WO2023005614A1 - Frame structure-based nanoparticle used for mass cytometry and preparation method therefor - Google Patents

Frame structure-based nanoparticle used for mass cytometry and preparation method therefor Download PDF

Info

Publication number
WO2023005614A1
WO2023005614A1 PCT/CN2022/103548 CN2022103548W WO2023005614A1 WO 2023005614 A1 WO2023005614 A1 WO 2023005614A1 CN 2022103548 W CN2022103548 W CN 2022103548W WO 2023005614 A1 WO2023005614 A1 WO 2023005614A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
metal
antibody
organic
linker
Prior art date
Application number
PCT/CN2022/103548
Other languages
French (fr)
Chinese (zh)
Inventor
陈缘
赵俊杰
罗英武
曾浔
Original Assignee
浙江大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江大学 filed Critical 浙江大学
Publication of WO2023005614A1 publication Critical patent/WO2023005614A1/en

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N2015/1486—Counting the particles

Definitions

  • the invention relates to the technical field of cell biology, in particular to a nanoparticle material with a metal-binding framework structure of a labeled antibody based on a single-cell protein detection technology combined with flow cytometry and ICP-MS.
  • Mass cytometry technology integrates the principles of mass spectrometry and flow cytometry, and uses metal-labeled antibodies to specifically recognize cell surface antigen proteins, realizing multi-parameter, multi-parameter, and quantitative analysis of the type and quantity of antigen proteins expressed by each cell.
  • High-throughput qualitative analysis provides an important analytical tool for precise disease diagnosis at the gene level and biomedical research and development testing. It inherits the high-speed analysis characteristics of traditional flow cytometry, and has the technical advantages of high-resolution mass spectrometry detection and diversified data processing.
  • the current flow mass spectrometry technology still has the problems of a small number of available metal isotope channels and insufficient sensitivity.
  • the main reason is that the number of metals that can be loaded by existing commercial metal antibody labels is less than 40 kinds of lanthanide metals.
  • a metal isotope corresponds to a combination of labeled antibodies and antigens, so cells with a larger number of characteristic protein types cannot meet a higher level of multi-parameter detection; on the other hand, due to the existing single metal antibody label
  • the number of metal atoms is only about 100, and one antibody binds 2-4 metal tags, while the detection of flow mass spectrometer needs to reach tens of thousands of metal atoms to have better sensitivity, which means that the existing metal tags only It has a good detection effect on cells with high protein expression (the number of certain protein antigens on the surface of a cell reaches hundreds), but there are still limitations in the detection of low-expression antigenic proteins.
  • the purpose of the present invention is to provide a new type of metal antibody-labeled nanoparticle for mass spectrometry flow cytometry and its preparation method. It is a nanoparticle with a framework structure that is grafted on the surface and combined with metal elements. It can currently be combined with non-lanthanide Metal elements, and the metal ion loading capacity of a single nanoparticle can reach 10 3 -10 4 , which solves the problem that the number of available metal channels for existing commercial metal antibody labels is small and the sensitivity of a single metal label is not high enough, and the number of detection channels is extended to atomic weight 135 channels of 75-210.
  • a nanoparticle based on a framework structure capable of linking antibodies for mass spectrometry flow cytometry contains metal ions, antibodies and a coordination network framework formed by connecting organic ligands to each other through linkers, wherein the metal Ions bind to organic ligands.
  • the linker is an organic linker with a functionality of n or a metal cluster with a coordination number of n, where n is greater than or equal to 2.
  • the antibody is connected to an organic ligand or a linker through a grafting agent.
  • the coordination network framework of the nanoparticles of the present invention is a porous network formed by self-assembly of organic ligands and linkers through covalent bonds or coordination bonds.
  • Each structural unit has a functional group that can bind metals, and its structural unit
  • the number of metal atoms can ensure a high loading of metal atoms, and the variability of its structural units can be applied to the loading of various types of metal elements in the framework; in addition, the surface of the framework is grafted with functional groups through chemical modification, which can be compatible with the corresponding antibodies on the antibody. The groups form bonds to achieve grafting.
  • organic ligand is composed of one or more of the following structures:
  • organic ligands are in sequence o-phenylenedithiol, bipyridine, 2,2'-diphenolic hydroxyl-biphenyl, disalicylaldehyde diamine, porphyrin, dehexahydrotriphenylcyclene and other structures, wherein, A The substituents participate in the connection to form a coordination network, and R1-R8 are other substituents.
  • the end of the A substituent of the organic ligand is a carboxyl group, a hydroxyl group on catechol, a pyridyl group, a pyrazinyl group, an imidazolyl group, a triazolyl group, a tetrazolyl group, pyrazolyl, etc.
  • the combination of the paired reactive group at the end of the A substituent of the organic ligand and the end of the organic linker includes amino+aldehyde group, catechol hydroxyl group+boronic acid group, amino+nitro group, aldehyde group+phthalate group Amino group, acid chloride group (-COCl)+amino group, phthalic anhydride group+amino group, cyano group+aldehyde group, o-benzoquinone group+o-phenylenediamino group, catechol hydroxyl group+o-difluorophenyl group, etc.
  • phthalic anhydride group o-benzoquinone group, o-phenylenediamino group and o-difluorophenyl group are as follows:
  • the A substituent end of the organic ligand is combined with the reactive group at the end of the linker bonding form Metal clusters of carboxyl groups + transition metal ions Formation of metal-organic coordination bonds Metal clusters of catechol hydroxyl + transition metal ions Formation of metal-organic coordination bonds Metal clusters of pyridyl + transition metal ions Formation of metal-organic coordination bonds Metal clusters of pyrazinyl + transition metal ions Formation of metal-organic coordination bonds Metal clusters of imidazolyl + transition metal ions Formation of metal-organic coordination bonds Metal clusters of triazolyl + transition metal ions Formation of metal-organic coordination bonds Metal clusters of tetrazolyl + transition metal ions Formation of metal-organic coordination bonds Metal clusters of pyrazolyl + transition metal ions Formation of metal-organic coordination bonds Amino + aldehyde carbon nitrogen double bond Catechol hydroxyl group + boric acid group borate formation amino + nitro nitrogen nitrogen double bond Aldehyde + o-phenylen
  • the atomic weight of the metal ion is 75-210.
  • the grafting agent is a bifunctional linear structure molecule, one end of the head is bonded with the corresponding substituent group on the organic ligand or linker through a click reaction, and the end end is bonded with the amino group or disulfide group on the antibody.
  • the bond-reduced sulfhydryl group is bonded by a click reaction.
  • Click reaction is a type of reaction with mild reaction conditions and high reaction efficiency in a short period of time.
  • the group at the end of the grafting agent is N-hydroxysuccinimide group, pentafluorophenol ester group, carboxyl group, aldehyde group, maleimide group, vinyl sulfone group and the like.
  • the structures of N-hydroxysuccinimide group, pentafluorophenol ester group, maleimide group and vinyl sulfone group are as follows:
  • Substituents for organic ligands or linkers grafting agent head Amino N-hydroxysuccinimide group, pentafluorophenol ester group, carboxyl group, aldehyde group Mercapto Maleimide group, vinyl sulfone group carboxyl Amino Azido Alkynyl Alkynyl Azido hydroxyl Chlorosiloxane Cl-Si(CH 3 ) 2 -
  • Antibody group grafting agent tail group Amino N-hydroxysuccinimide group, pentafluorophenol ester group, carboxyl group, aldehyde group Mercapto Maleimide group, vinyl sulfone group
  • the diameter of the coordination network framework is 20-200nm.
  • the invention synthesizes and prepares the frame structure nanoparticle material capable of binding metals through rational design, which is used as the metal marker of the antibody and is used as a marker material for single-cell protein detection of flow mass spectrometry.
  • the present invention also provides a method for preparing the above nanoparticles, comprising the following steps:
  • organic solvent is a reagent that can dissolve the organic ligands and linkers participating in the reaction.
  • High loading capacity By loading metals with nanoframes, compared with the existing metal carriers of polymer linear chains, the metal loading capacity can be greatly increased, thereby improving the detection sensitivity.
  • commercial polymer antibody tags can only carry about 100 metal ions on one chain, and an antibody is connected to three chains on average.
  • a cell needs at least tens of thousands of metal ion signals , corresponding to a cell with at least hundreds of antibodies to have a labeling effect, which means that there is a great limit to the detection of low-expression proteins; and after the three-dimensional framework structure of the present invention greatly improves the metal loading, the metal loading of a single label is at least Increased by an order of magnitude, the same sensitivity can be obtained for a cell combined with a dozen or even fewer framework antibody tags, which facilitates the detection of low-abundance protein expression.
  • the frame material prepared by the present invention facilitates the loading of more kinds of metals: the frame material prepared by the present invention, first of all, the type combination of the frame structure is flexible and changeable, and structural units containing suitable functional groups can be designed for different metals, so as to adapt to various metal elements; Secondly, since the metal is combined inside the framework structure, it is impossible to form cross-linking points between nanoparticles, thereby avoiding the problem of cross-linking and aggregation caused by the metal carrier of the existing polymer linear chain combined with certain metal ions.
  • the structure of the internal organic ligand has a strong binding ability for a variety of metal elements, on the one hand, it can greatly increase the metal loading and improve the signal sensitivity, on the other hand, it can facilitate Loading different metal isotopes in a frame structure, especially metal elements other than lanthanides, greatly expands the available detection channels of metal isotopes, and provides key new technologies for the multi-channel high-precision detection of single-cell proteins by flow cytometry. Material.
  • Fig. 1 is a schematic diagram of the preparation process of the metal antibody tag capable of binding to the metal frame structure of the present invention
  • Fig. 2 is the organic ligand binding metal schematic diagram of o-phenylenedithiol structure
  • Figure 3 is a schematic diagram of the organic ligand binding metal of the bipyridine structure
  • Fig. 4 is the organic ligand binding metal schematic diagram of 2,2'-diphenolic hydroxyl-biphenyl structure
  • Fig. 5 is the schematic diagram of the organic ligand binding metal of the double salicylaldehyde diamine structure
  • Fig. 6 is the organic ligand binding metal schematic diagram of porphyrin structure
  • Figure 7 is a schematic diagram of the organic ligand binding metal of the hexahydrotriphenylcycloene structure
  • Example 8 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, grafting agent, and framework nanoparticles of Example 1;
  • FIG. 9 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, grafting agent, and framework nanoparticles of Example 2;
  • FIG. 10 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, grafting agent, and framework nanoparticles of Example 3;
  • FIG. 11 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, linker, grafting agent, and framework nanoparticles of Example 4;
  • FIG. 12 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, linker, grafting agent, and framework nanoparticles of Example 5;
  • FIG. 13 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, linker, grafting agent, and framework nanoparticles of Example 6;
  • Figure 14 is a diagram of the staining and grouping effect of Pt-195-labeled antibody PD-1 on yin and yang cells in Example 1;
  • Figure 15 is a diagram of the staining and grouping effect of Sn-120-labeled antibody CD-4 on yin and yang cells in Example 2;
  • Figure 16 is a diagram of the staining and grouping effect of Zr-90-labeled antibody IgD on yin and yang cells in Example 3;
  • Figure 17 is a diagram of the staining and grouping effect of Tm-169-labeled antibody ICOS on yin and yang cells in Example 4;
  • Figure 18 is a diagram of the staining and grouping effect of Pd-104-labeled antibody TCR Va7.2 on yin and yang cells in Example 5;
  • Fig. 19 is a graph showing the effect of staining and grouping of Yin and Yang cells by Yb-171-labeled antibody CCR7 in Example 6.
  • the framework nanoparticle material containing metal elements provided by the present invention can first realize the effective loading of various metal isotopes by flexibly changing the functional groups of the structural units of the framework nanoparticles, so that the available detection channels can be extended to mass spectrometry. Range, that is, more than one hundred isotope channels with a molecular weight of 75-210, to achieve a higher level of multi-parameter simultaneous label detection.
  • the antibody is coupled through surface chemical modification, which can efficiently and firmly graft the antibody without affecting the specific recognition of the target cell population by the antibody.
  • the number of structural units can be adjusted by controlling the size of the nanoparticles, thereby adjusting the number of metal ions loaded, so that the number of metal ions that can be loaded on a nanoparticle can reach 10 3 -10 4 , which greatly improves the metal density of a single metal label.
  • the signal strength is conducive to the sensitive detection of low-expressed antigenic protein markers on cells.
  • the nanoframe material provided by the present invention is synthesized by a two-step method, as shown in Figure 1, the first step is to form a framework structure by coordinating and self-assembling the organic ligand and the linker, and then loading the metal, or the first step is directly
  • the framework structure is obtained by self-assembly of metal-containing organic ligands and linkers; the second step is to introduce a grafting agent on the surface of the framework structure, and then graft antibodies at the end of the grafting agent to obtain nanoparticles as metal antibody labels.
  • the organic ligand in the first step its main metal-binding part is shown in Figure 2-7, and there are many combinations of the bonding between the end of the A substituent and the terminal group of the linker, as shown in Table 1 , so as to obtain a variety of self-assembled framework structures; and the metal ions can be all metals with an atomic weight of 75-210.
  • the grafting agent is introduced on the surface of the framework through the click reaction bonding between the head group of the grafting agent and the substituent of the organic ligand on the framework or the substituent of the linker.
  • the tail group of the agent is bonded to the group on the antibody through a click reaction, and finally a nanoframe material that binds the metal and connects the antibody is obtained.
  • groups in the two-step click reaction as shown in Table 2, which can provide a variety of connection strategies.
  • Metal-bonded framework materials (1) based on o-phthalic dimercapto-dimercapto-6-azido-1,4-terephthalic acid organic ligands coordinated with linkers of zirconium metal clusters from Assembled to form framework structure nanoparticles, using PtCl 4 as the metal reagent to load Pt into the framework, using CH ⁇ C-CH 2 -CH 2 -O-CH 2 -MAH as the grafting agent, through the head group carbon-carbon triple bond and The azido group on the substituent of the organic ligand is bonded to the -MAH of the tail group and the sulfhydryl group obtained by reducing the disulfide bond on the antibody.
  • the organic ligands, grafting agents, and repeating structural units used to obtain framework nanoparticles are shown in FIG. 8 .
  • the specific preparation method is as follows:
  • Solvent molecules and metal ions then add 10mg of grafting agent CH ⁇ C-CH 2 -CH 2 -O-CH 2 -MAH, fully mix and dissolve, and react at room temperature for 1 hour; use a dialysis bag with a molecular weight cut-off of 300KD, remove Dialyze 3 times in deionized water, at least 2 hours each time; prepare for the reduction of antibody disulfide bonds while dialysis: For 100 ⁇ g of antibody, add 100 ⁇ L of 4 mM TCEP to react in PBS buffer for 0.5 hours, and then use a 3KD retention tube Centrifuge at 12000xg for 30min to separate TCEP, then add 300 ⁇ L of PBS buffer to resuspend.
  • the organic ligands, grafting agents, and repeating structural units used to obtain framework nanoparticles are shown in FIG. 9 .
  • the specific preparation method is as follows:
  • 2,2'-diphenolic hydroxyl-biphenyl-bonded metal-based framework material (3) composed of 2,2-diphenolic hydroxyl-5,5-diynyl-1,1'-biphenyl-4,4' - Bipyridine and zinc ions coordinate self-assembled to form nanoparticles with a framework structure, use ZrCl 4 as a metal reagent to load Zr into the framework, and use N 3 -CH 2 -CH 2 -O-CH 2 -NHS as a grafting agent , bonded to the carbon-carbon triple bond on the substituent of the organic ligand through the azido group of the head group, and bonded to the amino group on the antibody through the N-hydroxysuccinimide group of the tail group.
  • the organic ligands, grafting agents, and repeating structural units used to obtain framework nanoparticles are shown in FIG. 10 .
  • the specific preparation method is as follows:
  • Metal-bonded framework material (4) based on bis-salicylaldehyde diamine, formed from tetrakis(3-formyl-4-phenolhydroxyphenyl)methane and 4-mercapto-1,2-diphenylamine via condensation of aldehyde groups with amino groups
  • Nanoparticles with framework structure were obtained by reactive self-assembly, Tm was loaded into the framework using TmCl 3 as the metal reagent, MAH-CH 2 -CH 2 -O-CH 2 -COOH was used as the grafting agent, and the head group -MAH was connected with The sulfhydryl group on the substituent is bonded and connected, and the carboxyl group of the tail group is bonded to the amino group of the antibody under the catalysis of N,N'-diisopropylcarbodiimide.
  • the used organic ligands, linkers, grafting agents, and repeating structural units to obtain framework nanoparticles are shown in FIG. 11
  • the azido group is bonded to the carbon-carbon triple bond on the substituent of the linker, and the -MAH of the tail group is bonded to the sulfhydryl group obtained by reducing the disulfide bond on the antibody.
  • the used organic ligands, linkers, grafting agents, and repeating structural units to obtain framework nanoparticles are shown in FIG. 12 .
  • the specific preparation method is as follows:
  • Solvent molecules and metal ions then add 10mg of grafting agent N 3 -CH 2 -CH 2 -O-CH 2 -MAH, fully mix and dissolve, and react at room temperature for 1 hour; then use a dialysis bag with a molecular weight cut-off of 300KD, Dialyze three times in deionized water, at least 2 hours each time; prepare for the reduction of antibody disulfide bonds while dialysis, add 100 ⁇ L of 4 mM TCEP to PBS buffer for half an hour for 100 ⁇ g of antibody, and then use 3KD Centrifuge the retention tube at 12000xg for 30 minutes to separate TCEP, then add 300 ⁇ L of PBS buffer to resuspend.
  • a dehexahydrotriphenylcyclene-bonded metal-based framework material (6) composed of dehydrobenzocyclopentene (DBA) and tetrakis-(3-mercapto-4-dihydroxyborylphenyl)methane (TBPM) via The phenolic hydroxyl group and boronic acid group condensation reaction self-assembled to obtain the framework structure nanoparticles, YbCl 3 was used as the metal reagent to load Yb into the framework, and MAH-CH 2 -CH 2 -O-CH 2 -NHS was used as the grafting agent, by The -MAH group of the head group is bonded to the sulfhydryl group of the substituent on the linker, and is bonded to the amino group of the antibody through the epoxy group of the tail group.
  • the used organic ligands, linkers, grafting agents, and repeating structural units to obtain framework nanoparticles are shown in FIG. 13 .
  • the specific preparation method is as follows
  • DBA dehydrobenzocyclopentene
  • TBPM tetrakis-(2-mercapto-4-dihydroxyborylphenyl) methane
  • Nanoparticle size potential analyzer Zetasizer Nano-ZS Utilize the nanoparticle size potential analyzer Zetasizer Nano-ZS to measure the particle size and particle size distribution of the nanoparticles obtained in embodiment 1-6, and observe the aqueous dispersion of the nanoparticles obtained in embodiments 1-6 after standing for six months The stability is judged by the presence or absence of aggregation and sedimentation, and the results are shown in Table 4, which shows that the nanoparticles under each particle size have a narrow size distribution and good dispersion stability.
  • Example Average particle size/nm Particle size distribution (Cv/%) dispersion stability 1 100 6% good 2 40 8% good 3 60 7% good 4 80 6% good 5 20 10% good 6 200 8% good
  • the metal ion concentration in the sample liquid was tested by ICP-MS, and then the metal loading amount of a single nanoparticle was calculated. The results are shown in Table 5, indicating that the nanoparticle can successfully load the target metal ion and the loading amount can be increased by increasing the particle size. to 10 3 -10 4 pcs.
  • the results of the staining effects of the six antibodies are shown in Figure 14-19.
  • the abscissa Ir191 indicates the staining of DNA, which proves to be a cell signal; the ordinate is the intensity of the corresponding isotope signal.
  • the ordinate value of the background signal is obtained according to the control group.
  • the dividing line of yin and yang cells is obtained, that is, the circled in the box is the percentage of the positive cell population of each type of antibody in the total number of cells.
  • the immune cells added with the antibody were divided into negative and positive groups, which proved that the antibody labeling effect is good, and the target cells can be separated from non-target cells, and the antibody can be used for subsequent experiments.

