may form an eight-coordinate complex with the side groups of the polymer.

[0093] In some aspects, the chelating agent of the polymer includes an isohydroxamic acid group, such as in DFO and / or its derivatives. Alternatively or additionally, the polymer may include a aza-macrocycle, such as a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelating agent or a derivative thereof. In some embodiments, the chelating agent may include one of DOTAM, DOTP, and DOTA (e.g., supported with zirconium or hafnium isotopes or provided separately from zirconium or hafnium isotopes). In some aspects, the chelating agent is a DOTA derivative that has improved zirconium or hafnium binding (and potentially reduced binding with lanthanides) compared to DOTA. For example, the DOTA derivative may have eight sites coordinated on zirconium and / or hafnium atoms and may optionally include spacers between ligands that contribute to (e.g., stabilize) the binding of zirconium and / or hafnium. For example, the binding of the DOTA derivative to zirconium and / or hafnium is enhanced compared to lanthanide isotopes.

[0094] Nanoparticle synthesis on nanoparticles and polymers

[0095] Metal nanoparticles (such as nanoscale metal clusters) offer high densities of labeled atoms, but they also have several drawbacks. Functionalization of nanoparticles with inert surfaces to connect to SBPs is important and often results in multiple connection sites for the SBP. The synthesis of small nanoparticles (e.g., less than 10 nm or less than 5 nm) can be challenging, leading to steric hindrance, poor solubility, poor colloidal stability (aggregation), and nonspecific binding. The synthesis of metal cluster nanoparticles can also be difficult (e.g., may require high temperatures and may be sensitive to synthesis conditions). The size of the nanoparticles may be non-uniform.

[0096] In some respects, metal nanoparticles can be synthesized at moderate temperatures (e.g., below 100°C, below 50°C, or below 37°C) in the presence of stabilizers (such as organic stabilizers). For example, the metal nanoparticles can be quantum dots. In some respects, the organic stabilizers can contain thiol groups, such as cysteine.

[0097] In some respects, stabilizers can act as end-capping agents. Stabilizers can be on the polymer, and nanoparticles can be synthesized on the polymer. The size of the polymer can limit (e.g., control) the size of the nanoparticles. The particles can include linear or branched portions exhibiting multiple instances of the stabilizer. The particles can also include linking groups for attaching the polymer (including nanoparticles synthesized on the polymer) to a single SBP. The quality label can have low polydispersity, such as a polydispersity index less than 1.5, 1.2, or 1.1. Therefore, the size of the nanoparticles can be uniform (e.g., can have a polydispersity index less than 1.5, 1.2, or 1.1). Most nanoparticles can have small diameters, such as between 1 nm and 10 nm, 1 nm and 5 nm, 1 nm and 3 nm, 1 nm and 2 nm, 2 nm and 5 nm, or 2 nm and 3 nm. Nanoparticles can be elements with multiple isotopes, such as cadmium or tellurium, but can have non-natural isotopic compositions (such as enriched isotopes of cadmium or tellurium). Nanoparticles can be monodisperse. In some aspects, the polymer may include multiple nanoparticles. In some aspects, the rate at which nanoparticles are inoculated onto the polymer and grown may be slower than the growth rate. Rapid growth of nanoparticles can consume stabilizing groups on the polymer, preventing the polymer from associating with nanoparticles grown on other polymers. The polymer can be dispersed to reduce the rate at which multiple polymers associate with the same nanoparticles as the nanoparticles grow. In some aspects, pre-formed (pre-inoculated) nanoparticles can be mixed with the polymer before growing nanoparticles on the polymer. In some aspects, the polymer may have between 10 and 10,000, between 10 and 1,000, between 10 and 100, between 10 and 50, between 20 and 500, or between 20 and 100 stabilizer instances. In some aspects, the polymer may have fewer than 10, or even only a single, stabilizer instance, and the presence of the stabilizer in solution allows nanoparticles to grow on the polymer. During the synthesis of nanoparticles on the polymer, the same or different stabilizers may be provided in solution.