Abstract

A frame structure-based nanoparticle used for mass cytometry and a preparation method therefor. The nanoparticle comprises metal ions, an antibody, and a coordination network framework formed by connecting organic ligands to each other by means of a linker, wherein the metal ions are bonded to the organic ligands; the linker is an organic linker having the functionality of n or a metal cluster having a coordination number of n, and n is greater than or equal to 2; and the antibody is connected to the organic ligands or the linker by means of a grafting agent. The nanoparticle can load a plurality of metal isotopes, the loading capacity is high, and a high-sensitivity metal detection signal having a plurality of available metal isotope channels can be provided in flow-type mass spectrometry detection, thereby achieving single-cell protein analysis and detection of simultaneous measurement of more cell parameters, and providing an important analysis tool for accurate diagnosis of diseases at the gene level and research and development detection of biomedicine.

Description

一种用于质谱流式细胞技术的基于框架结构的纳米颗粒及其制备方法A framework-based nanoparticle for mass flow cytometry and its preparation method 技术领域technical field
本发明涉及细胞生物学技术领域，具体涉及一种基于流式结合ICP-MS的单细胞蛋白检测技术的标记抗体的结合金属的框架结构的纳米颗粒材料。The invention relates to the technical field of cell biology, in particular to a nanoparticle material with a metal-binding framework structure of a labeled antibody based on a single-cell protein detection technology combined with flow cytometry and ICP-MS.
背景技术Background technique
质谱流式技术(Mass Cytometry)集成了质谱和流式细胞仪的原理，通过金属标记的抗体对细胞表面抗原蛋白进行特异性识别，实现对每一个细胞表达的抗原蛋白种类和数量进行多参数、高通量的定性分析，进而为基因层面的疾病精确诊断、生物医学的研发检测提供重要的分析工具。它继承了传统流式细胞仪的高速分析的特点，又具有质谱检测的高分辨能力以及多样化数据处理等技术优势，由于质谱细胞仪较传统荧光流式细胞仪测量的参数更多且通道之间没有干扰，并且不需要进行荧光补偿计算，因此大幅拓展了单样本的数据产出量和检测范围，使数据更全面，结果更可靠，已成为单细胞蛋白表达分析的一个新方向，在临床医学与生物研究领域里得到了越来越广泛的应用。Mass cytometry technology (Mass Cytometry) integrates the principles of mass spectrometry and flow cytometry, and uses metal-labeled antibodies to specifically recognize cell surface antigen proteins, realizing multi-parameter, multi-parameter, and quantitative analysis of the type and quantity of antigen proteins expressed by each cell. High-throughput qualitative analysis provides an important analytical tool for precise disease diagnosis at the gene level and biomedical research and development testing. It inherits the high-speed analysis characteristics of traditional flow cytometry, and has the technical advantages of high-resolution mass spectrometry detection and diversified data processing. Since mass spectrometry cytometry measures more parameters than traditional fluorescence flow cytometry and the channel There is no interference between them, and there is no need for fluorescence compensation calculations, so the data output and detection range of a single sample are greatly expanded, the data is more comprehensive, and the results are more reliable. It has become a new direction for single-cell protein expression analysis. It has been widely used in the fields of medicine and biological research.
然而目前的流式质谱技术还存在可用的金属同位素通道数少、灵敏度不够高的问题，主要原因一方面在于现有商用的金属抗体标签能够负载的金属种类数仅有不到40种镧系金属同位素，而一种金属同位素对应一种标记抗体和抗原的组合，因此对于特征蛋白种类数更多的细胞无法满足更高水平的多参数检测；另一方面，由于现有单个的金属抗体标签的金属原子个数仅有100左右，一个抗体结合2-4个金属标签，而流式质谱仪的检测需要达到上万个金属原子数才会有较好的灵敏度，意味着现有的金属标记只对蛋白含量高表达(一个细胞表面的某种蛋白抗原数量达到上百个)的细胞有很好的检测效果，对于低表达的抗原蛋白的标记检测还存在限制。However, the current flow mass spectrometry technology still has the problems of a small number of available metal isotope channels and insufficient sensitivity. The main reason is that the number of metals that can be loaded by existing commercial metal antibody labels is less than 40 kinds of lanthanide metals. isotope, and a metal isotope corresponds to a combination of labeled antibodies and antigens, so cells with a larger number of characteristic protein types cannot meet a higher level of multi-parameter detection; on the other hand, due to the existing single metal antibody label The number of metal atoms is only about 100, and one antibody binds 2-4 metal tags, while the detection of flow mass spectrometer needs to reach tens of thousands of metal atoms to have better sensitivity, which means that the existing metal tags only It has a good detection effect on cells with high protein expression (the number of certain protein antigens on the surface of a cell reaches hundreds), but there are still limitations in the detection of low-expression antigenic proteins.
发明内容Contents of the invention
本发明的目的是提供一种用于质谱流式细胞技术的新型金属抗体标记纳米颗粒及其制备方法，是表面接枝抗体、结合金属元素的框架结构的纳米颗粒，目前能够结合非镧系的金属元素、同时单个纳米颗粒的金属离子负载量可达到10 3-10 4个，解决现有商用金属抗体标签可用金属通道数少、单个金属标签灵敏度不够高的问题，将检测通道数扩展至原子量75-210的135种通道。 The purpose of the present invention is to provide a new type of metal antibody-labeled nanoparticle for mass spectrometry flow cytometry and its preparation method. It is a nanoparticle with a framework structure that is grafted on the surface and combined with metal elements. It can currently be combined with non-lanthanide Metal elements, and the metal ion loading capacity of a single nanoparticle can reach 10 3 -10 4 , which solves the problem that the number of available metal channels for existing commercial metal antibody labels is small and the sensitivity of a single metal label is not high enough, and the number of detection channels is extended to atomic weight 135 channels of 75-210.
本发明采用的技术方案具体如下：The technical scheme that the present invention adopts is specifically as follows:
一种用于质谱流式细胞技术的能够连接抗体的基于框架结构的纳米颗粒，所述纳米颗粒包含金属离子、抗体和通过连接子将有机配体相互连接形成的配位网络框架，其中，金属离子结合于有机配体上。所述连接子是官能度为n的有机连接子或者配位数为n的金属簇，n大于等于2。所述抗体通过接枝剂与有机配体或者连接子连接。A nanoparticle based on a framework structure capable of linking antibodies for mass spectrometry flow cytometry, the nanoparticle contains metal ions, antibodies and a coordination network framework formed by connecting organic ligands to each other through linkers, wherein the metal Ions bind to organic ligands. The linker is an organic linker with a functionality of n or a metal cluster with a coordination number of n, where n is greater than or equal to 2. The antibody is connected to an organic ligand or a linker through a grafting agent.
本发明的纳米颗粒的配位网络框架是有机配体与连接子通过共价键或者配位键自组装形成的多孔网络，每一个结构单元都有可以结合金属的功能基团，并且其结构单元的数量可以保证金属原子的高负载量，其结构单元的可变性可适用于各种不同类型的金属元素在框架中的负载；此外，框架表面通过化学修饰接枝官能团，能够与抗体上的相应基团成键实现接枝。The coordination network framework of the nanoparticles of the present invention is a porous network formed by self-assembly of organic ligands and linkers through covalent bonds or coordination bonds. Each structural unit has a functional group that can bind metals, and its structural unit The number of metal atoms can ensure a high loading of metal atoms, and the variability of its structural units can be applied to the loading of various types of metal elements in the framework; in addition, the surface of the framework is grafted with functional groups through chemical modification, which can be compatible with the corresponding antibodies on the antibody. The groups form bonds to achieve grafting.
进一步地，所述有机配体由如下中的一种或几种结构混合组成：Further, the organic ligand is composed of one or more of the following structures:
Figure PCTCN2022103548-appb-000001
Figure PCTCN2022103548-appb-000001
上述有机配体依次为邻苯二巯基、联吡啶、2，2’-二酚羟基-联苯、双水杨醛缩二胺、卟啉、脱六氢三苯环烯等结构，其中，A取代基参与连接形成配位网络，R1～R8为其它取代基。The above-mentioned organic ligands are in sequence o-phenylenedithiol, bipyridine, 2,2'-diphenolic hydroxyl-biphenyl, disalicylaldehyde diamine, porphyrin, dehexahydrotriphenylcyclene and other structures, wherein, A The substituents participate in the connection to form a coordination network, and R1-R8 are other substituents.
进一步地，所述连接子为金属簇时，有机配体的A取代基末端为羧基、位于邻苯二酚上的羟基、吡啶基、吡嗪基、咪唑基、***基、四唑基、吡唑基等。Further, when the linker is a metal cluster, the end of the A substituent of the organic ligand is a carboxyl group, a hydroxyl group on catechol, a pyridyl group, a pyrazinyl group, an imidazolyl group, a triazolyl group, a tetrazolyl group, pyrazolyl, etc.
具体地，优选为如下所示结构：Specifically, the structure shown below is preferred:
Figure PCTCN2022103548-appb-000002
Figure PCTCN2022103548-appb-000002
进一步地，所述有机配体的A取代基末端与有机连接子末端的配对反应基团组合包括氨基+醛基、邻苯二酚羟基+硼酸基、氨基+硝基、醛基+邻苯二氨基、酰氯基(-COCl)+氨基、邻苯二酸酐基+氨基、氰基+醛基、邻苯醌基+邻苯二氨基、邻苯二酚羟基+邻二氟苯基等。Further, the combination of the paired reactive group at the end of the A substituent of the organic ligand and the end of the organic linker includes amino+aldehyde group, catechol hydroxyl group+boronic acid group, amino+nitro group, aldehyde group+phthalate group Amino group, acid chloride group (-COCl)+amino group, phthalic anhydride group+amino group, cyano group+aldehyde group, o-benzoquinone group+o-phenylenediamino group, catechol hydroxyl group+o-difluorophenyl group, etc.
其中，邻苯二酸酐基、邻苯醌基、邻苯二氨基、邻二氟苯基结构如下所示：Among them, the structures of phthalic anhydride group, o-benzoquinone group, o-phenylenediamino group and o-difluorophenyl group are as follows:
Figure PCTCN2022103548-appb-000003
Figure PCTCN2022103548-appb-000003
具体的基团组合及成键连接形式如表1所示，表1所示组合形式能够形成稳固的纳米框架结构。The specific group combinations and bonding forms are shown in Table 1, and the combination forms shown in Table 1 can form a stable nanoframe structure.
表1有机配体的A取代基末端与连接子末端的反应基团配对组合Table 1 The A substituent end of the organic ligand and the reactive group pairing combination at the linker end
有机配体的A取代基末端与连接子末端的反应基团配对组合The A substituent end of the organic ligand is combined with the reactive group at the end of the linker 成键连接形式bonding form
羧基+过渡金属离子的金属簇Metal clusters of carboxyl groups + transition metal ions 形成金属-有机配位键Formation of metal-organic coordination bonds
邻苯二酚羟基+过渡金属离子的金属簇Metal clusters of catechol hydroxyl + transition metal ions 形成金属-有机配位键Formation of metal-organic coordination bonds
吡啶基+过渡金属离子的金属簇Metal clusters of pyridyl + transition metal ions 形成金属-有机配位键Formation of metal-organic coordination bonds
吡嗪基+过渡金属离子的金属簇Metal clusters of pyrazinyl + transition metal ions 形成金属-有机配位键Formation of metal-organic coordination bonds
咪唑基+过渡金属离子的金属簇Metal clusters of imidazolyl + transition metal ions 形成金属-有机配位键Formation of metal-organic coordination bonds
***基+过渡金属离子的金属簇Metal clusters of triazolyl + transition metal ions 形成金属-有机配位键Formation of metal-organic coordination bonds
四唑基+过渡金属离子的金属簇Metal clusters of tetrazolyl + transition metal ions 形成金属-有机配位键Formation of metal-organic coordination bonds
吡唑基+过渡金属离子的金属簇Metal clusters of pyrazolyl + transition metal ions 形成金属-有机配位键Formation of metal-organic coordination bonds
氨基+醛基Amino + aldehyde 形成碳氮双键carbon nitrogen double bond
邻苯二酚羟基+硼酸基Catechol hydroxyl group + boric acid group 形成硼酸酯borate formation
氨基+硝基amino + nitro 氮氮双键nitrogen nitrogen double bond
醛基+邻苯二氨基Aldehyde + o-phenylenediamino 咪唑imidazole
酰氯+氨基Acid chloride + amino 酰胺Amide
邻苯二酸酐基+氨基Phthalic anhydride group + amino group 邻苯二酰亚胺Phthalimide
氰基+醛基cyano + aldehyde 碳碳双键carbon carbon double bond
邻苯醌基+邻苯二胺基o-benzoquinone group + o-phenylenediamine group 吩嗪Phenazine
邻苯二酚羟基+邻二氟苯基Catechol hydroxyl + o-difluorophenyl 二恶英Dioxins
进一步地，所述金属离子的原子量为75-210。Further, the atomic weight of the metal ion is 75-210.
进一步地，所述接枝剂是双官能度的线性结构分子，头部一端与有机配体或者连接子上的相应取代基团通过点击反应成键结合，末尾一端与抗体上的氨基或者二硫键还原的巯基通过点击反应成键结合。点击反应是一类反应条件温和、在较短时间内有高反应效率的反应，Further, the grafting agent is a bifunctional linear structure molecule, one end of the head is bonded with the corresponding substituent group on the organic ligand or linker through a click reaction, and the end end is bonded with the amino group or disulfide group on the antibody. The bond-reduced sulfhydryl group is bonded by a click reaction. Click reaction is a type of reaction with mild reaction conditions and high reaction efficiency in a short period of time.
进一步地，所述接枝剂末尾一端的基团为N-羟基琥珀酰亚胺基，五氟苯酚酯基、羧基、醛基、马来酰亚胺基，乙烯基砜基等。其中，N-羟基琥珀酰亚胺基、五氟苯酚酯基、马来酰亚胺基，乙烯基砜基结构如下所示：Further, the group at the end of the grafting agent is N-hydroxysuccinimide group, pentafluorophenol ester group, carboxyl group, aldehyde group, maleimide group, vinyl sulfone group and the like. Among them, the structures of N-hydroxysuccinimide group, pentafluorophenol ester group, maleimide group and vinyl sulfone group are as follows:
Figure PCTCN2022103548-appb-000004
Figure PCTCN2022103548-appb-000004
进一步地，所述接枝剂头部与有机配体或者连接子的基团组合如表2所示。Further, the combination of the head of the grafting agent and the organic ligand or linker is shown in Table 2.
表2接枝剂头部与框架表面点击反应的基团组合Table 2 Group combination of grafting agent head and framework surface click reaction
有机配体或者连接子的取代基Substituents for organic ligands or linkers 接枝剂头部grafting agent head
氨基Amino N-羟基琥珀酰亚胺基，五氟苯酚酯基、羧基、醛基N-hydroxysuccinimide group, pentafluorophenol ester group, carboxyl group, aldehyde group
巯基Mercapto 马来酰亚胺基，乙烯基砜基Maleimide group, vinyl sulfone group
羧基carboxyl 氨基Amino
叠氮基Azido 炔基Alkynyl
炔基Alkynyl 叠氮基Azido
羟基hydroxyl 氯代硅氧烷基Cl-Si(CH 3) 2- Chlorosiloxane Cl-Si(CH 3 ) 2 -
所述接枝剂头部与抗体点击反应的基团组合如表3所示The group combination of the grafting agent head and the antibody click reaction is shown in Table 3
表3接枝剂尾部与抗体点击反应的基团组合Table 3 Group combination of grafting agent tail and antibody click reaction
抗体基团Antibody group 接枝剂尾部基团grafting agent tail group
氨基Amino N-羟基琥珀酰亚胺基，五氟苯酚酯基、羧基、醛基N-hydroxysuccinimide group, pentafluorophenol ester group, carboxyl group, aldehyde group
巯基Mercapto 马来酰亚胺基，乙烯基砜基Maleimide group, vinyl sulfone group
进一步地，所述配位网络框架直径为20-200nm。Further, the diameter of the coordination network framework is 20-200nm.
本发明通过理性设计合成制备出能够结合金属的框架结构纳米颗粒材料，作为抗体的金属标记物，用于流式质谱的单细胞蛋白检测的标记材料。The invention synthesizes and prepares the frame structure nanoparticle material capable of binding metals through rational design, which is used as the metal marker of the antibody and is used as a marker material for single-cell protein detection of flow mass spectrometry.
本发明还提供了一种上述纳米颗粒的制备方法，包括以下步骤：The present invention also provides a method for preparing the above nanoparticles, comprising the following steps:
(1)合成纳米框架结构：将1-30重量份的有机配体和0.5-10重量份的连接子、500-3000重量份的有机溶剂混合，加热到35-150℃，搅拌反应0.5-24小时，得到含有纳米框架结构的分散液，再往合成好的纳米框架分散液中加入0.1-1重量份的金属盐，加热到35-120℃，搅拌反应0.5-24小时，得到含有金属的纳米框架材料分散液；(1) Synthesis of nanoframe structure: Mix 1-30 parts by weight of organic ligands, 0.5-10 parts by weight of linkers, and 500-3000 parts by weight of organic solvents, heat to 35-150 ° C, and stir for 0.5-24 hours, to obtain a dispersion containing a nanoframe structure, and then add 0.1-1 parts by weight of a metal salt to the synthesized nanoframe dispersion, heat to 35-120 ° C, stir and react for 0.5-24 hours, and obtain a metal-containing nano frame material dispersion;
或直接将1-30重量份的含有金属离子的有机配体和0.5-10重量份的连接子、500-3000重量份的有机溶剂混合，加热到35-150℃，搅拌反应0.5-24小时，得到含有金属的纳米框架材料分散液。其中，有机溶剂为可以溶解参与反应的有机配体和连接子的试剂。Or directly mix 1-30 parts by weight of organic ligands containing metal ions, 0.5-10 parts by weight of linkers, and 500-3000 parts by weight of organic solvents, heat to 35-150 ° C, and stir for 0.5-24 hours to react, A metal-containing nanoframe material dispersion is obtained. Wherein, the organic solvent is a reagent that can dissolve the organic ligands and linkers participating in the reaction.
(2)连接抗体：将含有金属的纳米框架材料分散液离心分离去除有机溶剂，加20-100重量份的去离子水重分散，再往含有金属的纳米框架材料的水分散液中加入0.2-5重量份的接枝剂，室温下反应0.5-12小时，透析分离游离的接枝剂，再加入0.01-0.1重量份的抗体，室温下反应0.5-2小时，再透析分离游离的抗体，得到所述纳米颗粒。(2) Connected antibody: centrifuge the metal-containing nanoframe material dispersion to remove the organic solvent, add 20-100 parts by weight of deionized water to redisperse, and then add 0.2- 5 parts by weight of grafting agent, reacted at room temperature for 0.5-12 hours, dialyzed to separate the free grafting agent, then added 0.01-0.1 parts by weight of antibody, reacted at room temperature for 0.5-2 hours, and then dialyzed to separate the free antibody to obtain the nanoparticles.
本发明的创新点及有益效果：Innovations and beneficial effects of the present invention:
1.高负载量：通过纳米框架负载金属，相比现有的聚合物线性链的金属载体能够极大提高金属负载量，进而提高检测灵敏度。目前商用的聚合物抗体标签，一条链上最多仅能负载100个左右金属离子，而一个抗体平均连接3条链，根据流式质谱的检测限，一个细胞上至少需要上万个金属离子的信号，对应一个细胞至少结合上百个抗体才能有标记效果，意味着对于低表达蛋白的检测有着很大限制；而本发明的三维框架结构极大提高金属负载量后，单个标签的金属负载量至少提高一个数量级，对于一个细胞结合十几个甚至更少的框架结构抗体标签就能得到同等的灵敏度，为低丰度蛋白表达的检测提供了便利。1. High loading capacity: By loading metals with nanoframes, compared with the existing metal carriers of polymer linear chains, the metal loading capacity can be greatly increased, thereby improving the detection sensitivity. At present, commercial polymer antibody tags can only carry about 100 metal ions on one chain, and an antibody is connected to three chains on average. According to the detection limit of flow cytometry, a cell needs at least tens of thousands of metal ion signals , corresponding to a cell with at least hundreds of antibodies to have a labeling effect, which means that there is a great limit to the detection of low-expression proteins; and after the three-dimensional framework structure of the present invention greatly improves the metal loading, the metal loading of a single label is at least Increased by an order of magnitude, the same sensitivity can be obtained for a cell combined with a dozen or even fewer framework antibody tags, which facilitates the detection of low-abundance protein expression.
2.方便更多种类金属的负载：本发明制备的框架材料，首先框架结构的类型组合灵活多变，可以针对不同的金属设计采用含有合适功能基团的结构单元，从而适应多种金属元素；其次，由于金属是在框架结构内部结合，不可能在纳米颗粒之间形成交联点，从而避免了现有的聚合物线性链的金属载体结合某些金属离子造成的交联聚集问题。2. Facilitate the loading of more kinds of metals: the frame material prepared by the present invention, first of all, the type combination of the frame structure is flexible and changeable, and structural units containing suitable functional groups can be designed for different metals, so as to adapt to various metal elements; Secondly, since the metal is combined inside the framework structure, it is impossible to form cross-linking points between nanoparticles, thereby avoiding the problem of cross-linking and aggregation caused by the metal carrier of the existing polymer linear chain combined with certain metal ions.
综上所述，本发明的框架结构纳米颗粒，内部有机配体的结构对于多种金属元素都有很强的结合能力，一方面能够极大增加金属负载量提高信号灵敏度，另一方面能够方便的以一种框架结构负载不同的金属同位素，尤其是镧系以外的金属元素，极大拓展现有可用的金属 同位素的检测通道，为流式质谱单细胞蛋白的多通道高精度检测提供关键新材料。To sum up, the frame structure nanoparticles of the present invention, the structure of the internal organic ligand has a strong binding ability for a variety of metal elements, on the one hand, it can greatly increase the metal loading and improve the signal sensitivity, on the other hand, it can facilitate Loading different metal isotopes in a frame structure, especially metal elements other than lanthanides, greatly expands the available detection channels of metal isotopes, and provides key new technologies for the multi-channel high-precision detection of single-cell proteins by flow cytometry. Material.
附图说明Description of drawings
图1为本发明能够结合金属的框架结构的金属抗体标签的制备过程示意图；Fig. 1 is a schematic diagram of the preparation process of the metal antibody tag capable of binding to the metal frame structure of the present invention;
图2为邻苯二巯基结构的有机配体结合金属示意图；Fig. 2 is the organic ligand binding metal schematic diagram of o-phenylenedithiol structure;
图3为联吡啶结构的有机配体结合金属示意图；Figure 3 is a schematic diagram of the organic ligand binding metal of the bipyridine structure;
图4为2，2’-二酚羟基-联苯结构的有机配体结合金属示意图；Fig. 4 is the organic ligand binding metal schematic diagram of 2,2'-diphenolic hydroxyl-biphenyl structure;
图5为双水杨醛缩二胺结构的有机配体结合金属示意图；Fig. 5 is the schematic diagram of the organic ligand binding metal of the double salicylaldehyde diamine structure;
图6为卟啉结构的有机配体结合金属示意图；Fig. 6 is the organic ligand binding metal schematic diagram of porphyrin structure;
图7为脱六氢三苯环烯结构的有机配体结合金属示意图；Figure 7 is a schematic diagram of the organic ligand binding metal of the hexahydrotriphenylcycloene structure;
图8为实施例1的有机配体、接枝剂、框架纳米颗粒的重复结构单元的化学结构示意图；8 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, grafting agent, and framework nanoparticles of Example 1;
图9为实施例2的有机配体、接枝剂、框架纳米颗粒的重复结构单元的化学结构示意图；9 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, grafting agent, and framework nanoparticles of Example 2;
图10为实施例3的有机配体、接枝剂、框架纳米颗粒的重复结构单元的化学结构示意图；10 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, grafting agent, and framework nanoparticles of Example 3;
图11为实施例4的有机配体、连接子、接枝剂、框架纳米颗粒的重复结构单元的化学结构示意图；11 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, linker, grafting agent, and framework nanoparticles of Example 4;
图12为实施例5的有机配体、连接子、接枝剂、框架纳米颗粒的重复结构单元的化学结构示意图；12 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, linker, grafting agent, and framework nanoparticles of Example 5;
图13为实施例6的有机配体、连接子、接枝剂、框架纳米颗粒的重复结构单元的化学结构示意图；13 is a schematic diagram of the chemical structure of the repeating structural unit of the organic ligand, linker, grafting agent, and framework nanoparticles of Example 6;
图14为实施例1，Pt-195标记的抗体PD-1对阴阳细胞的染色分群效果图；Figure 14 is a diagram of the staining and grouping effect of Pt-195-labeled antibody PD-1 on yin and yang cells in Example 1;