[0098] As previously mentioned, small cadmium (CdSe, CdS, and CdTe) nanoparticles can be formed in the presence of thiols or thiolic acid stabilizers. Cysteine-stabilized monodisperse CdSS nanoparticles can be synthesized by seeding these nanoparticles. While the synthesis and association of gold nanoparticles on large poly(cysteine) polymers have been reported, uniform size, monodispersity, or cysteine ​​as a stabilizer or blocking agent for gold nanoparticles have not been demonstrated. Notably, the synthesis of these nanoparticles was carried out at moderate temperatures.

[0099] Aspects of the invention include Cd or CdTe nanoparticles containing isotopes and synthesized on a thiol-presenting polymer such as a polycysteine ​​polymer, and the use of such nanoparticles as a quality tag for SBPs. In some aspects, the SBP itself can provide stabilizers, such as thiol groups (e.g., provided by a reduced antibody), and the nanoparticles can be synthesized directly on the SBP. Direct synthesis can prevent the nanoparticles from spatially interfering with binding if the thiol groups are not located near the binding site of the SBP.

[0100] Hybridization schemes for signal amplification

[0101] Quality-tagged oligonucleotides can hybridize directly or indirectly with target oligonucleotides. For example, one or more intermediate oligonucleotides can provide a scaffold on which multiple quality-tagged oligonucleotides can hybridize, thereby amplifying the signal. Therefore, aspects of this application include oligonucleotides for hybridization-based signal amplification.

[0102] The target oligonucleotide can be an endogenous DNA or RNA molecule (such as encoding RNA, small interfering RNA, or microRNA). The target oligonucleotide can be single-stranded. The target oligonucleotide can have a known specific sequence (or be homologous to a known specific sequence). In some respects, SBPs (such as antibodies or their derivatives) can be conjugated to the target oligonucleotide, such as to a synthetic single-stranded DNA oligonucleotide containing a known sequence. In this case, both the antibody and the oligonucleotide can be referred to as SBPs.

[0103] After SBP binds to the analyte in the sample, multiple quality-tagged oligonucleotides can hybridize directly or indirectly with the first oligonucleotide. Hybridization can be branched or linear. In some respects, the polymerase can extend the first oligonucleotide along the template to provide additional hybridization sites. Quality-tagged oligonucleotides can include a single labeled atom or can include a polymer containing multiple labeled atoms. Quality-tagged oligonucleotides may include labeled atoms, such as heavy metal atoms, in the chemical structure of the oligonucleotide itself.

[0104] In some respects, quality-labeled oligonucleotides can be quality-labeled using the highly sensitive polymers or nanoparticles described herein.

[0105] Massive Tag Particles

[0106] Contrary to intuition, large-mass-tagged particles (e.g., those with diameters greater than 50 nm, 100 nm, 200 nm, 500 nm, or 1 μm) can provide reduced background and / or SBP binding disruption compared to smaller particle (e.g., nanoparticle) mass tags.

[0107] For example, in larger mass tag particles, nonspecific (and non-covalent) binding of the mass tag to the sample or underlying substrate can be reduced compared to the force applied to remove unbound tags by washing the mass tag. Specifically, nonspecific binding can be proportional to the surface area of ​​the particle, while the force that disrupts nonspecific binding under active mass tag washing can increase proportionally to the weight of the mass tag (increasing exponentially with respect to surface area as particle diameter increases).

[0108] Furthermore, SBPs can present multiple groups that bind to the mass tag (e.g., reducing antibodies presenting multiple thiol groups), and the particle mass tag can provide multiple SBP linker groups. Smaller mass tag particles (e.g., less than 10 nm, less than 50 nm, or less than 100 nm) can crosslink with such SBPs. In contrast, larger mass tag particles (e.g., greater than 200 nm, 500 nm, or 1 μm) can bind to most of the linker groups on the SBP and / or prevent other large mass tag particles from binding to the same SBP molecule through steric hindrance.