图15为实施例2，Sn-120标记的抗体CD-4对阴阳细胞的染色分群效果图；Figure 15 is a diagram of the staining and grouping effect of Sn-120-labeled antibody CD-4 on yin and yang cells in Example 2;
图16为实施例3，Zr-90标记的抗体lgD对阴阳细胞的染色分群效果图；Figure 16 is a diagram of the staining and grouping effect of Zr-90-labeled antibody IgD on yin and yang cells in Example 3;
图17为实施例4，Tm-169标记的抗体ICOS对阴阳细胞的染色分群效果图；Figure 17 is a diagram of the staining and grouping effect of Tm-169-labeled antibody ICOS on yin and yang cells in Example 4;
图18为实施例5，Pd-104标记的抗体TCR Va7.2对阴阳细胞的染色分群效果图；Figure 18 is a diagram of the staining and grouping effect of Pd-104-labeled antibody TCR Va7.2 on yin and yang cells in Example 5;
图19为实施例6，Yb-171标记的抗体CCR7对阴阳细胞的染色分群效果图。Fig. 19 is a graph showing the effect of staining and grouping of Yin and Yang cells by Yb-171-labeled antibody CCR7 in Example 6.
具体实施方式Detailed ways
本发明提供的含有金属元素的框架结构纳米颗粒材料，首先能够通过灵活的变换框架纳米颗粒的结构单元的功能基团，实现多种金属同位素的有效负载，使得可用的检测通道扩展到质谱可检测范围，即分子量75-210的一百多种同位素通道，实现更高水平的多参数同步标记检测。同时通过表面化学修饰偶联抗体，能够高效牢固的接枝抗体，并且不影响抗体特异性识别目标细胞群。此外，可以通过控制纳米颗粒的尺寸来调节结构单元数，从而调节金属离子负载的数量，使得一个纳米颗粒可以负载的金属离子数量能够达到10 3-10 4个，极大提高 单个金属标签的金属信号强度，有利于实现细胞上低表达的抗原蛋白标记结合后的灵敏检测。 The framework nanoparticle material containing metal elements provided by the present invention can first realize the effective loading of various metal isotopes by flexibly changing the functional groups of the structural units of the framework nanoparticles, so that the available detection channels can be extended to mass spectrometry. Range, that is, more than one hundred isotope channels with a molecular weight of 75-210, to achieve a higher level of multi-parameter simultaneous label detection. At the same time, the antibody is coupled through surface chemical modification, which can efficiently and firmly graft the antibody without affecting the specific recognition of the target cell population by the antibody. In addition, the number of structural units can be adjusted by controlling the size of the nanoparticles, thereby adjusting the number of metal ions loaded, so that the number of metal ions that can be loaded on a nanoparticle can reach 10 3 -10 4 , which greatly improves the metal density of a single metal label. The signal strength is conducive to the sensitive detection of low-expressed antigenic protein markers on cells.
具体地，本发明提供的纳米框架材料通过两步法合成得到，如图1所示，第一步是有机配体与连接子配位自组装形成框架结构，再负载金属，或者第一步直接用含有金属的有机配体与连接子自组装得到框架结构；第二步是在框架结构表面引入接枝剂，再在接枝剂末端接枝抗体，得到作为金属抗体标签的纳米颗粒。Specifically, the nanoframe material provided by the present invention is synthesized by a two-step method, as shown in Figure 1, the first step is to form a framework structure by coordinating and self-assembling the organic ligand and the linker, and then loading the metal, or the first step is directly The framework structure is obtained by self-assembly of metal-containing organic ligands and linkers; the second step is to introduce a grafting agent on the surface of the framework structure, and then graft antibodies at the end of the grafting agent to obtain nanoparticles as metal antibody labels.
其中第一步中的有机配体，其主要结合金属的部分如图2-7所示，其A取代基末端与连接子的末端基团的键合有很多种组合形式，如表1所示，从而得到多种自组装的框架结构；而金属离子可以是原子量75-210的所有金属。Among them, the organic ligand in the first step, its main metal-binding part is shown in Figure 2-7, and there are many combinations of the bonding between the end of the A substituent and the terminal group of the linker, as shown in Table 1 , so as to obtain a variety of self-assembled framework structures; and the metal ions can be all metals with an atomic weight of 75-210.
第二步中在框架表面引入接枝剂，是通过接枝剂的头基与框架上的有机配体取代基或者连接子的取代基之间的点击反应键合实现的，然后再利用接枝剂尾基与抗体上的基团通过点击反应键合，最终得到结合金属并且连接抗体的纳米框架材料。其中两步的点击反应的基团也有很多组合形式，如表2所示，能够提供很多种连接策略。In the second step, the grafting agent is introduced on the surface of the framework through the click reaction bonding between the head group of the grafting agent and the substituent of the organic ligand on the framework or the substituent of the linker. The tail group of the agent is bonded to the group on the antibody through a click reaction, and finally a nanoframe material that binds the metal and connects the antibody is obtained. There are also many combinations of groups in the two-step click reaction, as shown in Table 2, which can provide a variety of connection strategies.
下面结合具体实施例和附图对本发明作进一步说明：Below in conjunction with specific embodiment and accompanying drawing, the present invention will be further described:
实施例1Example 1
基于邻苯二巯基结合金属的框架材料(1)，由2，3-二巯基-6-叠氮基-1，4-对苯二甲酸的有机配体与锆金属簇的连接子配位自组装形成框架结构纳米颗粒，使用PtCl 4作为金属试剂将Pt负载到框架中，使用CH≡C-CH 2-CH 2-O-CH 2-MAH作为接枝剂，通过头基碳碳三键与有机配体取代基上的叠氮基键合连接，通过尾基的-MAH与抗体上经还原二硫键得到的巯基键合连接。所用的有机配体、接枝剂、得到框架纳米颗粒的重复结构单元如图8所示。具体制备方法如下： Metal-bonded framework materials (1) based on o-phthalic dimercapto-dimercapto-6-azido-1,4-terephthalic acid organic ligands coordinated with linkers of zirconium metal clusters from Assembled to form framework structure nanoparticles, using PtCl 4 as the metal reagent to load Pt into the framework, using CH≡C-CH 2 -CH 2 -O-CH 2 -MAH as the grafting agent, through the head group carbon-carbon triple bond and The azido group on the substituent of the organic ligand is bonded to the -MAH of the tail group and the sulfhydryl group obtained by reducing the disulfide bond on the antibody. The organic ligands, grafting agents, and repeating structural units used to obtain framework nanoparticles are shown in FIG. 8 . The specific preparation method is as follows:
1)将50mg的2，3-二巯基-6-叠氮基-1，4-对苯二甲酸与150mg的八水合氯氧化锆一起加入到50g的DMF溶液中充分搅拌溶解，加热至90℃，反应2小时后，再将混合液在15000xg下离心30min，吸取去掉上清液后得到框架结构的纳米颗粒。1) Add 50mg of 2,3-dimercapto-6-azido-1,4-terephthalic acid and 150mg of zirconium oxychloride octahydrate into 50g of DMF solution, stir and dissolve, and heat to 90°C After reacting for 2 hours, the mixture was centrifuged at 15000×g for 30 minutes, and the supernatant was removed by suction to obtain framework-structured nanoparticles.
2)加5g DMF重悬分散离心后收取的纳米颗粒，再加入500μL浓度为20mg/ml的PtCl 4的DMF溶液，加热至120℃，反应24小时后，再将混合液在15000xg下离心20min，吸取去掉上清液后得到负载了金属Pt的框架结构纳米颗粒。 2) Add 5 g of DMF to resuspend the collected nanoparticles after dispersing and centrifuging, then add 500 μL of PtCl 4 DMF solution with a concentration of 20 mg/ml, heat to 120 ° C, react for 24 hours, and then centrifuge the mixture at 15000 x g for 20 min, After sucking and removing the supernatant, the framework structure nanoparticles loaded with metal Pt were obtained.
3)加入2g去离子水重悬分散收取的负载了Pt的框架结构纳米颗粒，用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，彻底分离游离的有机溶剂分子和金属离子；然后加入10mg接枝剂CH≡C-CH 2-CH 2-O-CH 2-MAH，充分混合溶解后室温反应1小时；用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时；在透析的同时准备好抗体二硫键的还原：对100μg的抗体，加入100μL的4mM 的TCEP在PBS buffer中反应0.5小时，再用3KD的截留管12000xg离心30min分离TCEP，再加入300μL的PBS buffer重悬。 3) Add 2 g of deionized water to resuspend and disperse the collected Pt-loaded framework structure nanoparticles, and use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400 ml of deionized water for at least 2 hours each time to completely separate free organic matter. Solvent molecules and metal ions; then add 10mg of grafting agent CH≡C-CH 2 -CH 2 -O-CH 2 -MAH, fully mix and dissolve, and react at room temperature for 1 hour; use a dialysis bag with a molecular weight cut-off of 300KD, remove Dialyze 3 times in deionized water, at least 2 hours each time; prepare for the reduction of antibody disulfide bonds while dialysis: For 100 μg of antibody, add 100 μL of 4 mM TCEP to react in PBS buffer for 0.5 hours, and then use a 3KD retention tube Centrifuge at 12000xg for 30min to separate TCEP, then add 300μL of PBS buffer to resuspend.
4)将透析除去多余接枝剂的框架结构纳米颗粒和还原了二硫键的抗体混合，室温下反应1小时，最后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，分离游离抗体，最终得到连接了抗体的负载Pt的框架结构纳米颗粒。4) Mix the framework-structured nanoparticles that have been dialyzed to remove excess grafting agent and the antibody that has reduced the disulfide bond, react at room temperature for 1 hour, and finally use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400ml of deionized water, Each time for at least 2 hours, the free antibody was separated to finally obtain the antibody-linked Pt-loaded framework nanoparticle.
实施例2Example 2
基于联吡啶结合金属的框架材料(2)，由2,2'-联吡啶-5,5'-对二苯甲酸与锆金属簇的连接子配位自组装形成框架结构纳米颗粒，使用SnCl 2·2H 2O作为金属试剂将Sn负载到框架中，使用Cl-Si(CH 3) 2-CH 2-CH 2-O-CH 2-NHS作为接枝剂，通过头基Cl-Si(CH 3) 2-与锆金属簇上的羟基键合连接，通过尾基的-NHS基团与抗体上的氨基键合连接。所用的有机配体、接枝剂、得到框架纳米颗粒的重复结构单元如图9所示。具体制备方法如下： Framework materials based on bipyridyl-bonded metals (2), self-assembled by linkers of 2,2'-bipyridine-5,5'-terephthalic acid and zirconium metal clusters to form framework-structured nanoparticles, using SnCl 2 2H 2 O was used as the metal reagent to load Sn into the framework, using Cl-Si(CH 3 ) 2 -CH 2 -CH 2 -O-CH 2 -NHS as the grafting agent, through the head group Cl-Si(CH 3 ) 2 - bonded to the hydroxyl group on the zirconium metal cluster, and bonded to the amino group on the antibody through the -NHS group of the tail group. The organic ligands, grafting agents, and repeating structural units used to obtain framework nanoparticles are shown in FIG. 9 . The specific preparation method is as follows:
1)将30mg的2,2'-联吡啶-5,5'-对二苯甲酸与150mg的八水合氯氧化锆一起加入到50g的DMF溶液中充分搅拌溶解，加热至90℃，反应5小时后，再将混合液在15000xg下离心30min，吸取去掉上清液后得到框架结构的纳米颗粒。1) Add 30mg of 2,2'-bipyridine-5,5'-tere-dibenzoic acid and 150mg of zirconium oxychloride octahydrate into 50g of DMF solution, stir and dissolve, heat to 90°C, and react for 5 hours Finally, the mixture was centrifuged at 15000×g for 30 min, and the supernatant was removed by suction to obtain framework-structured nanoparticles.
2)加5g DMF重悬分散离心后收取的纳米颗粒，再加入500μL浓度为20mg/ml的SnCl 2.2H 2O的DMF溶液，加热至35℃，反应0.5小时后，再将混合液在15000xg下离心20min，吸取去掉上清液后得到负载了金属Sn的框架结构纳米颗粒。 2) Add 5 g of DMF to resuspend the collected nanoparticles after dispersing and centrifuging, then add 500 μL of a DMF solution of SnCl 2 .2H 2 O with a concentration of 20 mg/ml, heat to 35 ° C, react for 0.5 hours, and then put the mixture at 15000 xg After centrifuging for 20 min, absorbing and removing the supernatant, the framework structure nanoparticles loaded with metal Sn were obtained.
3)加入2g去离子水重悬分散收取的负载了Sn的框架结构纳米颗粒，用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，彻底分离游离的有机溶剂分子和金属离子；然后加入10mg接枝剂Cl-Si(CH 3) 2-CH 2-CH 2-O-CH 2-NHS，充分混合溶解后室温反应12小时；然后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时；。 3) Add 2 g of deionized water to resuspend and disperse the collected Sn-loaded framework structure nanoparticles, and use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400 ml of deionized water for at least 2 hours each time to completely separate the free organic matter. Solvent molecules and metal ions; then add 10mg of grafting agent Cl-Si(CH 3 ) 2 -CH 2 -CH 2 -O-CH 2 -NHS, fully mix and dissolve, and react at room temperature for 12 hours; then use a molecular weight cut-off of 300KD Dialysis bag, dialyze 3 times in 400ml of deionized water, at least 2 hours each time;
4)将透析除去多余接枝剂的框架结构纳米颗粒和100μg抗体在100μL PBS buffer中混合，室温下反应1小时，最后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，分离游离抗体，最终得到连接了抗体的负载Sn的框架结构纳米颗粒。4) Mix the framework-structured nanoparticles and 100 μg antibody in 100 μL PBS buffer with dialysis to remove excess grafting agent, react at room temperature for 1 hour, and finally use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400ml deionized water, Each time for at least 2 hours, the free antibody was separated to finally obtain the Sn-loaded framework structure nanoparticle linked to the antibody.
实施例3Example 3
基于2，2’-二酚羟基-联苯结合金属的框架材料(3)，由2,2-二酚羟基-5,5-二炔基-1,1’-联苯-4,4’-联吡啶与锌离子配位自组装形成框架结构的纳米颗粒，使用ZrCl 4作为金属试剂将Zr负载到框架中，使用N 3-CH 2-CH 2-O-CH 2-NHS作为接枝剂，通过头基叠氮基与有机配体取代基上的碳碳三键进行键合连接，通过尾基的N-羟基琥珀酰亚胺基与抗体上的氨基键合连接。所用的有机配体、接枝剂、得到框架纳米颗粒的重复结构单元如图10所示。具体制备方法如 下： 2,2'-diphenolic hydroxyl-biphenyl-bonded metal-based framework material (3), composed of 2,2-diphenolic hydroxyl-5,5-diynyl-1,1'-biphenyl-4,4' - Bipyridine and zinc ions coordinate self-assembled to form nanoparticles with a framework structure, use ZrCl 4 as a metal reagent to load Zr into the framework, and use N 3 -CH 2 -CH 2 -O-CH 2 -NHS as a grafting agent , bonded to the carbon-carbon triple bond on the substituent of the organic ligand through the azido group of the head group, and bonded to the amino group on the antibody through the N-hydroxysuccinimide group of the tail group. The organic ligands, grafting agents, and repeating structural units used to obtain framework nanoparticles are shown in FIG. 10 . The specific preparation method is as follows:
1)将50mg的2,2-二酚羟基-5,5-二炔基-1,1’-联苯-4,4’-联吡啶与150mg的硝酸锌一起加入到50ml的甲醇溶液中充分搅拌溶解，加热至35℃，反应0.5小时后，再将混合液在15000xg下离心30min，吸取去掉上清液后得到框架结构的纳米颗粒。1) Add 50mg of 2,2-diphenol hydroxy-5,5-diynyl-1,1'-biphenyl-4,4'-bipyridyl together with 150mg of zinc nitrate to 50ml of methanol solution Stir to dissolve, heat to 35° C., react for 0.5 hours, then centrifuge the mixture at 15,000×g for 30 minutes, absorb and remove the supernatant to obtain framework-structured nanoparticles.
2)加5g DMSO重悬分散离心后收取的纳米颗粒，再加入500μL浓度为20mg/ml的ZrCl 4的DMF溶液，加热至45℃，反应12小时后，再将混合液在15000xg下离心20min，吸取去掉上清液后得到负载了金属Zr的框架结构纳米颗粒。 2) Add 5 g of DMSO to resuspend and disperse the collected nanoparticles after centrifugation, then add 500 μL of a DMF solution of ZrCl 4 with a concentration of 20 mg/ml, heat to 45° C., react for 12 hours, and then centrifuge the mixture at 15,000×g for 20 min. After sucking and removing the supernatant, the framework structure nanoparticles loaded with metal Zr were obtained.
3)加入2g去离子水重悬分散收取的负载了Zr的框架结构纳米颗粒，用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，彻底分离游离的有机溶剂分子和金属离子；然后加入10mg接枝剂N 3-CH 2-CH 2-O-CH 2-NHS，充分混合溶解后室温反应2小时；然后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时； 3) Add 2g of deionized water to resuspend and disperse the collected Zr-loaded framework structure nanoparticles, use a dialysis bag with a molecular weight cut-off of 300KD, dialyze in 400ml of deionized water for 3 times, at least 2 hours each time, and completely separate the free organic matter. Solvent molecules and metal ions; then add 10mg of grafting agent N 3 -CH 2 -CH 2 -O-CH 2 -NHS, fully mix and dissolve, and react at room temperature for 2 hours; then use a dialysis bag with a molecular weight cut-off of 300KD to Dialysis in deionized water 3 times, at least 2 hours each time;
将透析除去多余接枝剂的框架结构纳米颗粒和100μg抗体在100μL PBS buffer中混合，室温下反应1小时，最后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，分离游离抗体，最终得到连接了抗体的负载Zr的框架结构纳米颗粒。Mix the framework-structured nanoparticles and 100 μg antibody in 100 μL PBS buffer with dialysis to remove excess grafting agent, react at room temperature for 1 hour, and finally use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400ml deionized water, each time For at least 2 hours, free antibody was isolated, resulting in antibody-attached Zr-loaded framework nanoparticles.
实施例4Example 4
基于双水杨醛缩二胺结合金属的框架材料(4)，由四(3-醛基-4-酚羟基苯基)甲烷和4-巯基-1,2二苯胺通过醛基与氨基的缩合反应自组装得到框架结构的纳米颗粒，使用TmCl 3作为金属试剂将Tm负载到框架中，使用MAH-CH 2-CH 2-O-CH 2-COOH作为接枝剂，通过头基-MAH与连接子取代基上的巯基进行键合连接，通过尾基的羧基在N,N'-二异丙基碳二亚胺催化下与抗体的氨基键合连接。所用的有机配体、连接子、接枝剂、得到框架纳米颗粒的重复结构单元如图11所示。具体制备方法如下： Metal-bonded framework material (4) based on bis-salicylaldehyde diamine, formed from tetrakis(3-formyl-4-phenolhydroxyphenyl)methane and 4-mercapto-1,2-diphenylamine via condensation of aldehyde groups with amino groups Nanoparticles with framework structure were obtained by reactive self-assembly, Tm was loaded into the framework using TmCl 3 as the metal reagent, MAH-CH 2 -CH 2 -O-CH 2 -COOH was used as the grafting agent, and the head group -MAH was connected with The sulfhydryl group on the substituent is bonded and connected, and the carboxyl group of the tail group is bonded to the amino group of the antibody under the catalysis of N,N'-diisopropylcarbodiimide. The used organic ligands, linkers, grafting agents, and repeating structural units to obtain framework nanoparticles are shown in FIG. 11 . The specific preparation method is as follows:
1)将50mg的四(3-醛基-4-酚羟基苯基)甲烷与120mg的4-巯基-1,2二苯胺一起加入到100g的DMF溶液中充分搅拌溶解，加热至120℃，反应12小时后，再将混合液在15000xg下离心30min，吸取去掉上清液后得到框架结构的纳米颗粒。1) Add 50mg of tetrakis(3-formyl-4-phenolhydroxyphenyl)methane and 120mg of 4-mercapto-1,2-diphenylamine into 100g of DMF solution, stir and dissolve, heat to 120°C, react After 12 hours, the mixture was centrifuged at 15000×g for 30 min, and the supernatant was removed by suction to obtain nanoparticles with a framework structure.
2)加5g水重悬分散离心后收取的纳米颗粒，再加入500μL浓度为20mg/ml的TmCl 3的水溶液，加热至60℃，反应6小时后，再将混合液在15000xg下离心20min，吸取去掉上清液后得到负载了金属Tm的框架结构纳米颗粒。 2) Add 5 g of water to resuspend the collected nanoparticles after dispersing and centrifuging, then add 500 μL of an aqueous solution of TmCl 3 with a concentration of 20 mg/ml, heat to 60° C., and react for 6 hours, then centrifuge the mixture at 15,000×g for 20 minutes, absorb After removing the supernatant, the framework structure nanoparticles loaded with metal Tm were obtained.
3)加入2g去离子水重悬分散收取的负载了Tm的框架结构纳米颗粒，用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，彻底分离游离的有机溶剂分子和金属离子；然后加入10mg接枝剂MAH-CH 2-CH 2-O-CH 2-COOH，充分混合溶解 后室温反应1小时；然后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时。 3) Add 2 g of deionized water to resuspend and disperse the collected Tm-loaded framework structure nanoparticles, and use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400 ml of deionized water for at least 2 hours each time to completely separate free organic matter. Solvent molecules and metal ions; then add 10mg of grafting agent MAH-CH 2 -CH 2 -O-CH 2 -COOH, fully mix and dissolve, and react at room temperature for 1 hour; then use a dialysis bag with a molecular weight cut-off of 300KD to remove Dialyze in deionized water 3 times, at least 2 hours each time.
将透析除去多余接枝剂的框架结构纳米颗粒和100μg抗体在100μL PBS buffer中混合，并加入2μL 0.2M的N,N'-二异丙基碳二亚胺的DMSO溶液，混匀后在室温下反应2小时，最后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，分离游离抗体，最终得到连接了抗体的负载Tm的框架结构纳米颗粒。Mix the framework-structured nanoparticles that were dialyzed to remove excess grafting agent and 100 μg antibody in 100 μL PBS buffer, and add 2 μL 0.2M N,N'-diisopropylcarbodiimide in DMSO solution, mix well and then store at room temperature After reacting for 2 hours, finally use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400ml of deionized water for at least 2 hours each time to separate the free antibody, and finally obtain the Tm-loaded framework structure nanoparticles connected to the antibody.
实施例5Example 5
基于卟啉结合金属的框架材料(5)，由钯负载的5,10,15,20-四(4-氨基苯基)卟啉和四(3-炔基-4-醛基苯基)甲烷自组装得到框架结构的纳米颗粒，使用Pd(NO 3) 3作为金属试剂将Pd负载到框架中，使用N 3-CH 2-CH 2-O-CH 2-MAH作为接枝剂，通过头基叠氮基与连接子取代基上的碳碳三键进行键合连接，通过尾基的-MAH与抗体上经还原二硫键得到的巯基键合连接。所用的有机配体、连接子、接枝剂、得到框架纳米颗粒的重复结构单元如图12所示。具体制备方法如下： Porphyrin-bound metal-based framework materials (5), 5,10,15,20-tetrakis(4-aminophenyl)porphyrin and tetrakis(3-alkynyl-4-formylphenyl)methane supported on palladium Self-assembled nanoparticles with a framework structure, using Pd(NO 3 ) 3 as the metal reagent to load Pd into the framework, using N 3 -CH 2 -CH 2 -O-CH 2 -MAH as the grafting agent, through the head group The azido group is bonded to the carbon-carbon triple bond on the substituent of the linker, and the -MAH of the tail group is bonded to the sulfhydryl group obtained by reducing the disulfide bond on the antibody. The used organic ligands, linkers, grafting agents, and repeating structural units to obtain framework nanoparticles are shown in FIG. 12 . The specific preparation method is as follows:
1)将50mg的钯负载的5,10,15,20-四(4-氨基苯基)卟啉与120mg的四(3-炔基-4-醛基苯基)甲烷一起加入到100ml的DMF溶液中充分搅拌溶解，加热至150℃，反应24小时后，再将混合液在15000xg下离心30min，吸取去掉上清液后得到负载了金属Pd的框架结构的纳米颗粒。1) 50 mg of palladium-loaded 5,10,15,20-tetrakis(4-aminophenyl) porphyrin and 120 mg of tetrakis(3-alkynyl-4-formylphenyl)methane were added to 100 ml of DMF Fully stir and dissolve in the solution, heat to 150° C., react for 24 hours, then centrifuge the mixture at 15,000×g for 30 minutes, absorb and remove the supernatant to obtain metal Pd-loaded nanoparticles with a framework structure.
2)加入2g去离子水重悬分散收取的负载了Pd的框架结构纳米颗粒，用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，彻底分离游离的有机溶剂分子和金属离子；然后加入10mg的接枝剂N 3-CH 2-CH 2-O-CH 2-MAH，充分混合溶解后室温反应1小时；然后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时；在透析的同时准备好抗体二硫键的还原，对100μg的抗体，加入100μL的4mM的TCEP在PBS buffer中反应半小时，再用3KD的截留管12000xg离心30min分离TCEP，再加入300μL的PBS buffer重悬。 2) Add 2 g of deionized water to resuspend and disperse the collected Pd-loaded framework structure nanoparticles, use a dialysis bag with a molecular weight cut-off of 300KD, dialyze 3 times in 400 ml of deionized water, at least 2 hours each time, and completely separate the free organic matter. Solvent molecules and metal ions; then add 10mg of grafting agent N 3 -CH 2 -CH 2 -O-CH 2 -MAH, fully mix and dissolve, and react at room temperature for 1 hour; then use a dialysis bag with a molecular weight cut-off of 300KD, Dialyze three times in deionized water, at least 2 hours each time; prepare for the reduction of antibody disulfide bonds while dialysis, add 100 μL of 4 mM TCEP to PBS buffer for half an hour for 100 μg of antibody, and then use 3KD Centrifuge the retention tube at 12000xg for 30 minutes to separate TCEP, then add 300 μL of PBS buffer to resuspend.