[0109] Massive tag particles can comprise colloidal metal clusters (e.g., having a surface that is coated / capped and functionalized to bind SBPs), such as large nanoparticles, branched or hyperbranched polymers or matrices containing metal chelating groups, or polymers that trap metal atoms (such as polystyrene).

[0110] blocking reagents

[0111] Blocking agents (i.e., one or more blocking agents) can be used to prevent the nonspecific binding of quality-labeled SBPs. Blocking agents can be added to cell samples, such as cells in suspension or cell samples on solid supports, such as tissue sections on slides. Quality-labeled SBPs can be added to cell samples after the addition of the blocking agent and / or after mixing with the blocking agent. In some respects, the concentration (e.g., molar concentration) of the blocking agent used can exceed the amount of the quality-labeled SBP (e.g., at least 5, 10, 20, 50, or 100 times). As described herein, quality-labeled polymers (especially highly sensitive polymers) may have some background (e.g., signals on cells that do not express the target analyte, and / or artificial features such as edge effects and dots).

[0112] Traditional blocking agents include serum (e.g., BSA), gelatin, milk (or casein), typically at concentrations exceeding 1% or even 5% in buffer solutions. Alternatively, or in addition to one or more traditional blocking agents, one or more polymeric blocking agents (e.g., those not conjugated to SBP) may be used. Polymeric blocking agents can be linear or branched polymers and may include polar solubility-enhancing groups and / or charged groups. Polymeric blocking agents can be used to prevent nonspecific binding of SBP labeled with highly sensitive polymers as described herein. For example, highly sensitive polymers (e.g., with an average of more than 30 labeled atoms) can increase nonspecific binding, such as nonspecific binding to the surface of tissue slides (e.g., glass surfaces), compared to polymers with fewer labeled atoms.

[0113] In some respects, polymer blocking agents may comprise a polymer backbone that is the same as or similar to the mass tag attached to the SBP, and / or may have metal chelating groups (e.g., other than conventional blocking agents such as BSA), such as DOTA, DTPA, EDTA, and / or derivatives thereof. Polymer blocking agents may be polymer mass tags that are not bound to the SBP, such as those not bound to any biomolecule or bound to nonspecific biomolecules (such as oligonucleotide random bodies) or proteins that do not specifically label the target analyte (such as BSA). Polymer mass tag blocking agents may be loaded with metal isotopes that are not used as labeling atoms for any SBP, such as isotopes outside the detection range or isotopes that are already endogenous in the cell. Loading polymer mass tags with metal isotopes makes them chemically similar to the mass tag of the SBP and allows them to bind nonspecifically in a manner similar to that of a mass-labeled SBP. Blocking with polymer mass tags can reduce the nonspecific binding of mass-labeled SBPs. In some respects, the distribution of polymer mass tag blocking agents (e.g., determined by mass channels of metal loaded on the mass tag polymer across pixels by IMC measurement) can be used to reduce the reported amount of mass-labeled SBPs. For example, the signal from the mass-labeled SBP can be normalized to the signal from the polymer mass-labeled blocking agent.

[0114] In some respects, polymer blocking agents can include hydrophilic polymers that do not contain chelating groups, such as charged polymers. Hydrophilic polymers contain polar or charged functional groups, making them soluble in water. In this section, most hydrophilic polymers are grouped according to the chemical properties of their structure. For example, acrylic resins include polymers and copolymers of acrylic acid, acrylamide, and maleic anhydride. Amine-functionalized polymers include allylamine, ethyleneimine, oxazoline, and other polymers containing amine groups on their main chain or side chains.