将透析除去多余接枝剂的框架结构纳米颗粒和还原了二硫键的抗体混合，室温下反应1小时，最后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，分离游离抗体，最终得到连接了抗体的负载Tm的框架结构纳米颗粒。Mix the framework-structured nanoparticles that have been dialyzed to remove the excess grafting agent and the antibody that has reduced the disulfide bond, react at room temperature for 1 hour, and finally use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400ml of deionized water, each time For at least 2 hours, free antibody was isolated, resulting in antibody-linked Tm-loaded framework nanoparticles.
实施例6Example 6
基于脱六氢三苯环烯结合金属的框架材料(6)，由脱氢苯并环戊烯(DBA)与四-(3-巯基-4-二羟基硼基苯基)甲烷(TBPM)通过酚羟基与硼酸基缩合反应自组装得到框架结构的纳米颗粒，使用YbCl 3作为金属试剂将Yb负载到框架中，使用MAH-CH 2-CH 2-O-CH 2-NHS 作为接枝剂，通过头基的-MAH基团与连接子上的取代基巯基键合连接，通过尾基的环氧基与抗体上的氨基键合连接。所用的有机配体、连接子、接枝剂、得到框架纳米颗粒的重复结构单元如图13所示。具体制备方法如下： A dehexahydrotriphenylcyclene-bonded metal-based framework material (6), composed of dehydrobenzocyclopentene (DBA) and tetrakis-(3-mercapto-4-dihydroxyborylphenyl)methane (TBPM) via The phenolic hydroxyl group and boronic acid group condensation reaction self-assembled to obtain the framework structure nanoparticles, YbCl 3 was used as the metal reagent to load Yb into the framework, and MAH-CH 2 -CH 2 -O-CH 2 -NHS was used as the grafting agent, by The -MAH group of the head group is bonded to the sulfhydryl group of the substituent on the linker, and is bonded to the amino group of the antibody through the epoxy group of the tail group. The used organic ligands, linkers, grafting agents, and repeating structural units to obtain framework nanoparticles are shown in FIG. 13 . The specific preparation method is as follows:
1)将50mg的脱氢苯并环戊烯(DBA)与100mg的四-(2-巯基-4-二羟基硼基苯基)甲烷(TBPM)一起加入到150g的DMF溶液中充分搅拌溶解，加热至150℃，反应24小时后，再将混合液在15000xg下离心30min，吸取去掉上清液后得到框架结构的纳米颗粒。1) 50 mg of dehydrobenzocyclopentene (DBA) and 100 mg of tetrakis-(2-mercapto-4-dihydroxyborylphenyl) methane (TBPM) were added to 150 g of DMF solution and fully stirred to dissolve, After heating to 150° C. and reacting for 24 hours, the mixture was centrifuged at 15,000×g for 30 minutes, and the supernatant was removed by suction to obtain framework-structured nanoparticles.
2)加5g水重悬分散离心后收取的纳米颗粒，再加入500μL浓度为20mg/ml的YbCl 3的水溶液，加热至60℃，反应6小时后，再将混合液在15000xg下离心20min，吸取去掉上清液后得到负载了金属Yb的框架结构纳米颗粒。 2) Add 5 g of water to resuspend the collected nanoparticles after dispersing and centrifuging, then add 500 μL of an aqueous solution of YbCl 3 with a concentration of 20 mg/ml, heat to 60° C., and react for 6 hours. After removing the supernatant, the framework structure nanoparticles loaded with metal Yb were obtained.
3)加入2g去离子水重悬分散收取的负载了Yb的框架结构纳米颗粒，用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，彻底分离游离的有机溶剂分子和金属离子；然后加入10mg接枝剂MAH-CH 2-CH 2-O-CH 2-NHS，充分混合溶解后室温反应1小时；然后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时。 3) Add 2 g of deionized water to resuspend and disperse the collected Yb-loaded framework structure nanoparticles, and use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400 ml of deionized water for at least 2 hours each time to completely separate free organic matter. Solvent molecules and metal ions; then add 10mg of grafting agent MAH-CH 2 -CH 2 -O-CH 2 -NHS, fully mix and dissolve, and react at room temperature for 1 hour; then use a dialysis bag with a molecular weight cut-off of 300KD to remove Dialyze in deionized water 3 times, at least 2 hours each time.
4)将透析除去多余接枝剂的框架结构纳米颗粒和100μg抗体在100μL PBS buffer中混合，室温下反应1小时，最后再用截留分子量300KD的透析袋，在400ml的去离子水中透析3次，每次至少2小时，分离游离抗体，最终得到连接了抗体的负载Yb的框架结构纳米颗粒。4) Mix the framework-structured nanoparticles and 100 μg antibody in 100 μL PBS buffer with dialysis to remove excess grafting agent, react at room temperature for 1 hour, and finally use a dialysis bag with a molecular weight cut-off of 300KD to dialyze 3 times in 400ml deionized water, Each time for at least 2 hours, the free antibody was separated to finally obtain the antibody-linked Yb-loaded framework nanoparticle.
利用纳米粒度电位分析仪Zetasizer Nano-ZS测实施例1-6得到的纳米颗粒的粒径和粒径分布，并将实施例1-6得到的纳米颗粒的水分散液静置六个月后观察其有无聚集和沉降判断稳定性好坏，结果如表4所示，表明：各个粒径下的纳米颗粒都具有较窄的尺寸分布和良好的分散稳定性。Utilize the nanoparticle size potential analyzer Zetasizer Nano-ZS to measure the particle size and particle size distribution of the nanoparticles obtained in embodiment 1-6, and observe the aqueous dispersion of the nanoparticles obtained in embodiments 1-6 after standing for six months The stability is judged by the presence or absence of aggregation and sedimentation, and the results are shown in Table 4, which shows that the nanoparticles under each particle size have a narrow size distribution and good dispersion stability.
表4框架结构的纳米颗粒的粒径、分布和分散稳定性The particle size, distribution and dispersion stability of the nanoparticles of the framework structure in Table 4
实施例Example 平均粒径/nmAverage particle size/nm 粒径分布(Cv/％)Particle size distribution (Cv/%) 分散稳定性 dispersion stability
11 100100 6％6% 好good
22 4040 8％8% 好good
33 6060 7％7% 好good
44 8080 6％6% 好good
55 2020 10％10% 好good
66 200200 8％8% 好good
利用ICP-MS测试样品液中的金属离子浓度，进而计算得到单个纳米颗粒的金属负载量，结果如表5所示，表明纳米颗粒能够成功负载目标金属离子并且能够通过增加粒径将负载数 量提高到10 3-10 4个。 The metal ion concentration in the sample liquid was tested by ICP-MS, and then the metal loading amount of a single nanoparticle was calculated. The results are shown in Table 5, indicating that the nanoparticle can successfully load the target metal ion and the loading amount can be increased by increasing the particle size. to 10 3 -10 4 pcs.
表5框架结构的纳米颗粒金属抗体标签的金属负载量Table 5 The metal loading capacity of the nanoparticle metal antibody label of the framework structure
Figure PCTCN2022103548-appb-000005
Figure PCTCN2022103548-appb-000005
利用表6中的6种胞外抗体使用6个同位素检测通道，通过对1：1混合的细胞进行标记染色检测分群效果。Use the 6 kinds of extracellular antibodies in Table 6 and use 6 isotope detection channels to detect the grouping effect by staining the 1:1 mixed cells.
表6金属抗体标签标记结合细胞染色的实验组Table 6 Experimental groups of metal antibody labeling combined with cell staining
实施例Example 抗体Antibody 克隆号clone number 标记金属同位素labeled metal isotope
11 PD-1PD-1 EH12.2H7EH12.2H7 Pt-195Pt-195
22 CD4CD4 RPA-T4RPA-T4 Sn-120Sn-120
33 lgDlm w lA6-2lA6-2 Zr-90Zr-90
44 TCR Va7.2TCR Va7.2 3C103C10 Tm-169Tm-169
55 ICOSICOS C398.4AC398.4A Pd-104Pd-104
66 CCR7CCR7 G043H7G043H7 Yb-171Yb-171
标记细胞染色方法如下：The staining method of labeled cells is as follows:
1、准备新鲜的正常人外周血，提取免疫细胞。1. Prepare fresh normal human peripheral blood and extract immune cells.
2、将免疫细胞平均分成对照组和实验组两组，每组约2×10^6个细胞，其中对照组是2×10^6个未表达目标蛋白的阴性细胞，实验组是1×10^6个表达目标蛋白的阳性细胞与1×10^6个不表达目标蛋白的阴性细胞混合。分别用PBS重悬，调节体积至1mL，加入Rh-103，室温染色5min，区分细胞死活。2. Divide the immune cells into two groups on average, the control group and the experimental group, with about 2×10^6 cells in each group, in which the control group is 2×10^6 negative cells that do not express the target protein, and the experimental group is 1×10 ^6 positive cells expressing the target protein were mixed with 1×10^6 negative cells not expressing the target protein. Resuspend with PBS, adjust the volume to 1mL, add Rh-103, stain at room temperature for 5min, and distinguish between dead and alive cells.
3、每组加入2mL 0.5-5mg/ml牛血清白蛋白溶液，500xg离心5min，吸除上清，加入50μL封闭液(0.5μL 0.5-1.5mg/ml人免疫球蛋白溶液、0.5μL 0.5-1.5mg/ml小鼠免疫球蛋白溶液、0.5μL  0.5-1.5mg/ml大鼠免疫球蛋白溶液、0.5μL 0.5-1.5mg/ml仓鼠免疫球蛋白溶液、48μL 0.5-5mg/ml牛血清蛋白溶液)，冰上封闭20min。3. Add 2mL 0.5-5mg/ml bovine serum albumin solution to each group, centrifuge at 500xg for 5min, remove the supernatant, add 50μL blocking solution (0.5μL 0.5-1.5mg/ml human immunoglobulin solution, 0.5μL 0.5-1.5 mg/ml mouse immunoglobulin solution, 0.5μL 0.5-1.5mg/ml rat immunoglobulin solution, 0.5μL 0.5-1.5mg/ml hamster immunoglobulin solution, 48μL 0.5-5mg/ml bovine serum albumin solution) , sealed on ice for 20 minutes.
4、对照组加入50μL0.5-5mg/ml牛血清蛋白溶液作为空白对照，实验组加入50μL胞外抗体混合液(本发明的纳米粒子，抗体浓度为0.1-1mg/ml)，重悬细胞，冰上染色30min。4. Add 50 μL 0.5-5 mg/ml bovine serum albumin solution to the control group as a blank control, add 50 μL extracellular antibody mixture (nanoparticles of the present invention, antibody concentration is 0.1-1 mg/ml) to the experimental group, resuspend the cells, Stain on ice for 30min.
5、加入2mL0.5-5mg/ml牛血清白蛋白溶液，500xg离心5min，吸除上清，加入含有0.5v/v‰单细胞指示剂191/193Ir(201192B，fluidigm)的固定-破膜混合液(fix and perm buffer，fluidigm，201067)1ml，重悬细胞，4℃过夜。5. Add 2mL of 0.5-5mg/ml bovine serum albumin solution, centrifuge at 500xg for 5min, remove the supernatant, and add the fixation-permeabilization mixture containing 0.5v/v‰ single cell indicator 191/193Ir (201192B, fluidigm) Solution (fix and perm buffer, fluidigm, 201067) 1ml, resuspended cells, 4 ℃ overnight.
6、加入2mL0.5-5mg/ml牛血清白蛋白溶液，800xg离心5min，吸除上清，重复2次。6. Add 2mL of 0.5-5mg/ml bovine serum albumin solution, centrifuge at 800xg for 5min, remove the supernatant, and repeat twice.
7、加入2mL去离子水，800xg离心5min，吸除上清，重复2次。7. Add 2 mL of deionized water, centrifuge at 800xg for 5 min, remove the supernatant, and repeat 2 times.
8、样品过滤，细胞计数，调整体积，准备上机，进行质谱流式检测。8. Filter the sample, count the cells, adjust the volume, prepare for the machine, and perform mass spectrometry flow detection.
6种抗体染色效果结果如图14-19所示，横坐标Ir191表示染色DNA的情况，证明是细胞信号；纵坐标是对应的同位素信号强度，根据对照组得到背景信号的纵坐标值强弱，由此得到阴阳细胞的分界线，即框中圈出来的是每一类抗体的阳性细胞群占细胞总数的百分比。与对照组相比，加入抗体的免疫细胞分成了阴性和阳性两个群，证明抗体标记效果好，可将目的细胞与非目的细胞分开，抗体可用于后续实验。The results of the staining effects of the six antibodies are shown in Figure 14-19. The abscissa Ir191 indicates the staining of DNA, which proves to be a cell signal; the ordinate is the intensity of the corresponding isotope signal. The ordinate value of the background signal is obtained according to the control group. Thus, the dividing line of yin and yang cells is obtained, that is, the circled in the box is the percentage of the positive cell population of each type of antibody in the total number of cells. Compared with the control group, the immune cells added with the antibody were divided into negative and positive groups, which proved that the antibody labeling effect is good, and the target cells can be separated from non-target cells, and the antibody can be used for subsequent experiments.
以上实施例仅用于说明本发明，但并不用来限定本发明的实施范围。The above embodiments are only used to illustrate the present invention, but are not intended to limit the implementation scope of the present invention.

Claims (9)

  1. 一种用于质谱流式细胞技术的基于框架结构的纳米颗粒，其特征在于，所述纳米颗粒包含金属离子、抗体和通过连接子将有机配体相互连接形成的配位网络框架，其中，金属离子结合于有机配体上；所述连接子是官能度为n的有机连接子或者配位数为n的金属簇，n大于等于2；所述抗体通过接枝剂与有机配体或者连接子连接；所述有机配体由如下中的一种或几种结构混合组成：A framework-based nanoparticle for mass spectrometry flow cytometry, characterized in that the nanoparticle contains metal ions, antibodies and a coordination network framework formed by linking organic ligands to each other, wherein the metal The ion is bound to the organic ligand; the linker is an organic linker with a functionality of n or a metal cluster with a coordination number of n, and n is greater than or equal to 2; the antibody is connected to the organic ligand or linker through a grafting agent connection; the organic ligand is composed of one or more of the following structures:
    Figure PCTCN2022103548-appb-100001
    Figure PCTCN2022103548-appb-100001
    其中，A取代基为参与连接形成配位网络的官能团，R1～R8为其它取代基。Wherein, the A substituent is a functional group participating in connection to form a coordination network, and R1-R8 are other substituents.
  2. 根据权利要求1所述的纳米颗粒，其特征在于，所述连接子为金属簇时，有机配体的A取代基末端为羧基、邻苯二酚羟基、吡啶基、吡嗪基、咪唑基、***基、四唑基或吡唑基。The nanoparticle according to claim 1, wherein when the linker is a metal cluster, the A substituent end of the organic ligand is carboxyl, catechol hydroxyl, pyridyl, pyrazinyl, imidazolyl, Triazolyl, tetrazolyl or pyrazolyl.
  3. 根据权利要求1所述的纳米颗粒，其特征在于，所述有机配体通过A取代基末端与有机连接子末端的配对反应基团组合包括氨基+醛基、邻苯二酚羟基+硼酸基、氨基+硝基、醛基+邻苯二氨基、酰氯基+氨基、邻苯二酸酐基+氨基、氰基+醛基、邻苯醌基+邻苯二氨基、邻苯二酚羟基+邻二氟苯基。The nanoparticle according to claim 1, wherein the organic ligand comprises amino+aldehyde group, catechol hydroxyl+boronic acid group, Amino + nitro, aldehyde + o-phenylenediamino, acid chloride + amino, phthalic anhydride + amino, cyano + aldehyde, o-quinone + o-phenylenediamino, catechol hydroxyl + o-di Fluorophenyl.
  4. 根据权利要求1所述的纳米颗粒，其特征在于，所述金属离子的原子量为75-210。The nanoparticle according to claim 1, characterized in that the atomic weight of the metal ion is 75-210.
  5. 根据权利要求1所述的纳米颗粒，其特征在于，所述接枝剂是双官能度的线性结构分子， 头部一端与有机配体或者连接子上的相应取代基团通过点击反应成键结合，末尾一端与抗体上的氨基或者二硫键还原的巯基通过点击反应成键结合。The nanoparticle according to claim 1, wherein the grafting agent is a bifunctional linear structure molecule, and one end of the head is bonded with the corresponding substituent group on the organic ligand or linker through a click reaction , and the end end is bonded to the amino group on the antibody or the sulfhydryl group reduced by the disulfide bond through a click reaction.
  6. 根据权利要求5所述的纳米颗粒，其特征在于，所述接枝剂末尾一端的基团为N-羟基琥珀酰亚胺基，五氟苯酚酯基、羧基、醛基、马来酰亚胺基，乙烯基砜基或氯代硅氧烷基。The nanoparticle according to claim 5, wherein the group at one end of the grafting agent is N-hydroxysuccinimide, pentafluorophenol ester, carboxyl, aldehyde, maleimide group, vinylsulfone group or chlorosiloxane group.
  7. 根据权利要求6所述的纳米颗粒，其特征在于，所述接枝剂头部与有机配体或者连接子的基团组合为The nanoparticle according to claim 6, wherein the grafting agent head is combined with an organic ligand or a linker group as
    有机配体或者连接子的取代基 接枝剂头部 氨基 N-羟基琥珀酰亚胺基，五氟苯酚酯基、羧基、醛基 巯基 马来酰亚胺基，乙烯基砜基 羧基 氨基 叠氮基 炔基 炔基 叠氮基 羟基 氯代硅氧烷基Cl-Si(CH 3) 2-
    。     Substituents for organic ligands or linkers grafting agent head Amino N-hydroxysuccinimide group, pentafluorophenol ester group, carboxyl group, aldehyde group Mercapto Maleimide group, vinyl sulfone group carboxyl Amino Azido Alkynyl Alkynyl Azido hydroxyl Chlorosiloxane Cl-Si(CH 3 ) 2 -
    .
  8. 根据权利要求1所述的纳米颗粒，其特征在于，所述配位网络框架直径为20-200nm。The nanoparticle according to claim 1, characterized in that, the diameter of the coordination network framework is 20-200nm.
  9. 一种权利要求1-8任一项所述纳米颗粒的制备方法，其特征在于，包括以下步骤：A method for preparing nanoparticles according to any one of claims 1-8, characterized in that it comprises the following steps:
    (1)合成纳米框架结构：将1-30重量份的有机配体和0.5-10重量份的连接子、500-3000重量份的有机溶剂混合，加热到35-150℃，搅拌反应0.5-24小时，得到含有纳米框架结构的分散液，再往合成好的纳米框架分散液中加入0.1-1重量份的金属盐，加热到35-120℃，搅拌反应0.5-24小时，得到含有金属的纳米框架材料分散液；(1) Synthesis of nanoframe structure: Mix 1-30 parts by weight of organic ligands, 0.5-10 parts by weight of linkers, and 500-3000 parts by weight of organic solvents, heat to 35-150 ° C, and stir for 0.5-24 hours, to obtain a dispersion containing a nanoframe structure, and then add 0.1-1 parts by weight of a metal salt to the synthesized nanoframe dispersion, heat to 35-120 ° C, and stir for 0.5-24 hours to obtain a metal-containing nano frame material dispersion;
    或直接将1-30重量份的含有金属离子的有机配体和0.5-10重量份的连接子、500-3000重量份的有机溶剂混合，加热到35-150℃，搅拌反应0.5-24小时，得到含有金属的纳米框架材料分散液；Or directly mix 1-30 parts by weight of organic ligands containing metal ions, 0.5-10 parts by weight of linkers, and 500-3000 parts by weight of organic solvents, heat to 35-150 ° C, and stir for 0.5-24 hours to react, obtaining a metal-containing nanoframe material dispersion;
    (2)连接抗体：将含有金属的纳米框架材料分散液离心分离去除有机溶剂，加20-100重量份的去离子水重分散，再往含有金属的纳米框架材料的水分散液中加入0.2-5重量份的接枝剂，室温下反应0.5-12小时，透析分离游离的接枝剂，再加入0.01-0.1重量份的抗体，室温下反应0.5-2小时，再透析分离游离的抗体，得到所述纳米颗粒。(2) Connected antibody: centrifuge the metal-containing nanoframe material dispersion to remove the organic solvent, add 20-100 parts by weight of deionized water to redisperse, and then add 0.2- 5 parts by weight of grafting agent, reacted at room temperature for 0.5-12 hours, dialyzed to separate the free grafting agent, then added 0.01-0.1 parts by weight of antibody, reacted at room temperature for 0.5-2 hours, and then dialyzed to separate the free antibody to obtain the nanoparticles.
PCT/CN2022/103548 2021-07-26 2022-07-04 Frame structure-based nanoparticle used for mass cytometry and preparation method therefor WO2023005614A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110843965.7 2021-07-26
CN202110843965.7A CN113588523B (en) 2021-07-26 2021-07-26 Frame structure-based nano-particles for mass flow cytometry and preparation method thereof