[0115] In some respects, the charged polymer may have multiple negatively charged groups (and optionally a net negative charge), multiple positively charged groups (and optionally a net positive charge), or may be zwitterionic (having both positively and negatively charged groups, such as strong acid / strong base groups at neutral pH, for example, very close to each other, e.g., within 20, 10, 5, 3, or 2 bonds), and optionally a net neutral charge. The zwitterionic polymer may be a copolymer of positively and negatively charged subunits and may contain one or more acrylic monomers. The zwitterionic polymer may be a polyamphoteric electrolyte or a polybetaine. Amphoteric polymers may include zwitterionic groups such as ammonium phosphate (phosphobetaine or lecithin analogs), ammonium phosphonate (phosphonyl betaine), ammonium phosphonite (phosphonyl betaine), ammonium sulfonate (sulfobetaine, such as methacrylate sulfobetaine), ammonium sulfate, ammonium carboxylate (carbetaine or carboxybetaine), ammonium sulfonamide, ammonium sulfonylimide, guanidinocarboxylate (asparagine analogs), pyridinecarboxylate, ammonium (alkoxy)dicyanovinyl alcohol, ammonium borate, sulfocarboxylate, phosphonosulfonate, phosphonocarboxylate, and oxypyridine betaine.

[0116] Suitable hydrophilic polymers may include poly(N-isopropylacrylamide) (PNIPAM) and polyacrylamide (PAM), poly(oxazoline) and polyethyleneimine (PEI), poly(acrylic acid), polymethacrylate and other acrylic polymers, poly(ethylene glycol) and poly(ethylene oxide), poly(vinyl alcohol) (PVA) and copolymers, poly(vinylpyrrolidone) (PVP) and copolymers, polyelectrolytes and / or their derivatives. Suppliers such as Sigma offer a wide catalog of such hydrophilic polymers.

[0117] This polymer can be synthesized using a variety of polymerization techniques, including free radical polymerization (FRP), step-growth polymerization, and / or ring-opening metathesis polymerization (ROMP). Polymerization allows for the alternating incorporation of different monomers. Polymer blocking agents can be provided in the form of salts or solutions.

[0118] Charged polymers can possess chemical properties similar to polymer quality tags. For example, the chelating groups of a polymer quality tag may have a negative charge when not bound to a metal, and / or a slight positive charge when loaded with a metal. Metal-loaded polymer quality tags may not have each chelating group loaded with the metal (e.g., loading efficiency may be less than 100%, such as less than 90%, less than 80%, or less than 70%). Therefore, metal-loaded polymer quality tags can have charged groups and can be zwitterionic. Thus, charged polymer blocking agents can block surfaces on a sample (e.g., certain types of biomolecules and / or carriers of the sample itself) from which a particular polymer quality tag would otherwise nonspecifically bind non-specifically. Charged polymer blocking agents can be used at low concentrations, such as less than 10 wt%, 5 wt%, 1 wt%, 0.5 wt%, or 0.1 wt%.

[0119] The blocking agents used in this study can reduce nonspecific binding of quality-labeled SBP by more than 50%, more than 75%, or more than 90%. The reduction in nonspecific binding can be determined by staining two consecutive tissue sections with the same quality-labeled SBP, where only one section is blocked, and by assessing the area of ​​reduced signal from the quality-labeled SBP. Alternatively or additionally, when nonspecific binding has a characteristic pattern (e.g., edge effect or blotch), the reduction in nonspecific binding can be assessed as a decrease in the appearance and / or intensity of the pattern.

[0120] In some respects, the kit may include a quality label (e.g., optionally linked to an SBP) and the polymer blocking agent described herein.

[0121] In some respects, metal nanoparticles (e.g., as discussed herein) can be used as quality tags and can be blocked (e.g., passivated) with the polymer blocking agents discussed herein. The nanoparticles can be silica-coated lanthanides or non-lanthanide nanoparticles, such as non-lanthanide metal oxide nanoparticles. Challenges of metal nanoparticle quality tags (e.g., metal nanoparticles with solid metal clusters) may include surface functionalization for linking SBPs, non-specific binding to samples, non-specific adsorption of SBPs (such as antibodies or their derivatives), poor solubility, and / or monodispersity. Coating metal nanoparticle quality tags with the polymer blocking agents described herein can mitigate one or more of these characteristics. Polymer blocking agents can be added to nanoparticles before surface functionalization, during surface functionalization (e.g., the polymer blocking agent can crosslink with a molecule containing a linking group, or it may itself include a group for linking SBP), after surface functionalization (e.g., to block any remaining surface of the exposed metal cluster core), before linking SBP (e.g., to reduce nonspecific adsorption of SBP), and / or after linking SBP (e.g., to reduce nonspecific binding with the sample).

[0122] Combination with secondary affinity SBP

[0123] For example, different secondary antibodies derived from primary antibodies of different species can allow for signal amplification of a variety of target analytes. Secondary antibodies can bind to multiple sites on the Fc portion of the primary antibody. Alternatively, secondary SBPs can bind to fluorophores or other tags used on the primary SBP.

[0124] As discussed in this paper, signal amplification methods may play different roles for different SBPs, or even for a certain type of SBP (such as an antibody). Therefore, secondary SBPs can be prepared for (or used for) signal amplification methods and validated (e.g., to determine low background and strong signal) and then used (e.g., in different experiments) to detect a variety of different primary SBP analytes.

[0125] Biotin-avidin can be used as a secondary affinity. For example, a primary SBP bound to streptavidin will provide four sites for attaching biotinylated quality tags. Biotin and avidin derivatives are within the scope of this application.

[0126] Combined with enzyme deposition

[0127] In some respects, SBPs can be conjugated to enzymes such as HRP. The quality tags described herein (such as quality tag polymers (e.g., high-sensitivity polymers)) can be conjugated to water-soluble substrates that are rendered insoluble by an enzyme and / or modified by an enzyme to bind to a biological sample. In some respects, enzymatic cleavage can render the substrate insoluble, thereby causing deposition of the associated quality tag. Suitable enzymes include oxidoreductases (e.g., peroxidases), phosphatases (e.g., alkaline phosphatases), lactamases (e.g., β-lactamases), and galactosidases (e.g., β-D-galactosidase, β-galactosidase). HRP substrates include 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), o-phenylenediamine dihydrochloride (OPD), and their derivatives. AP substrates include nitrotetrazole blue chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and / or p-nitrophenyl phosphate (PNPP) and their derivatives. Glucose oxidase substrates include nitrotetrazole blue chloride (NBT). β-galactosidase substrates include 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (BCIG or X-Gal). For example, the enzyme can be horseradish peroxidase (HRP) or a derivative thereof, and the substrate can be a tyrosamide moiety or a derivative thereof, such as a metal-chelated tyrosamide moiety. For example, the substrate can be a tyrosamide derivative comprising a tyrosamide moiety and a chelating agent (such as DTPA, DOTA, or a derivative thereof) that binds to a metal (such as a lanthanide or its isotopes). The enzyme can be β-galactosidase (Gal), alkaline phosphatase (AP), and / or horseradish peroxidase (HRP). In some respects, the enzyme can be inactivated, and then a new SBP with the same enzyme can be used to deposit different quality tags (e.g., with different labeled atoms).

[0128] The substrate (e.g., a DTPA or DOTA chelator of the substrate) can be provided separately from the metal to be loaded onto the substrate. Alternatively, a substrate pre-loaded with metal can be provided.

[0129] In some respects, substrates (such as metal-chelated (e.g., DTPA or DOTA-modified) tyrosine derivatives) can form aggregates (e.g., substrates that nonspecifically adhere to cell or tissue samples and are immobilized thereon, and / or prevent enzyme deposition). Such aggregates may form if multiple instances of the tyrosine derivative are partially coordinated with a metal in multiple instances of the tyrosine derivative. Therefore, the substrate can be filtered before application to a sample labeled with the corresponding enzyme (e.g., less than one day, less than 4 hours, less than 2 hours, less than 1 hour, or less than 30 minutes prior), so that the aggregates are removed. The tyrosine derivative can be less than 1 kDa, but the filter can be 50 kDa or greater.

[0130] Alternatively or additionally, a metal (e.g., a lanthanide) or its isotope may be loaded onto the substrate (e.g., loaded onto a chelating agent, such as DTPA or DOTA of the substrate) before the substrate is applied to a sample labeled with the corresponding enzyme (e.g., less than one day, less than four hours, less than two hours, less than one hour, or less than 30 minutes prior).

[0131] In some respects, catalysts (i.e., enhancers) can be added along the substrate to increase the rate and / or extent of deposition. In other respects, terminating agents (e.g., in wash buffer) can be added to terminate enzyme deposition of the substrate in a controlled manner. The use of catalysts and / or terminating agents can provide further control over the extent of the reaction to improve signal enhancement and / or provide better target quantification. In some respects, terminating agents or other agents inactivate the enzyme, allowing another instance of the enzyme to associate with a different target, and substrates containing different metals (e.g., lanthanides) or their isotopes can be applied. Thus, enzyme deposition against multiple different targets can be achieved by sequentially depositing substrates with different metals (e.g., lanthanides) or their isotopes.

[0132] For example, catalysts used for HRP deposition of tyrosine derivatives can increase the rate of free radical formation (e.g., they can be electron mediators). For instance, GHGThorpe and LJKricka, Methods Enzymol. 1986; 133:331 describe 6-hydroxybenzotriazole, iodophenol (such as p-iodophenol), coumaric acid (such as p-coumaric acid); aromatic amines in U.S. Patent No. 4,279,950; acetanilide in European Patent Application No. 603,953 (1994); indophenol and phenothiazine N-substituted and indophenol in U.S. Patent No. 5,171,668; and substituted boric acids in U.S. Patent No. 5,629,168. Such mediators have been described for chemiluminescence, such as by U.S. Patent No. 7,803,573. In some aspects, the catalyst can be applied to buffer solutions containing one or more borates (e.g., perborate or isoborate), acetate buffers, and / or sulfonates.

[0133] In some aspects, a terminating agent may be provided (e.g., used within minutes of the catalyst-enhanced reaction described above), such as for catalytic HRP deposition in less than 5 minutes or less than 3 minutes (e.g., at room temperature or higher). The terminating agent may contain a peroxide, such as hydrogen peroxide, in an amount sufficient to temporarily or permanently inactivate HRP. Alternatively or additionally, the terminating agent may include components that quench free radical formation, such as azides. Alternatively or additionally, conditions such as microwave treatment, elevated temperatures (e.g., above 50 degrees Celsius, such as above 60 degrees Celsius), or low pH (e.g., below pH 4) may be applied to temporarily or permanently inactivate HRP. In some aspects, the terminating agent may be applied to a wash buffer and / or may be incubated for one minute or more. In some aspects, another SBP conjugated to HRP may be applied after the terminating reaction, thereby allowing multiplex reactions in which different tyrosine derivative mass tags associate with different targets. After one or more rounds of enzyme-based mass tag deposition, multiplex and simultaneous staining steps can be performed using multiple SBPs with different mass tags. The sample can then be analyzed by imaging mass spectrometry, such as by LA-ICP-MS or by SIMS.

[0134] In some respects, multiplexing can be performed by hybridizing an oligonucleotide conjugated to HRP directly or indirectly with an oligonucleotide conjugated to SBP (such as an antibody). This allows for a faster sequence of HRP associating with the target, depositing a new quality-labeled tyrosine, inactivating it, and then repeating it with a new oligonucleotide-HRP conjugate. In this system, multiple oligonucleotide-SBPs can be applied to the sample simultaneously at an earlier stage. Hybridization to bind HRP to the target may be faster than antibody staining and may be less susceptible to sample damage after successive inactivation steps.

[0135] Therefore, kits for enzyme deposition may include substrates, such as tyrosine or derivatives thereof, and metals, such as lanthanides or isotopes thereof. The metals may be pre-loaded onto the substrate or may be provided separately for loading onto the substrate. In some aspects, the kit may provide a mass-labeled tyrosine derivative and may further include catalyst reagents, termination reagents, buffers, and / or filters as described above. The kit may further include additional components discussed in other embodiments herein.

[0136] Double marking

[0137] Signal amplification for imaging mass spectrometry cytometry can be combined with fluorescent labeling, allowing for the identification of regions of interest via fluorescence microscopy and / or co-registration of fluorescence images with mass spectrometry cytometry images. In some respects, the SBP can be mass-labeled as described herein and also linked to a fluorophore tag. This dual labeling can be achieved via the same linker attached to the same linking group on the SBP.

[0138] Single molecule detection

[0139] In some respects, signal amplification can allow for the detection of a single SBP at once. For example, imaging mass spectrometry cytometry, which provides high sensitivity, high resolution, and a fast pixel acquisition rate, can allow for the detection of individual SBP masses labeled with signal amplification reagents and / or the methods described herein. Therefore, signal amplification mass labels can contain target barcodes (unique combinations) of isotopes, thereby allowing for a significant increase in target multiplicity. The combination of isotopes can include at least two, at least three, at least four, or at least five different isotopes that are not naturally present in mixtures. For example, a target barcode mass label having a unique combination of six of 20 different isotopes allows for 20 choices of six (38,760) unique barcodes, any of which can be used to label different SBPs. Additionally, target barcode SBPs can be distinguished based on different isotope ratios. In some respects, the target barcode mass label can be a hybridization scheme of oligonucleotides that together provide a mass label of a unique combination of isotopes.

[0140] Reagent test kit

[0141] Kits for mass spectrometry cytology may include one or more of the reagents described herein. Any combination of the above components may be provided in the kit. Kits may include mass labels (e.g., polymer mass labels), isotope compositions, polymers loaded with isotope compositions, polymers and isotope compositions provided separately, or polymers loaded with isotope compositions and conjugated with antibodies.

[0142] The kit may further include any additional components (e.g., buffers, filters, etc.) for loading the isotopic composition onto the polymer and / or binding the loaded polymer to the bioactive material. Alternatively or additionally, the kit may include additional reagents for mass spectrometry cytometry, such as buffers, standards, cell barcodes, and / or reagents comprising heavy atoms of varying masses (e.g., mass tags attached to the bioactive material, or providing mass tags for attachment to the bioactive material).

[0143] The kit may include additional isotopic compositions that are separate from and distinguishable from the aforementioned isotopic compositions (e.g., enriched isotopes of different masses). The additional isotopic compositions may include zirconium, hafnium, and / or lanthanide isotopes. For example, the kit may further include additional polymers comprising multiple side groups that chelate (e.g., stably chelate) lanthanides but not zirconium or hafnium. In some aspects, the kit may include multiple antibodies (e.g., antibodies targeting different targets) covalently bound to polymers loaded with different isotopic compositions. Such antibody sets may be provided together in a single group. The groups may be provided as solutions or lyophilized mixtures comprising less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% water by weight.

Claims

1. A mass spectrometry cytometry method for signal amplification of at least one target analyte, the method comprising: Cell samples were blocked using a polymer blocking reagent containing chelating groups loaded with metals. The cell sample is brought into contact with a specific binding partner (SBP) of the target analyte in the sample; The signal is amplified by associating more than 100 labeled atoms with the target analyte bound by the SBP, wherein: The SBP is attached to one or more branched heterofunctional connectors. Each of one or more branched heterofunctional connectors is attached to multiple quality labels. Each branch heterofunctional connector is connected to the plurality of quality tags via a click chemical, and the click chemical is opposite Mode - The reaction between the cyclooctene (TCO) moiety and the tetrazine moiety or the dibenzocyclooctene (DBCO) moiety and the azide moiety. Each branched heterofunctional linker includes a solubility-enhancing subunit, and Each of the plurality of quality tags is a polymer quality tag containing a metal chelating group, the polymer quality tag having fewer than 30 tag atoms; The labeled atoms were detected by mass spectrometry.

2. The method of claim 1, wherein the background of the labeled atoms is less than 10%.

3. The method of claim 1, wherein the plurality of quality labels comprises four or more quality labels.

4. The method of claim 1, wherein the one or more branched heterofunctional connectors are click-chemically linked to the SBP or the quality label.

5. The method of any one of claims 1 to 4, wherein the polymer blocking agent is not conjugated with SBP.

6. The method of claim 5, wherein the polymer blocking agent comprises positively charged and / or negatively charged groups.

7. The method according to any one of claims 1 to 6, wherein the molecular weight of the SBP is less than 50 kDa.

8. The method of any one of claims 1 to 7, wherein the signal amplification includes a hybridization scheme.

9. The method of claim 8, wherein signal amplification comprises direct or indirect hybridization of quality-labeled oligonucleotides with single-stranded oligonucleotides.

10. The method of claim 9, wherein the single-stranded oligonucleotide is a single-stranded DNA oligonucleotide conjugated to an antibody or a derivative thereof bound to the target analyte.

11. The method of any one of claims 1 to 10, further comprising signal amplification for more than five different target analytes, wherein the labeled atoms associated with different target analytes can be distinguished by mass spectrometry.

12. The method of claim 11, wherein at least one target analyte is a protein.

13. The method of claim 11 or 12, wherein at least one target analyte is an oligonucleotide.

14. The method of any one of claims 1 or 2, wherein multiple SBPs combining the same target analyte are conjugated with the same high-mass tag particles.

15. A signal amplification kit for performing the method as described in any one of claims 1 to 14.

16. A signal amplification kit for mass spectrometry cytology, comprising: Mass tags containing fewer than 30 labeled atoms, of which: The quality tag is either linked to a specific binding partner SBP or functionalized to link to the SBP. The quality label is a polymer quality label; One or more branched heterofunctional junctions; and Polymer blocking agents containing chelating groups and loaded with metals. Each of the one or more branched heterofunctional connectors includes a solubility-enhancing subunit and multiple instances of click chemical groups for attachment to the mass label, wherein the click chemical groups include a TCO portion or a DBCO portion.

17. The kit of claim 16, wherein the quality label is attached to the SBP.

18. The kit of claim 17, wherein a plurality of quality tags are connected to the same connection site of the SBP via a branched heterofunctional connector in one or more of the branched heterofunctional connectors.

19. The kit according to any one of claims 16 to 18, further comprising an isotopic composition.

20. The kit of claim 19, wherein the isotopic composition is isotopically enriched.

21. The kit of claim 19 or 20, wherein the isotopic composition is loaded onto the mass label.

22. The kit according to any one of claims 16 to 21, further comprising oligonucleotides for hybridization-based signal amplification.

23. A method for preparing a signal amplification-specific binding partner body (SBP), comprising: Connect one or more branched heterofunctional connectors to one or more connection sites on the SBP; Connect multiple quality labels to each of the one or more branched heterofunctional connectors; Each of the plurality of quality tags is a polymer quality tag containing a metal chelating group, the polymer quality tag binding fewer than 30 tag atoms, and The connection method between the one or more branched heterofunctional connectors and the SBP is different from the connection method between the multiple quality labels and the one or more branched heterofunctional connectors. The connection between the branched heterofunctional connector and the SBP or the quality label is performed via click chemistry, and the click chemistry is... trans - The reaction between the cyclooctene (TCO) moiety and the tetrazine moiety or the dibenzocyclooctene (DBCO) moiety and the azide moiety.

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