Publications (1)

Publication Number Publication Date
WO2023005614A1 true WO2023005614A1 (en) 2023-02-02

Family

ID=78249932

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/103548 WO2023005614A1 (en) 2021-07-26 2022-07-04 Frame structure-based nanoparticle used for mass cytometry and preparation method therefor

Country Status (2)

Country Link
CN (1) CN113588523B (en)
WO (1) WO2023005614A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113588523B (en) * 2021-07-26 2022-03-29 浙江大学 Frame structure-based nano-particles for mass flow cytometry and preparation method thereof

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080095857A1 (en) * 2004-03-09 2008-04-24 Lts Lohmann Therapie-Systeme Ag Lohmannstrasse 2 Carrier system in the form of protein-based nanoparticles for the cell-specific enrichment of pharmaceutically active substances
US20110135571A1 (en) * 2008-02-22 2011-06-09 Wenbin Lin Hybrid nanoparticles as anti-cancer therapeutic agents and dual therapeutic/imaging contrast agents
CN107796748A (en) * 2017-09-28 2018-03-13 上海交通大学 A kind of detection method for unicellular mass spectrum flow cytometry
CN112683985A (en) * 2020-12-28 2021-04-20 中国科学院苏州生物医学工程技术研究所 Metal label based on rare earth doped nano metal organic framework material
CN112778474A (en) * 2020-12-28 2021-05-11 中国科学院苏州生物医学工程技术研究所 Rare earth cage-shaped coordination compound metal fluorescence dual-mode label for flow cytometry and preparation method thereof
CN112834411A (en) * 2021-01-07 2021-05-25 上海交通大学 Metal nanoprobe applied to mass flow technology, preparation method and application
CN113075114A (en) * 2019-12-17 2021-07-06 北京大学 Organic mass spectrometry flow cytometry analysis technology
CN113588523A (en) * 2021-07-26 2021-11-02 浙江大学 Frame structure-based nano-particles for mass flow cytometry and preparation method thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2021793B1 (en) * 2006-05-27 2017-04-26 Fluidigm Canada Inc. Polymer backbone element tags
CN109323908A (en) * 2018-10-10 2019-02-12 浙江大学 A kind of detection method for mass spectrum streaming technology
CN110987914B (en) * 2019-11-19 2022-09-16 江苏大学 Method for detecting and distinguishing phosphorylated protein based on Zr-MOF nanoenzyme and alpha-casein quantitative detection
US10989711B1 (en) * 2020-11-25 2021-04-27 King Abdulaziz University Gold@zinc salen-based metal organic framework composite and methods of use thereof
CN112604659B (en) * 2020-11-27 2022-07-05 苏州优特创优新材料科技有限公司 Chiral ligand exchange type COF @ MOF/L composite material and preparation method thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080095857A1 (en) * 2004-03-09 2008-04-24 Lts Lohmann Therapie-Systeme Ag Lohmannstrasse 2 Carrier system in the form of protein-based nanoparticles for the cell-specific enrichment of pharmaceutically active substances
US20110135571A1 (en) * 2008-02-22 2011-06-09 Wenbin Lin Hybrid nanoparticles as anti-cancer therapeutic agents and dual therapeutic/imaging contrast agents
CN107796748A (en) * 2017-09-28 2018-03-13 上海交通大学 A kind of detection method for unicellular mass spectrum flow cytometry
CN113075114A (en) * 2019-12-17 2021-07-06 北京大学 Organic mass spectrometry flow cytometry analysis technology
CN112683985A (en) * 2020-12-28 2021-04-20 中国科学院苏州生物医学工程技术研究所 Metal label based on rare earth doped nano metal organic framework material
CN112778474A (en) * 2020-12-28 2021-05-11 中国科学院苏州生物医学工程技术研究所 Rare earth cage-shaped coordination compound metal fluorescence dual-mode label for flow cytometry and preparation method thereof
CN112834411A (en) * 2021-01-07 2021-05-25 上海交通大学 Metal nanoprobe applied to mass flow technology, preparation method and application
CN113588523A (en) * 2021-07-26 2021-11-02 浙江大学 Frame structure-based nano-particles for mass flow cytometry and preparation method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHO, H. ET AL.: "A metal-chelating polymer for chelating zirconium and its use in mass cytometry", <EUROPEAN POLYMER JOURNAL>, vol. 120, 3 August 2019 (2019-08-03), XP085894756, DOI: 10.1016/j.eurpolymj.2019.08.002 *
LI, YU ET AL.: "Multicomponent zirconium-based metal-orangic frameworks for impedimetric aptasensing of living cancer cells", <SENSORS & ACTUATORS: B. CHEMICAL>, vol. 306, 20 December 2019 (2019-12-20), XP085995225, DOI: 10.1016/j.snb.2019.127608 *
YANG YU, CHUANQIANG SUN, ZISHAN GONG, RU YANG, YAN WANG: "Application of Mass Cytometry in Single-Cell Detection", CHINESE JOURNAL OF CELL BIOLOGY, vol. 42, no. 2, 29 February 2020 (2020-02-29), pages 358 - 368, XP093029265, DOI: 10.11844/cjcb.2020.02.0022 *

Also Published As

Publication number Publication date
CN113588523B (en) 2022-03-29
CN113588523A (en) 2021-11-02

Similar Documents

Publication Publication Date Title
Yang et al. Luminol/antibody labeled gold nanoparticles for chemiluminescence immunoassay of carcinoembryonic antigen
US11073517B1 (en) Method for preparing nanohybrid used for ratiometric fluorescence and ratiometric electrochemical sensing simultaneously
Song et al. Fluorescent-magnetic-biotargeting multifunctional nanobioprobes for detecting and isolating multiple types of tumor cells
US8092859B2 (en) Synthesis of highly luminescent colloidal particles
WO2023005614A1 (en) Frame structure-based nanoparticle used for mass cytometry and preparation method therefor
CN110146571B (en) Bimetallic organic framework composite material aptamer sensor and preparation method and application thereof
JP2007506084A (en) Nanoparticle conjugate and method for producing the same
CN108535342B (en) Preparation method and application of electrochemical immunosensor based on palladium-doped molybdenum disulfide nanospheres
Li et al. A label-free electrochemical aptasensor based on the core–shell Cu-MOF@ TpBD hybrid nanoarchitecture for the sensitive detection of PDGF-BB
Li et al. Selective Capture and Quick Detection of Targeting Cells with SERS‐Coding Microsphere Suspension Chip
Qin et al. Electrochemiluminescence immunoassay of human chorionic gonadotropin using silver carbon quantum dots and functionalized polymer nanospheres
CN109839501B (en) Electrochemiluminescence immunosensor for measuring circulating tumor cells and preparation method and application thereof
US20230333099A1 (en) Composite magnetic nanomaterial based on dna tetrahedron, preparation therefor and use thereof
Zhang et al. Tantalum oxide nanoparticle-based mass tag for mass cytometry
Roberts et al. Reproducible large-scale synthesis of surface silanized nanoparticles as an enabling nanoproteomics platform: enrichment of the human heart phosphoproteome
CN109613244B (en) Preparation method and application of Ag @ Pt-CuS labeled immunosensor
Ma et al. Multi-targeting magnetic hyaluronan capsules efficiently capturing circulating tumor cells
CN112240929A (en) Donor particle for homogeneous phase chemiluminescence analysis and application thereof
CN110687175B (en) Construction method of electrochemical luminescence sensor based on cerium dioxide and nano-silver dual-enhanced perylene tetracarboxylic acid luminescence
Liu et al. Nanovehicles based bioassay labels
CN106366195B (en) PD-L1 antibody immunomagnetic beads and preparation method thereof
Zhu et al. Immuno-affinitive supramolecular magnetic nanoparticles incorporating cucurbit [8] uril-mediated ternary host-guest complexation structures for high-efficient small extracellular vesicle enrichment
Noviana et al. Advances in nanomaterial-based biosensors for determination of glycated hemoglobin
Lyu et al. Enhanced Chemiluminescence under the Nanoconfinement of Covalent–Organic Frameworks and Its Application in Sensitive Detection of Cancer Biomarkers
CN110702758B (en) Method for enhancing luminous intensity of squamous cell carcinoma antigen in electrochemical luminescence detection

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22848219

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE